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Carriers of cystic fibrosis among sperm donors: complete CFTR gene analysis versus CFTR genotyping

      Objective

      To determine the frequency of cystic fibrosis (CF) carriers among sperm donors in Spain studied through a complete analysis of the CFTR gene and to compare the results with those that would have been obtained by the 4 genotyping panels of the CFTR gene most commonly used as a carrier test in the context of assisted reproduction in our country.

      Design

      Descriptive observational study.

      Setting

      Private center.

      Patients

      Nine hundred thirty-five sperm donors, from January 2014 to June 2019.

      Intervention

      None.

      Main Outcome Measure

      Presence of pathogenic variants in the CFTR gene.

      Results

      17% of the donors were carriers of at least 1 pathogenic variant in CFTR, with 39 different pathogenic variants detected. Only 4 of these 39 variants (10.27%) would have been detected by the 4 genotyping tests considered, and 22 variants (56.41%) would not have been detected by any of the genotyping tests. The pathogenic variants of the CFTR gene included in the different genotyping tests analyzed vary widely, and <50% are common to all of them.

      Conclusions

      Although the was not based in the general population, these results show that the use of genotyping tests is associated with a high reproductive risk, because the rate of detection of CF carriers was lower when these panels were applied, in comparison with the complete study of the CFTR gene. We recommend that complete sequencing of the CFTR gene by next-generation sequencing be performed as a screening method for CF in sperm donors.
      Portadores de fibrosis quística entre donantes de esperma: análisis completo del gen CFTR versus genotipado CFTR.

      Objetivo

      Determinar la frecuencia de portadores de fibrosis quística (FQ) entre los donantes de esperma en España estudiados mediante un análisis completo del gen CFTR, y comparar los resultados con los que se habrían obtenido mediante los 4 paneles de genotipado del gen CFTR más utilizados como test de portadores en el contexto de la reproducción asistida en nuestro país.

      Diseño

      Estudio observacional descriptivo.

      Entorno

      Centro privado.

      Pacientes

      Novecientos treinta y cinco donantes de esperma, desde enero de 2014 a junio de 2019.

      Intervención

      Ninguna.

      Principales medidas de resultado

      Presencia de variantes patogénicas en el gen CFTR.

      Resultados

      El 17% de los donantes eran portadores de al menos 1 variante patogénica en CFTR, con detección de 39 diferentes variantes patogénicas detectadas. Solamente 4 de estas 39 variantes (10.27%) habrían sido detectadas por las 4 pruebas de genotipado consideradas, y 22 variantes (56.41%) no habrían sido detectadas por ninguna de las pruebas de genotipado. Las variantes patogénicas del gen CFTR incluidas en las diferentes pruebas de genotipado analizadas varían ampliamente, y <50% son comunes a todos ellos.

      Conclusiones

      Aunque no se basaron en la población general, estos resultados muestran que el uso de pruebas de genotipado está asociado con un alto riesgo reproductivo, debido a que la tasa de detección de portadores de FQ fue menor cuando se aplicaron estos paneles, en comparación con el estudio completo del gen CFTR. Recomendamos que la secuenciación completa del gen CFTR mediante secuenciación de última generación (NGS) sea realizada como método de detección de la FQ en donantes de esperma.

      Key Words

      Discuss: You can discuss this article with its authors and other readers at https://www.fertstertdialog.com/users/16110-fertility-and-sterility/posts/65610-29532
      With an approximate incidence of 1 in 2500 live births, cystic fibrosis (CF) is one of the most common severe autosomal recessive diseases in the white population. It is caused by mutations in the cystic fibrosis transmembrane regulatory duct gene (CFTR, OMIM602421) (
      • Rowntree R.K.
      • Harris A.
      The phenotypic consequences of CFTR mutations.
      ).
      Since the CFTR gene, located on chromosome 7q31 (
      • Rommens J.M.
      • Iannuzzi M.C.
      • Kerem B.
      • Drumm M.L.
      • Melmer G.
      • Dean M.
      • et al.
      Identification of the cystic fibrosis gene: chromosome walking and jumping.
      ), was first cloned, >2,000 disease-causing sequence variants have been identified, thanks to major advances in molecular biology techniques (
      • Drumm M.L.
      • Ziady A.G.
      • Davis P.B.
      Genetic variation and clinical heterogeneity in cystic fibrosis.
      ). Most mutations are rare, with a population frequency <0.01%.
      The large number of mutations detected and their genetic and phenotypic variability make the study of carriers in a healthy population a complex task. There is a wide variety of mutation panels of the CFTR gene, each one studying a limited number of variants and targeting particular populations and ethnic groups. These panels usually incorporate variants based on criteria of population frequency and phenotypic severity. Owing to the great diversity of mutations described in the CFTR gene, the effective detection of CF carriers by means of such targeted panels is limited. This is especially apparent with respect to subpopulations such as Hispanic individuals, among whom the mutations are not sufficiently well characterized despite the great efforts that have been made in this respect (
      • Kammesheidt A.
      • Kharrazi M.
      • Graham S.
      • Young S.
      • Pearl M.
      • Duncop C.
      • et al.
      Comprehensive genetic analysis of the cystic fibrosis transmembrane conductance regulator from dried blood specimens: implications for newborn screening.
      ).
      The genetic heterogeneity of the disease is further complicated by the existence of different classifications of the recognized clinical forms of CF, such as the classic form, disorders related to CF (lung disease without pancreatic disease, isolated pancreatitis, chronic sinusitis and/or lung disease in adults), and isolated congenital bilateral absence of the vas deferens (
      • Rowntree R.K.
      • Harris A.
      The phenotypic consequences of CFTR mutations.
      ,
      • Strom C.M.
      • Redman J.B.
      • Peng M.
      The dangers of including nonclassical cystic fibrosis variants in population-based screening panels: p.L997F, further genotype/phenotype correlation data.
      ). Therefore, a given genetic test may present different detection rates, depending on whether its application is directed toward a specific clinical form (
      • Lucarelli M.
      • Bruno S.M.
      • Pierandrei S.
      • Ferraguti G.
      • Testino G.
      • Truglio G.
      • et al.
      The impact on genetic testing of mutational patterns of CFTR gene in different clinical macrocategories of cystic fibrosis.
      ).
      Nevertheless, owing to the high prevalence of CF, many scientific societies recommend screening for sperm donors (
      • Sims C.A.
      • Callum P.
      • Ray M.
      • Iger J.
      • Falk R.E.
      Genetic testing of sperm donors: survey of current practices.
      ). However, none has specified which variants should be included in the study and which should not, or whether the screening should be directed toward a specific clinical form or should address the entire spectrum of clinical symptoms associated with CF, or whether the CFTR gene should be studied by complete sequencing and nondirected analysis of variants or by using a panel of targeted variants, i.e., a genotyping test.
      The aim of this study was to determine, through a complete study of the CFTR gene by next-generation sequencing (NGS), the frequency of sperm donors carrying pathogenic variants of the CFTR gene and to analyze the detection rate of these carriers that would have been obtained by applying the genotyping tests for CFTR gene mutations that are most commonly used in Spain.

      Materials and methods

       Population

      From January 2014 to June 2019, 5,872 potential sperm donors were interviewed. A semen analysis was performed on the 3,604 men who’s personal and family medical histories met the criteria for continuing in the donation program. After the selection process established by the sperm bank was applied, 935 men were finally accepted as donors.
      The study was based on these 935 men, ages 18 to 35 years, who were accepted as donors in the framework of a sperm donation program conducted by the Ceifer Biobanco sperm bank (Granada, Spain) from January 2014 to June 2019. All the sperm donors included in the study were white. Of the 935 individuals, 6 were North African, 923 were of Mediterranean origin (from Spain, Italy, or France), and 6 were Nordic.
      Sperm donors were selected in accordance with the legal regulations in force in Spain (Act 14/2006, of May 26, on assisted human reproduction techniques) and following the sperm bank’s own criteria for minimum seminal quality (sperm concentration >50 × 106 spz/mL, progressive motility >50%, sperm morphology [normal forms] >4%, and semen volume >2 mL), which are much stricter than the reference limits of the World Health Organization (
      WHO laboratory manual for the examination and processing of human semen. Fifth edition. World Health Organization.
      ). An extended carrier test for recessive diseases was performed on all donors who were accepted, in accordance with applicable legal criteria and with the sperm parameters established. In addition, all donors underwent a pretest and a posttest genetic counseling session to discuss basic genetic concepts, technical aspects of mass sequencing, and the implications of possible outcomes from the study. Institutional Review Board approval for the study was not necessary because the donors underwent routine genetic screening by NGS in our center, and no additional intervention was applied. Written informed consent, which complies with the legal requirements in Spain (Act 14/2006, of May 26, on assisted human reproduction techniques) and with the recommendations of the Spanish Fertility Society, was provided by each subject before participation in the study, accepting the use of their genetic data for research purposes. Donor information and data were anonymized and deidentified before analysis.

       Assessment of Sperm Quality

      Donor candidates introduced the semen samples into sterile polypropylene containers by masturbation. After liquefaction, sperm volume, concentration, progressive motility, and morphology were determined by trained laboratory technicians following the guidelines of the World Health Organization (
      WHO laboratory manual for the examination and processing of human semen. Fifth edition. World Health Organization.
      ). Total motility count was calculated by multiplying sperm volume by concentration and the percentage of progressive motility. All semen determinations were performed by professional technicians using the same apparatus, and all took part in internal and external quality control programs. The values obtained from the first semen samples provided by each donor candidate were used for this study.

       CFTR gene study

      The CFTR gene study is included in the qCarrier Plus carrier test (qGenomics, Barcelona, Spain), which uses NGS to detect the presence of genetic variants related to >300 recessive inheritance disorders. Only high-quality readings are used, with an average sequencing error of <1 base in 1,000. This technique is capable of detecting single nucleotide variants and small insertions and deletions (in/dels) of ≤9 nucleotides, with high analytic sensitivity (single nucleotide variant >99%; in/dels >85%) and specificity (>99%) The Carrier Plus Test sequences the CFTR gene completely (exons and introns), analyzes the variants found in the exonic and intronic regions (±5 nucleotides), and the variants of the deep intronic regions that are classified in ClinVar (http://www.ncbi.nlm.nih.gov/clinvar) as pathogenic.
      To obtain this level of performance, the samples are sequenced at high average coverage values, generally >150×. This ensures that a large majority of the sequenced bases (>96%) are read at a minimum depth of 30×. At this depth, the probability of not identifying a heterozygous variant as such, i.e., of taking an alternative heterozygous variant as a reference, is very low (0.016%) in the detection of ≤5 alternative reads out of 30 within a heterozygous variant, corresponding to an allele balance of 20%. In other words, the sensitivity achieved is 99.984% at 30×. Moreover, with this coverage the probability of a sequencing error being observed in >20% of the reads is also very low (this value depends on the error rate of the sequencing platform, usually <1%), thus minimizing the possibility of false positives.
      The reading depth of each variant is taken into account to assess its potential accuracy, without hard filters. In regions that have been sequenced with bases <15×, 10 times below the minimum average coverage, the probability of a sequencing error being interpreted as a real variant (false positive, lack of specificity) or of a heterozygous position being seen as a homozygous reference (lack of sensitivity) increases substantially. At 15×, the probability of observing a real heterozygous variant with an allele balance of <20% (as if it were a reference) is very low, at 0.36%, i.e. the sensitivity is 99.64%. When we approach very low depth values (<10×), we try to maximize both sensitivity and specificity. To do so, the reading depth is taken into account so as to assess the potential accuracy of the variant, but only after the filtering and interpretation process has concluded. All the variants that are considered potentially pathogenic are inspected individually and manually. When a known pathogenic variant is identified at a very low depth (i.e., a doubtful case), it may be validated by an alternative technique if this is considered appropriate (Sanger).

       Interpretation of Variants

      All the variants found were classified as pathogenic (pathogenic and/or probably pathogenic), common variants (polymorphisms), or variants of uncertain significance (
      • Richards S.
      • Aziz N.
      • Bale S.
      • Bick D.
      • Das S.
      • Gastier-Foster J.
      • et al.
      Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology.
      ), according to the following criteria.
      Pathogenic variants (pathogenic and/or probably pathogenic variants): mutations classed as pathogenic or probably pathogenic in the ClinVar database, in the Human Gene Mutation Database (http://www.hgmd.cf.ac.uk), or so described in the literature associated with the disease. Also included in this group are the de novo truncating variants (nonsense, splicing, frameshift, and large copy number variations [CNVs]).
      Common variants/polymorphisms: these variants are present in the databases of control individuals (1,000 genomes (http://1000genomes.org), Exome Variant Server (http://evs.gs.washington.edu/EVS/), and Exome Aggregation Consortium (http://exac.broadinstitute.org). These variants appear more frequently in the population than in the expected frequency of carriers, are present in homozygous controls, are reported as benign or probably benign in the ClinVar database, and are not reported to be associated with the disease.
      Variants of uncertain significance: these variants have not previously been reported as pathogenic in the ClinVar or Human Gene Mutation Database databases and present frequencies in the population that are compatible with those of the disease.
      For each of the pathogenic variants found, we reviewed its classification in the Cystic Fibrosis Mutation Database (http://www.cftr2.org/) (categories: CF-causing variant, Non–CF-causing variant, Variant of varying clinical consequence, Still under evaluation). In addition, we conducted a bibliographic review of all these variants and determined the expected population frequency according to the GenomAD database (https://gnomad.broadinstitute.org/), the population frequency observed in the sperm donors studied, and the clinical classification of the variants according to the categories of Castellani et al. (
      • Castellani C.
      • Cuppens H.
      • Macek M.
      • Cassiman J.J.
      • Kerem E.
      • Durie P.
      • et al.
      Consensus on the use and interpretation of cystic fibrosis mutation analysis in clinical practice.
      ): class A: mutations that cause CF; class B: mutations that are associated with clinically related CF; class C: clinical consequence of the mutations not known; class D: mutations with unlikely or unknown clinical significance).

       Targeted Tests of Mutations of the CFTR Gene (Genotyping Test)

      The study included an analysis of the 4 genotyping tests most commonly used in assisted reproduction centers in Spain to detect carriers of recessive diseases: panel H (HERES-GEN [Full Genomics]: 149 variants associated with CF), panel I (CGT 600 [iGenomix]: 146 variants associated with CF), panel SG (Preconception Focus [Sistemas Genómicos]: 327 variants associated with CF, and panel E (CFEU2v1 + CF Iberian + CF Poly-T [Elucigene]: 69 variants associated with CF, targeting European and Iberian populations).
      The comparison with these 4 genotype tests was performed only at the theoretical level, checking whether or not the variants found in semen donors by NGS were included in the genotyping tests.
      The variants addressed in all 4 targeted tests were termed common variants, and those studied by a single targeted test were termed exclusive.

       Statistical Methods

      The frequencies of CF carrier status observed in the sperm donors and those described in GenomAD were compared by the Fisher test, assuming statistical significance at P<.05. The 4 genotyping tests were compared by using a Venn diagram to assess their similarities and differences. In addition, we wished to determine the diagnostic sensitivity of each genotyping test, i.e., the number of pathogenic variants found in the sperm donors and included in a specific genotyping panel, and the clinical sensitivity, i.e., the number of sperm donors bearing a pathogenic variant in the CFTR gene that was detectable by any of the genotyping tests. The diagnostic and clinical sensitivities of the different tests were compared by the χ2 test, with a level of statistical significance of P<.05.
      Following the international guidelines for semen quality studies (
      • Sánchez-Pozo M.C.
      • Mendiola J.
      • Serrano M.
      • Mozas J.
      • Björndahl L.
      • Menkveld R.
      • et al.
      Proposal of guidelines for the appraisal of SEMen QUAlity studies (SEMQUA).
      ), the semen quality parameters were analyzed on a logarithmic scale to normalize the distribution. Student’s t-test was used to compare the semen parameters obtained for carrier donors of at least 1 pathogenic variant in the CFTR gene with those of noncarrier donors Statistical significance was assumed at P<.05.

      Results

       Frequency of Sperm Donor Carriers of Pathogenic Variants in the CFTR Gene

      Of the 935 sperm donors, 159 (17%) were carriers of at least 1 pathogenic variant in the CFTR gene. The frequency, therefore, was approximately 1 in 6.
      In total, 39 different variants were found, of which the most frequent were c.1210-34TG(11)T(5) (26.4% of the carriers), c.1210-34TG(12)T(5) (15.1% of the carriers), R75Q (11.3% of the carriers), L997F (10.1% of the carriers), G576A (6.9% of the carriers), V754M, and F508del (1 each, 3.8% of the carriers). Of those 39 variants, 1 was a deletion, 31 were missense variants, 2 were nonsense variants, and 5 were abnormal splicing (Table 1). All of the mutations found were heterozygous. The individuals presenting >1 variant were all healthy.
      Table 1Pathogenic variants of the CFTR gene found in sperm donors.
      Variant cDNA name (Variant legacy name)No. of donor carriersGTMutationFrequency total GenomAD (10-4)Frequency CFTR+ Ceifer (10-3)
      c.1001G>A (R334Q)10Missense1.061.07
      c.1043T>A (M348K)10Missense1.271.07
      c.1052C>G (T351S)10Missense1.701.07
      c.1210-34TG(11)T(5)42Eintron variantN/S44.92
      c.1210-34TG(12)T(5)24Eintron variantN/S25.67
      c.1210-34TG(13)T(5)1Eintron variantN/S1.07
      c.1310G>A (G437D)10Missense0.121.07
      P<.05; †P<.01; ‡P<.0001; frequency total GenomAD vs. frequency CFTR+ Ceifer.
      c.1727G>C/c.2002C>T/c.1327G>T (G576A/R668C/D443Y)20Missense50.42/59.79/2.6011.77
      P<.05; †P<.01; ‡P<.0001; frequency total GenomAD vs. frequency CFTR+ Ceifer.
      / 9.63/2.14
      P<.05; †P<.01; ‡P<.0001; frequency total GenomAD vs. frequency CFTR+ Ceifer.
      c.1521_1523delCTT (F508del)7H-E-I-SGDeletion71.727.49
      c.1624G>T (G542X)1H-E-I-SGNonsense3.221.07
      c.1727G>C (G576A)20Missense50.4211.77
      c.1727G>C /c.2002C>T (G576A/R668C)70Missense50.42/59.7911.77/9.63
      c.202A>G (K68E)10Missense1.491.07
      c.3808G>A/c.220C>T (D1270N/R74W)20/SGMissense15.12/17.181.07/2.14
      c.221G>A (R74Q)1IMissense2.551.07
      c.224G>A (R75Q)180Missense156.519.25
      c.2260G>A (V754M)70Missense18.167.49
      P<.05; †P<.01; ‡P<.0001; frequency total GenomAD vs. frequency CFTR+ Ceifer.
      c.2855T>C (M952T)1IMissense2.511.07
      c.2856G>C (M952I)10Missense0.801.07
      c.2900T>C (L967S)10Missense7.041.07
      c.2991G>C (L997F)160Missense22.2217.11
      P<.05; †P<.01; ‡P<.0001; frequency total GenomAD vs. frequency CFTR+ Ceifer.
      c.3023T>A (V1008D)10Missense0.041.07
      P<.05; †P<.01; ‡P<.0001; frequency total GenomAD vs. frequency CFTR+ Ceifer.
      c.3041A>G (Y1014C)20Missense2.582.14
      P<.05; †P<.01; ‡P<.0001; frequency total GenomAD vs. frequency CFTR+ Ceifer.
      c.3154T>G (F1052V)1SGMissense6.281.07
      c.3276C>A (Y1092X)1I-E-SGNonsense0.181.07
      P<.05; †P<.01; ‡P<.0001; frequency total GenomAD vs. frequency CFTR+ Ceifer.
      c.3454G>C (D1152H)1H-E-SGMissense3.761.07
      c.3458T>A (V1153E)10Missense0.321.07
      P<.05; †P<.01; ‡P<.0001; frequency total GenomAD vs. frequency CFTR+ Ceifer.
      c.3705T>G (S1235R)20Missense50.392.14
      c.3718-2477C>T (3849+10kbC->T)1EIntron variantN/S1.07
      c.3909C>G (N1303K)2H-E-I-SGMissense1.392.14
      P<.05; †P<.01; ‡P<.0001; frequency total GenomAD vs. frequency CFTR+ Ceifer.
      c.4097T>C (I1366T)10Missense0.121.07
      P<.05; †P<.01; ‡P<.0001; frequency total GenomAD vs. frequency CFTR+ Ceifer.
      c.4333G>A (D1445N)1IMissense3.761
      c.580-1G>T (712-1G->T)1H-E-SGsplice acceptor variant0.041.07
      P<.05; †P<.01; ‡P<.0001; frequency total GenomAD vs. frequency CFTR+ Ceifer.
      c.601G>A (V201M)10Missense2.161.07
      c.617T>G (L206W)1H-E-I-SGMissense1.801.07
      c.772A>G (R258G)10Missense1.721.07
      c.91C>T (R31C)10Missense16.401.07
      c.958T>G (L320V)20Missense5.772.139037
      Note: 0 = none; E = panel E; GT = genotyping test, including study of the variant; H = panel H; I = panel I; SG = panel SG.
      P<.05; P<.01; P<.0001; frequency total GenomAD vs. frequency CFTR+ Ceifer.
      Of the spermiogram analyses performed on the 3,604 donor candidates, 43 (1.19%) presented azoospermia. Of the group of azoospermic candidates, 16 (37.20%) had a low seminal volume (<1.5 mL), and 27 (62.80%) had a normal volume (>1.5 mL). Of the 16 individuals with hypospermia, 4 presented pH<6, 1 had pH between 6.1 and 6.5, 2 had pH 6.5 to 7.1, and 9 had pH between 7.2 and 8. Among the donors there were no statistically significant differences in the semen parameters analyzed between carrier and noncarrier donors of CFTR variants (Supplemental Table 1, available online).

       Comparison of the Variants of the Genotyping Tests for the CFTR Gene

      Analysis of all the genotyping tests showed that only 32 variants were studied in all 4 tests. Panels E, I, H, and SG presented 15, 26, 27, and 148 variants exclusively, respectively (Fig. 1). The percentages corresponding to the common and exclusive variants in each panel are shown in Table 2.
      Figure thumbnail gr1
      Figure 1Number of variants of the CFTR gene included in 4 different genotyping panels (panel H: HERES-GEN; panel I: CGT 600; panel SG: Preconception Focus; panel E: CFEU2v1 + CF Iberian + CF Poly-T).
      Table 2Percentage of common-exclusive variants and clinical-diagnostic sensitivity in each of the genotyping tests.
      VariablePanel E (n = 69)Panel I (n = 146)Panel H (n = 149)Panel SG (n = 327)
      % Variants
       Common46.38% (32/69)21.92% (32/146)21.48% (32/149)9.79% (32/327)
       Specific21.74% (15/69)17.81 (26/146)18.12% (27/149)45.26% (148/327)
      Sensitivity
       Diagnostic28.2% (11/39)20.5% (8/39)15.4% (6/39)23.1% (9/39)
       Clinical51.6% (82/159)9.4% (15/159)
      Statistical differences in clinical sensitivity (P<.05) of the different panels vs. panel E.
      8.2% (13/159)
      Statistical differences in clinical sensitivity (P<.05) of the different panels vs. panel E.
      10.7% (17/159)
      Statistical differences in clinical sensitivity (P<.05) of the different panels vs. panel E.
      Note: Clinical sensitivity = number of carrier donors detected by the test / total number of carrier donors; Diagnostic sensitivity = number of variants detected by the test / total number of variants found in the study population; n = number of variants included in each genotyping panel.
      a Statistical differences in clinical sensitivity (P<.05) of the different panels vs. panel E.
      Of the 39 variants found in the sperm donors, 22 variants (56.41%) would not have been detected by any of the 4 genotyping tests, and only 4 of the 39 variants (10.27%) would have been detected by all of the genotyping tests (Table 1).
      No statistically significant differences were observed between the diagnostic sensitivities of the different genotyping tests evaluated. However, there were differences in the clinical sensitivities, with panel E being clearly more sensitive in this respect than the other 3 tests (Table 2).
      The pathogenic variants most frequently detected were c.1210-34TG(11,12,13)T(5), R75Q, L997F, R668C, G756A, and V754M. These variants were not detected by any of the 4 genotyping tests, with the exception of Poly 5T (c.1210-34TG(11,12,13)T(5)), which was detected only by panel E. Twelve of the 39 variants presented a statistically significantly higher frequency (P<.05) in the study population than those reported in GenomAD (Table 1).
      Regarding the clinical classification of the variants found, 7 of the 39 variants detected would be classified by CFTR2 as CF-causing variant (F508del, G542X, Y1092X, 3849+10kbC->T, N1303K, 712-1G->T, and L206W), and 12 as variants of varying clinical consequence or still under classification. Of those 19 variants associated with CF, 6 would not have been detected by any of the 4 genotyping tests. Of the 7 variants classified as causing CF by CFTR2, only 4 would have been detected by all 4 genotyping tests. Moreover, 12 of the 39 variants found in our study would be classified by Castellani et al. (
      • Castellani C.
      • Cuppens H.
      • Macek M.
      • Cassiman J.J.
      • Kerem E.
      • Durie P.
      • et al.
      Consensus on the use and interpretation of cystic fibrosis mutation analysis in clinical practice.
      ) as class A or class A–B (c.1210-34TG(12)T(5), c.1210-34TG(13)T(5), F508del, G542X, G576A, Y1092X, D1152H, S1235R, 3849+10kbC->T, N1303K, 712-1G->T, and L206W), whereas 6 would be classified as class B or class B–C. Of those 18 variants, 7 would not have been detected by any of the 4 genotyping tests (Table 3). Of the 12 variants classified by Castellani et al. (
      • Castellani C.
      • Cuppens H.
      • Macek M.
      • Cassiman J.J.
      • Kerem E.
      • Durie P.
      • et al.
      Consensus on the use and interpretation of cystic fibrosis mutation analysis in clinical practice.
      ) as class A or A–B, only 4 would have been detected by the 4 genotyping tests considered.
      Table 3Classification of the variants found in the sperm donors according to CFTR2, Castellani et al. (
      • Castellani C.
      • Cuppens H.
      • Macek M.
      • Cassiman J.J.
      • Kerem E.
      • Durie P.
      • et al.
      Consensus on the use and interpretation of cystic fibrosis mutation analysis in clinical practice.
      ), and their clinical expressiveness.
      Variant cDNA name (variant legacy name)CFTR2Castellani et al. (
      • Castellani C.
      • Cuppens H.
      • Macek M.
      • Cassiman J.J.
      • Kerem E.
      • Durie P.
      • et al.
      Consensus on the use and interpretation of cystic fibrosis mutation analysis in clinical practice.
      )
      Clinical expressiveness (reference)
      c.1001G>A (R334Q)VCN/S
      • LaRusch J.
      • Jung J.
      • General I.J.
      • Lewis M.D.
      • Woo Park H.
      • Brand R.E.
      • et al.
      Mechanisms of CFTR functional variants that impair regulated bicarbonate permeation and increase risk for pancreatitis but not for cystic fibrosis.
      ,
      • Havasi V.
      • Rowe S.M.
      • Kolettis P.N.
      • Dayangac D.
      • Sxahin A.
      • Grangeia A.
      • et al.
      Association of cystic fibrosis genetic modifiers with congenital bilateral absence of the vas deferens.
      ,
      • Behar D.M.
      • Inbar O.
      • Shteinberg M.
      • Gur M.
      • Mussaffi H.
      • Shoseyov D.
      • et al.
      Nationwide genetic analysis for molecularly unresolved cystic fibrosis patients in a multiethnic society: implications for preconception carrier screening.
      c.1043T>A (M348K)N/SN/S
      • Masson E.
      • Chen J.M.
      • Audrézet M.P.
      • Cooper D.N.
      • Férec C.
      A conservative assessment of the major genetic causes of idiopathic chronic pancreatitis: data from a comprehensive analysis of PRSS1, SPINK1, CTRC and CFTR genes in 253 young French patients.
      ,
      • Audrézet M.P.
      • Novelli G.
      • Mercier B.
      • Sangiuolo F.
      • Maceratesi P.
      • Férec C.
      • et al.
      Identification of three novel cystic fibrosis mutations in a sample of Italian cystic fibrosis patients.
      ,
      • Scotet V.
      • De Braekeleer M.
      • Audrézet M.P.
      • Lodé L.
      • Verlingue C.
      • Quéré I.
      • et al.
      Prevalence of CFTR mutations in hypertrypsinaemia detected through neonatal screening for cystic fibrosis.
      c.1052C>G (T351S)N/SN/S
      • Dörk T.
      • Dworniczak B.
      • Aulehla-Scholz C.
      • Wieczorek D.
      • Böhm I.
      • Mayerova A.
      • et al.
      Distinct spectrum of CFTR gene mutations in congenital absence of vas deferens.
      ,
      • Schrijver I.
      • Ramalingam S.
      • Sankaran R.
      • Swanson S.
      • Dunlop C.L.
      • Keiles S.
      • et al.
      Diagnostic testing by CFTR gene mutation analysis in a large group of Hispanics: novel mutations and assessment of a population-specific mutation spectrum.
      c.1210-34TG(11)T(5)VCB–C
      • Grody W.W.
      • Cutting G.R.
      • Klinger K.W.
      • Richards C.S.
      • Watson M.S.
      • Desnick R.J.
      Laboratory standards and guidelines for population-based cystic fibrosis carrier screening.
      c.1210-34TG(12)T(5)VCA–B
      • Grody W.W.
      • Cutting G.R.
      • Klinger K.W.
      • Richards C.S.
      • Watson M.S.
      • Desnick R.J.
      Laboratory standards and guidelines for population-based cystic fibrosis carrier screening.
      ,
      • Casals T.
      • De-Gracia J.
      • Gallego M.
      • Dorca J.
      • Rodríguez-Sanchón B.
      • Ramos M.D.
      • et al.
      Bronchiectasis in adult patients: an expression of heterozygosity for CFTR gene mutations?.
      c.1210-34TG(13)T(5)VCA–B
      • Grody W.W.
      • Cutting G.R.
      • Klinger K.W.
      • Richards C.S.
      • Watson M.S.
      • Desnick R.J.
      Laboratory standards and guidelines for population-based cystic fibrosis carrier screening.
      c.1310G>A (G437D)N/SN/S
      • Bergougnoux A.
      • Viart V.
      • Miro J.
      • Bommart S.
      • Molinari N.
      • des Georges M.
      • et al.
      Should diffuse bronchiectasis still be considered a CFTR-related disorder?.
      ,
      • Kolesár P.
      • Minárik G.
      • Baldovic M.
      • Ficek A.
      • Kovács L.
      • Kádasi L.
      Mutation analysis of the CFTR gene in Slovak cystic fibrosis patients by DHPLC and subsequent sequencing: identification of four novel mutations.
      c.1727G>C/ c.2002C>T/c.1327G>T (G576A/R668C/D443Y)NCC/NCC/VCB
      • El-Seedy A.
      • Girodon E.
      • Norez C.
      • Pajaud J.
      • Pasquet M.C.
      • de Becdelièvre A.
      • et al.
      CFTR mutation combinations producing frequent complex alleles with different clinical and functional outcomes.
      ,
      • Pagani F.
      • Stuani C.
      • Tzetis M.
      • Kanavakis E.
      • Efthymiadou A.
      • Doudounakis S.
      • et al.
      New type of disease causing mutations: the example of the composite exonic regulatory elements of splicing in CFTR exon 12.
      ,
      • Ziętkiewicz E.
      • Rutkiewicz E.
      • Pogorzelski A.
      • Klimek B.
      • Voelkel K.
      • Witt M.
      CFTR mutations spectrum and the efficiency of molecular diagnostics in Polish cystic fibrosis patients.
      ,
      • de Cid R.
      • Ramos M.D.
      • Aparisi L.
      • García C.
      • Mora J.
      • Estivill X.
      • et al.
      Independent contribution of common CFTR variants to chronic pancreatitis.
      c.1521_1523delCTT (F508del)CCA
      • Bombieri C.
      • Giorgi S.
      • Carles S.
      • de Cid R.
      • Belpinati F.
      • Tandoi C.
      • et al.
      A new approach for identifying non-pathogenic mutations: an analysis of the cystic fibrosis transmembrane regulator gene in normal individuals.
      ,
      • Padoan R.
      • Genoni S.
      • Moretti E.
      • Seia M.
      • Giunta A.
      • Corbetta C.
      Genetic and clinical features of false-negative infants in a neonatal screening programme for cystic fibrosis.
      ,
      • Casals T.
      • Ramos M.D.
      • Giménez J.
      • Larriba S.
      • Nunes V.
      • Estivill X.
      High heterogeneity for cystic fibrosis in Spanish families: 75 mutations account for 90% of chromosomes.
      c.1624G>T (G542X)CCA
      • Bombieri C.
      • Seia M.
      • Castellani C.
      Genotypes and phenotypes in cystic fibrosis and cystic fibrosis transmembrane regulator-related disorders.
      ,
      • Hamoir C.
      • Pepermans X.
      • Piessevaux H.
      • Jouret-Mourin A.
      • Weynand B.
      • Habyalimana J.B.
      • et al.
      Clinical and morphological characteristics of sporadic genetically determined pancreatitis as compared to idiopathic pancreatitis: higher risk of pancreatic cancer in CFTR variants.
      c.1727G>C (G576A)NCCA-B
      • Pagani F.
      • Stuani C.
      • Tzetis M.
      • Kanavakis E.
      • Efthymiadou A.
      • Doudounakis S.
      • et al.
      New type of disease causing mutations: the example of the composite exonic regulatory elements of splicing in CFTR exon 12.
      c.1727G>C/c.2002C>T (G576A/R668C)NCCB
      • El-Seedy A.
      • Girodon E.
      • Norez C.
      • Pajaud J.
      • Pasquet M.C.
      • de Becdelièvre A.
      • et al.
      CFTR mutation combinations producing frequent complex alleles with different clinical and functional outcomes.
      ,
      • Ziętkiewicz E.
      • Rutkiewicz E.
      • Pogorzelski A.
      • Klimek B.
      • Voelkel K.
      • Witt M.
      CFTR mutations spectrum and the efficiency of molecular diagnostics in Polish cystic fibrosis patients.
      c.202A>G (K68E)N/SN/S
      • Bombieri C.
      • Giorgi S.
      • Carles S.
      • de Cid R.
      • Belpinati F.
      • Tandoi C.
      • et al.
      A new approach for identifying non-pathogenic mutations: an analysis of the cystic fibrosis transmembrane regulator gene in normal individuals.
      ,
      • Kilinç M.O.
      • Ninis V.N.
      • Dağli E.
      • Demirkol M.
      • Ozkinay F.
      • Arikan Z.
      • et al.
      Highest heterogeneity for cystic fibrosis: 36 mutations account for 75% of all CF chromosomes in Turkish patients.
      ,
      • Kahnoski K.
      • Khoo S.K.
      • Nassif N.T.
      • Chen J.
      • Lobo G.P.
      • Segelov E.
      • et al.
      Alterations of the Birt-Hogg-Dubé gene (BHD) in sporadic colorectal tumours.
      c.3808G>A/c.220C>T (D1270N/R74W)VCB
      • Ratbi I.
      • Legendre M.
      • Niel F.
      • Martin J.
      • Soufir J.C.
      • Izard V.
      • et al.
      Detection of cystic fibrosis transmembrane conductance regulator (CFTR) gene rearrangements enriches the mutation spectrum in congenital bilateral absence of the vas deferens and impacts on genetic counselling.
      ,
      • de Prada A.
      • Bütschi F.N.
      • Bouchardy I.
      • Beckmann J.S.
      • Morris M.A.
      • Hafen G.M.
      • Fellmann F.
      [R74W;R1070W;D1270N]: a new complex allele responsible for cystic fibrosis.
      ,
      • de Gracia J.
      • Mata F.
      • Alvarez A.
      • Casals T.
      • Gatner S.
      • Vendrell M.
      • et al.
      Genotype-phenotype correlation for pulmonary function in cystic fibrosis.
      c.221G>A (R74Q)N/SN/S
      • LaRusch J.
      • Jung J.
      • General I.J.
      • Lewis M.D.
      • Woo Park H.
      • Brand R.E.
      • et al.
      Mechanisms of CFTR functional variants that impair regulated bicarbonate permeation and increase risk for pancreatitis but not for cystic fibrosis.
      c.224G>A (R75Q)NCCNCC
      • Tzetis M.
      • Efthymiadou A.
      • Strofalis S.
      • Psychou P.
      • Dimakou A.
      • Pouliou E.
      • et al.
      CFTR gene mutations–including three novel nucleotide substitutions–and haplotype background in patients with asthma, disseminated bronchiectasis and chronic obstructive pulmonary disease.
      ,
      • Martinez B.
      • Heller M.
      • Gaitch N.
      • Hubert D.
      • Burgel P.R.
      • Levy P.
      • et al.
      p.Arg75Gln, a CFTR variant involved in the risk of CFTR-related disorders?.
      ,
      • Divac A.
      • Nikolic A.
      • Mitic-Milikic M.
      • Nagorni-Obradovic L.
      • Petrovic-Stanojevic N.
      • Dopudja-Pantic V.
      • et al.
      High frequency of the R75Q CFTR variation in patients with chronic obstructive pulmonary disease.
      ,
      • Borowitz D.
      CFTR, bicarbonate, and the pathophysiology of cystic fibrosis.
      c.2260G>A (V754M)NCCN/S
      • Bergougnoux A.
      • Viart V.
      • Miro J.
      • Bommart S.
      • Molinari N.
      • des Georges M.
      • et al.
      Should diffuse bronchiectasis still be considered a CFTR-related disorder?.
      ,
      • Dal'Maso V.B.
      • Mallmann L.
      • Siebert M.
      • Simon L.
      • Saraiva-Pereira M.L.
      Dalcin P de T. Diagnostic contribution of molecular analysis of the cystic fibrosis transmembrane conductance regulator gene in patients suspected of having mild or atypical cystic fibrosis.
      ,
      • Grangeia A.
      • Sá R.
      • Carvalho F.
      • Martin J.
      • Girodon E.
      • Silva J.
      • et al.
      Molecular characterization of the cystic fibrosis transmembrane conductance regulator gene in congenital absence of the vas deferens.
      c.2855T>C (M952T)N/SN/S
      • Schneider A.
      • Larusch J.
      • Sun X.
      • Aloe A.
      • Lamb J.
      • Hawes R.
      • et al.
      Combined bicarbonate conductance-impairing variants in CFTR and SPINK1 variants are associated with chronic pancreatitis in patients without cystic fibrosis.
      ,
      • Mak V.
      • Zielenski J.
      • Tsui L.C.
      • Durie P.
      • Zini A.
      • Martin S.
      • et al.
      Proportion of cystic fibrosis gene mutations not detected by routine testing in men with obstructive azoospermia.
      c.2856G>C (M952I)N/SB
      • Desgeorges M.
      • Mégarbané A.
      • Guittard C.
      • Carles S.
      • Loiselet J.
      • Demaille J.
      • et al.
      Cystic fibrosis in Lebanon: distribution of CFTR mutations among Arab communities.
      c.2900T>C (L967S)VCN/S
      • Masson E.
      • Chen J.M.
      • Audrézet M.P.
      • Cooper D.N.
      • Férec C.
      A conservative assessment of the major genetic causes of idiopathic chronic pancreatitis: data from a comprehensive analysis of PRSS1, SPINK1, CTRC and CFTR genes in 253 young French patients.
      ,
      • Borowitz D.
      CFTR, bicarbonate, and the pathophysiology of cystic fibrosis.
      c.2991G>C (L997F)NCCB
      • Strom C.M.
      • Redman J.B.
      • Peng M.
      The dangers of including nonclassical cystic fibrosis variants in population-based screening panels: p.L997F, further genotype/phenotype correlation data.
      ,
      • Casals T.
      • De-Gracia J.
      • Gallego M.
      • Dorca J.
      • Rodríguez-Sanchón B.
      • Ramos M.D.
      • et al.
      Bronchiectasis in adult patients: an expression of heterozygosity for CFTR gene mutations?.
      ,
      • Hamoir C.
      • Pepermans X.
      • Piessevaux H.
      • Jouret-Mourin A.
      • Weynand B.
      • Habyalimana J.B.
      • et al.
      Clinical and morphological characteristics of sporadic genetically determined pancreatitis as compared to idiopathic pancreatitis: higher risk of pancreatic cancer in CFTR variants.
      ,
      • Borowitz D.
      CFTR, bicarbonate, and the pathophysiology of cystic fibrosis.
      ,
      • Gomez M.
      • Benetazzo M.G.
      • Marzari M.G.
      • Bombieri C.
      • Belpinati F.
      • Castellani C.
      • et al.
      High frequency of cystic fibrosis transmembrane regulator mutation L997F in patients with recurrent idiopathic pancreatitis and in newborns with hypertrypsinemia.
      ,
      • Keiles S.
      • Kammesheidt A.
      Identification of CFTR, PRSS1 and SPINK1 mutations in 381 patients with pancreatitis.
      c.3023T>A (V1008D)N/SN/S
      • Alonso M.J.
      • Heine-Suñer D.
      • Calvo M.
      • Rosell J.
      • Giménez J.
      • Ramos M.D.
      • et al.
      Spectrum of mutations in the CFTR gene in cystic fibrosis patients of Spanish ancestry.
      c.3041A>G (Y1014C)SUEN/S
      • Casals T.
      • De-Gracia J.
      • Gallego M.
      • Dorca J.
      • Rodríguez-Sanchón B.
      • Ramos M.D.
      • et al.
      Bronchiectasis in adult patients: an expression of heterozygosity for CFTR gene mutations?.
      ,
      • Alonso M.J.
      • Heine-Suñer D.
      • Calvo M.
      • Rosell J.
      • Giménez J.
      • Ramos M.D.
      • et al.
      Spectrum of mutations in the CFTR gene in cystic fibrosis patients of Spanish ancestry.
      ,
      • Casals T.
      • Bassas L.
      • Egozcue S.
      • Ramos M.D.
      • Giménez J.
      • Segura A.
      • et al.
      Heterogeneity for mutations in the CFTR gene and clinical correlations in patients with congenital absence of the vas deferens.
      c.3154T>G (F1052V)VCN/S
      • Divac A.
      • Nikolic A.
      • Mitic-Milikic M.
      • Nagorni-Obradovic L.
      • Petrovic-Stanojevic N.
      • Dopudja-Pantic V.
      • et al.
      High frequency of the R75Q CFTR variation in patients with chronic obstructive pulmonary disease.
      ,
      • LaRusch J.
      • Jung J.
      • General I.J.
      • Lewis M.D.
      • Woo Park H.
      • Brand R.E.
      • et al.
      Mechanisms of CFTR functional variants that impair regulated bicarbonate permeation and increase risk for pancreatitis but not for cystic fibrosis.
      ,
      • Gomez M.
      • Benetazzo M.G.
      • Marzari M.G.
      • Bombieri C.
      • Belpinati F.
      • Castellani C.
      • et al.
      High frequency of cystic fibrosis transmembrane regulator mutation L997F in patients with recurrent idiopathic pancreatitis and in newborns with hypertrypsinemia.
      ,
      • Van Goor F.
      • Yu H.
      • Burton B.
      • Hoffman B.J.
      Effect of ivacaftor on CFTR forms with missense mutations associated with defects in protein processing or function.
      ,
      • Puéchal X.
      • Bienvenu T.
      • Génin E.
      • Berthelot J.M.
      • Sibilia J.
      • Gaudin P.
      • et al.
      Mutations of the cystic fibrosis gene in patients with bronchiectasis associated with rheumatoid arthritis.
      c.3276C>A (Y1092X)CCA–B
      • De Braekeleer M.
      • Allard C.
      • Leblanc J.P.
      • Simard F.
      • Aubin G.
      Phenotypic variability in five cystic fibrosis patients compound heterozygous for the Y1092X mutation.
      ,
      • Schrijver I.
      • Oitmaa E.
      • Metspalu A.
      • Gardner P.
      Genotyping microarray for the detection of more than 200 CFTR mutations in ethnically diverse populations.
      c.3454G>C (D1152H)VCA–B
      • Terlizzi V.
      • Carnovale V.
      • Castaldo G.
      • Castellani C.
      • Cirilli N.
      • Colombo C.
      • et al.
      Clinical expression of patients with the D1152H CFTR mutation.
      ,
      • Peleg L.
      • Karpati M.
      • Bronstein S.
      • Berkenstadt M.
      • Frydman M.
      • Yonath H.
      • et al.
      The D1152H cystic fibrosis mutation in prenatal carrier screening, patients and prenatal diagnosis.
      c.3458T>A (V1153E)VCN/S
      • Dörk T.
      • Dworniczak B.
      • Aulehla-Scholz C.
      • Wieczorek D.
      • Böhm I.
      • Mayerova A.
      • et al.
      Distinct spectrum of CFTR gene mutations in congenital absence of vas deferens.
      ,
      • Padoan R.
      • Genoni S.
      • Moretti E.
      • Seia M.
      • Giunta A.
      • Corbetta C.
      Genetic and clinical features of false-negative infants in a neonatal screening programme for cystic fibrosis.
      c.3705T>G (S1235R)NCCA–B
      • Grangeia A.
      • Sá R.
      • Carvalho F.
      • Martin J.
      • Girodon E.
      • Silva J.
      • et al.
      Molecular characterization of the cystic fibrosis transmembrane conductance regulator gene in congenital absence of the vas deferens.
      ,
      • Puéchal X.
      • Bienvenu T.
      • Génin E.
      • Berthelot J.M.
      • Sibilia J.
      • Gaudin P.
      • et al.
      Mutations of the cystic fibrosis gene in patients with bronchiectasis associated with rheumatoid arthritis.
      ,
      • Feldmann D.
      • Couderc R.
      • Audrezet M.P.
      • Ferec C.
      • Bienvenu T.
      • Desgeorges M.
      • et al.
      CFTR genotypes in patients with normal or borderline sweat chloride levels.
      c.3718-2477C>T (3849+10kbC->T)CCA
      • de Gracia J.
      • Mata F.
      • Alvarez A.
      • Casals T.
      • Gatner S.
      • Vendrell M.
      • et al.
      Genotype-phenotype correlation for pulmonary function in cystic fibrosis.
      ,
      • Augarten A.
      • Kerem B.S.
      • Yahav Y.
      • Noiman S.
      • Rivlin Y.
      • Tal A.
      • et al.
      Mild cystic fibrosis and normal or borderline sweat test in patients with the 3849 + 10 kb C-->T mutation.
      ,
      • Duguépéroux I.
      • De Braekeleer M.
      The CFTR 3849+10kbC->T and 2789+5G->A alleles are associated with a mild CF phenotype.
      c.3909C>G (N1303K)CCA
      • Bombieri C.
      • Giorgi S.
      • Carles S.
      • de Cid R.
      • Belpinati F.
      • Tandoi C.
      • et al.
      A new approach for identifying non-pathogenic mutations: an analysis of the cystic fibrosis transmembrane regulator gene in normal individuals.
      ,
      • de Gracia J.
      • Mata F.
      • Alvarez A.
      • Casals T.
      • Gatner S.
      • Vendrell M.
      • et al.
      Genotype-phenotype correlation for pulmonary function in cystic fibrosis.
      ,
      • Ataseven F.
      • Ozer S.
      • Yilmaz R.
      • Senayli A.
      Bilateral spontaneous pneumothorax in a newborn with N1303K mutation of cystic fibrosis (CFTR) gene.
      ,
      • Osborne L.
      • Santis G.
      • Schwarz M.
      • Klinger K.
      • Dörk T.
      • McIntosh I.
      • et al.
      Incidence and expression of the N1303K mutation of the cystic fibrosis (CFTR) gene.
      ,
      • Chávez-Saldaña M.
      • Yokoyama E.
      • Lezana J.L.
      • Carnevale A.
      • Macías M.
      • Vigueras R.M.
      • et al.
      CFTR allelic heterogeneity in Mexican patients with cystic fibrosis: implications for molecular screening.
      c.4097T>C (I1366T)N/SN/S
      • Steiner B.
      • Rosendahl J.
      • Witt H.
      • Teich N.
      • Keim V.
      • Schulz H.U.
      • et al.
      Common CFTR haplotypes and susceptibility to chronic pancreatitis and congenital bilateral absence of the vas deferens.
      c.4333G>A (D1445N)N/SN/S
      • Schrijver I.
      • Ramalingam S.
      • Sankaran R.
      • Swanson S.
      • Dunlop C.L.
      • Keiles S.
      • et al.
      Diagnostic testing by CFTR gene mutation analysis in a large group of Hispanics: novel mutations and assessment of a population-specific mutation spectrum.
      ,
      • Claustres M.
      • Guittard C.
      • Bozon D.
      • Chevalier F.
      • Verlingue C.
      • Ferec C.
      • et al.
      Spectrum of CFTR mutations in cystic fibrosis and in congenital absence of the vas deferens in France.
      c.580-1G>T (712-1G->T)CCA–B
      • Casals T.
      • Ramos M.D.
      • Giménez J.
      • Larriba S.
      • Nunes V.
      • Estivill X.
      High heterogeneity for cystic fibrosis in Spanish families: 75 mutations account for 90% of chromosomes.
      ,
      • Casals T.
      • Bassas L.
      • Egozcue S.
      • Ramos M.D.
      • Giménez J.
      • Segura A.
      • et al.
      Heterogeneity for mutations in the CFTR gene and clinical correlations in patients with congenital absence of the vas deferens.
      ,
      • Gené G.G.
      • Llobet A.
      • Larriba S.
      • de Semir D.
      • Martínez I.
      • Escalada A.
      • et al.
      N-terminal CFTR missense variants severely affect the behavior of the CFTR chloride channel.
      c.601G>A (V201M)SUEN/S
      • Bernardino A.L.
      • Ferri A.
      • Passos-Bueno M.R.
      • Kim C.E.
      • Nakaie C.M.
      • Gomes C.E.
      • et al.
      Molecular analysis in Brazilian cystic fibrosis patients reveals five novel mutations.
      ,
      • Boudaya M.
      • Fredj S.H.
      • Haj R.B.
      • Khrouf M.
      • Bouker A.
      • Halouani L.
      • et al.
      Cystic fibrosis transmembrane conductance regulator mutations and polymorphisms associated with congenital bilateral absence of vas deferens in a restricted group of patients from North Africa.
      ,
      • Giusti R.
      • Badgwell A.
      • Iglesias A.D.
      New York State cystic fibrosis consortium: the first 2.5 years of experience with cystic fibrosis newborn screening in an ethnically diverse population.
      c.617T>G (L206W)CCA–B
      • Hamoir C.
      • Pepermans X.
      • Piessevaux H.
      • Jouret-Mourin A.
      • Weynand B.
      • Habyalimana J.B.
      • et al.
      Clinical and morphological characteristics of sporadic genetically determined pancreatitis as compared to idiopathic pancreatitis: higher risk of pancreatic cancer in CFTR variants.
      ,
      • Van Goor F.
      • Yu H.
      • Burton B.
      • Hoffman B.J.
      Effect of ivacaftor on CFTR forms with missense mutations associated with defects in protein processing or function.
      ,
      • Rozen R.
      • Ferreira-Rajabi L.
      • Robb L.
      • Colman N.
      L206W mutation of the cystic fibrosis gene, relatively frequent in French Canadians, is associated with atypical presentations of cystic fibrosis.
      ,
      • Clain J.
      • Lehmann-Che J.
      • Duguépéroux I.
      • Arous N.
      • Girodon E.
      • Legendre M.
      • et al.
      Misprocessing of the CFTR protein leads to mild cystic fibrosis phenotype.
      c.772A>G (R258G)N/SN/S
      • Grangeia A.
      • Sá R.
      • Carvalho F.
      • Martin J.
      • Girodon E.
      • Silva J.
      • et al.
      Molecular characterization of the cystic fibrosis transmembrane conductance regulator gene in congenital absence of the vas deferens.
      ,
      • De Wachter E.
      • Thomas M.
      • Wanyama S.S.
      • Seneca S.
      • Malfroot A.
      What can the CF registry tell us about rare CFTR-mutations? A Belgian study.
      ,
      • Masvidal L.
      • Giménez J.
      • Ramos M.D.
      • Domingo C.
      • Farré A.
      • Bassas L.
      • et al.
      The p.Arg258Gly mutation in intracellular loop 2 of CFTR is associated with CFTR-related disorders.
      c.91C>T (R31C)NCCN/S
      • Gomez M.
      • Patuzzo C.
      • Castellani C.
      • Bovo P.
      • Cavallini G.
      • Mastella G.
      • et al.
      CFTR and cationic trypsinogen mutations in idiopathic pancreatitis and neonatal hypertrypsinemia.
      ,
      • Nakano E.
      • Masamune A.
      • Niihori T.
      • Kume K.
      • Hamada S.
      • Aoki Y.
      • et al.
      Targeted next-generation sequencing effectively analyzed the cystic fibrosis transmembrane conductance regulator gene in pancreatitis.
      ,
      • Jurkuvenaite A.
      • Varga K.
      • Nowotarski K.
      • Kirk K.L.
      • Sorscher E.J.
      • Li Y.
      • et al.
      Mutations in the amino terminus of the cystic fibrosis transmembrane conductance regulator enhance endocytosis.
      c.958T>G (L320V)NCCN/S
      • Schrijver I.
      • Ramalingam S.
      • Sankaran R.
      • Swanson S.
      • Dunlop C.L.
      • Keiles S.
      • et al.
      Diagnostic testing by CFTR gene mutation analysis in a large group of Hispanics: novel mutations and assessment of a population-specific mutation spectrum.
      ,
      • Keiles S.
      • Kammesheidt A.
      Identification of CFTR, PRSS1 and SPINK1 mutations in 381 patients with pancreatitis.
      ,
      • Pelletier A.L.
      • Bienvenu T.
      • Rebours V.
      • O'Toole D.
      • Hentic O.
      • Maire F.
      • et al.
      CFTR gene mutation in patients with apparently idiopathic pancreatitis: lack of phenotype-genotype correlation.
      A = mutations that cause CF; C = clinical consequences of the mutations are unknown; CC = CF-causing variant; class B = mutations that are associated with CF-related clinical presentation; NCC = non CF-causing variant; NCF = not cause CF; N/S = not found in CFTR2 database; SUE = still under evaluation; VC = variant of varying clinical consequences;

      Discussion

      The carrier frequency of CF pathogenic variants (1 in 6) (17% carriers) found in sperm donors is well above that described in previous research focused on a white European population (1 in 29) (
      • Grody W.W.
      • Cutting G.R.
      • Klinger K.W.
      • Richards C.S.
      • Watson M.S.
      • Desnick R.J.
      Laboratory standards and guidelines for population-based cystic fibrosis carrier screening.
      ). This may be because our study was conducted by the complete sequencing of the CFTR gene and by nontargeted analysis of variants, whereas the population frequency described in the literature was based on the use of panels that included a limited number of variants of the CFTR gene.
      Some publications have reported rates of CF carriers in sperm donors, but those papers refer to studies conducted with genotype tests analyzing a limited number of variants. In consequence, the carrier rate obtained is lower than if the analysis had been based on NGS (
      • Silver A.J.
      • Larson J.L.
      • Silver M.J.
      • Lim R.M.
      • Borroto C.
      • Spurrier B.
      • et al.
      Carrier screening is a deficient strategy for determining sperm donor eligibility and reducing risk of disease in recipient children.
      ). According to Landaburu et al. (
      • Landaburu I.
      • Gonzalvo M.C.
      • Clavero A.
      • Ramírez J.P.
      • Zamora S.
      • Martinez L.
      • et al.
      Genetic testing of sperm donors for cystic fibrosis and spinal muscular atrophy: evaluation of clinical utility.
      ), the rate of CF carriers in sperm donors is 1.3%. However, another study of sperm donors reported a carrier rate of 4.6% when the Poly 5T variant was excluded from the analysis, and 7.8% when it was included (
      • Urbano A.
      • Montoya E.
      • Ochando I.
      • Sánchez M.
      • Rueda J.
      Ventajas del cribado de portadores en donantes de gametos con un panel de 15 genes mediante tecnología Next-Generation Sequencing.
      ).
      The high CF carrier frequency observed in our study means that if all donor carriers are rejected, problems of donor gamete supply will arise. One means of reducing the risk of having offspring affected by CF after assisted reproduction treatment with sperm donation is genetic matching, i.e., selecting a donor who does not carry pathogenic variants in the CFTR gene when the prospective recipient is a carrier of a pathogenic variant in the CFTR gene (
      • Martin J.
      • Asan Yi Y.
      • Alberola T.
      • Rodríguez-Iglesias B.
      • Jimenez-Almazán J.
      • et al.
      Comprehensive carrier genetic test using next-generation deoxyribonucleic acid sequencing in infertile couples wishing to conceive through assisted reproductive technology.
      ,
      • Abulí A.
      • Latre L.
      • Boada M.
      • Palacios-Verdú G.
      • Clua E.
      • Rodríguez-Santiago B.
      • et al.
      Cribado ampliado de portadores en un programa de donación de ovocitos: Implementación de un nuevo test y resultados tras dos años de experiencia.
      ,
      Cribado Genético en Donación de Gametos
      Recomendaciones sobre el cribado genético en donación de gametos de la Sociedad Española de Fertilidad.
      ).
      Between the 2 extremes that could be adopted to reduce the reproductive risk of CF—excluding all donor carriers of pathogenic variants in the CFTR gene, or including donors carrying CF only when security is assured by genetic matching—we propose an alternative intermediate measure, by which reproductive risk may be reduced. An “exclusion panel” of variants, considered to be at high risk for the classic form of CF or of a severe phenotype, is defined. Donors carrying any of these variants are excluded from the donation program. The sperm bank should inform the collaborating centers that donors may be carriers of other variants in the CFTR gene, not included in the “exclusion panel,” and that if they wish to reduce the risk of CF associated with a nonclassic or less severe phenotype, they should perform genetic matching. If the recipient is a carrier of CF, a semen donor who has been studied by complete sequencing and is not a carrier of any variant of the CFTR gene will be selected.
      The American Society for Reproductive Medicine emphasizes the importance of providing patients with adequate genetic counseling and informing them about the residual risks of the genetic test, about the population risks of being a carrier of a recessive disease, and about the possibility of reducing the reproductive risk by selecting a donor on the basis of genetic matching (
      • Pfeifer S.
      • Goldberg J.
      • McClure R.
      • Lobo R.
      • Thomas M.
      • Widra E.
      • et al.
      Recommendations for gamete and embryo donation: a committee opinion.
      ). The reproductive risk for a prospective recipient of sperm donors of having a child with CF depends on the ethnic origin of the recipient, the tests performed on the recipient and on the sperm donor, the results obtained in those tests, and the exclusion criteria applied to the donor. Accordingly, the reproductive risk should be assessed individually, case by case (
      • Zenke U.
      • Chetkowski R.J.
      Inclusion of heterozygotes for cystic fibrosis in the egg donor pool.
      ).
      Our study shows there is great heterogeneity in the pathogenic variants of the CFTR gene that are included in the genotyping tests most commonly used in the context of assisted reproduction. Fewer than 50% of the variants included in the panels are common to all these tests. The clinical and diagnostic sensitivity of the test results obtained highlights the limitations of the tests currently used to detect CF carriers, corroborating previous research findings (
      • Kammesheidt A.
      • Kharrazi M.
      • Graham S.
      • Young S.
      • Pearl M.
      • Duncop C.
      • et al.
      Comprehensive genetic analysis of the cystic fibrosis transmembrane conductance regulator from dried blood specimens: implications for newborn screening.
      ,
      • Simpson J.L.
      • Rechitsky S.
      • Kuliev A.
      Before the beginning: the genetic risk of a couple aiming to conceive.
      ). Except for panel E, which is aimed specifically at the European and Hispanic population, panels I, H, and SG have been validated in accordance with the standards and guidelines of the American College of Medical Genetics and Genomics. Perhaps this is why these 3 tests indicate a lower rate of CF carriers in our population than is reported by the Elucigene test, although the latter includes the smallest number of variants. Our study findings in this respect are in line with those obtained previously in southern European populations, which present high molecular heterogeneity and in which commercial panels cover 50% to 75% of the alleles (
      • Kanavakis E.
      • Efthymiadou A.
      • Strofalis S.
      • Duodounakis S.
      • Traeger-Synodinos J.
      • Tzetis M.
      Cystic fibrosis in Greece: molecular diagnosis, haplotypes, prenatal diagnosis and carrier identification amongst high-risk individuals.
      ).
      Only 4 of the 7 variants classified as CF-causing variants by CFTR2 (57.14% of CF-causing variants by CFTR2) would have been detected by all 4 genotyping tests. On the other hand, of the 12 variants classified by Castellani et al. (
      • Castellani C.
      • Cuppens H.
      • Macek M.
      • Cassiman J.J.
      • Kerem E.
      • Durie P.
      • et al.
      Consensus on the use and interpretation of cystic fibrosis mutation analysis in clinical practice.
      ) as class A or A–B, only 4 would have been detected by the 4 genotyping tests (33.33% of the A–B variants by Castellani et al. [10]). These results highlight the low detection rate of genotyping tests in comparison with the results obtained by complete sequencing of the CFTR gene by NGS. In addition, they show that detection rates vary depending on whether the test is focused on a specific clinical form (
      • Kammesheidt A.
      • Kharrazi M.
      • Graham S.
      • Young S.
      • Pearl M.
      • Duncop C.
      • et al.
      Comprehensive genetic analysis of the cystic fibrosis transmembrane conductance regulator from dried blood specimens: implications for newborn screening.
      ,
      • Lucarelli M.
      • Bruno S.M.
      • Pierandrei S.
      • Ferraguti G.
      • Testino G.
      • Truglio G.
      • et al.
      The impact on genetic testing of mutational patterns of CFTR gene in different clinical macrocategories of cystic fibrosis.
      ,
      • Simpson J.L.
      • Rechitsky S.
      • Kuliev A.
      Before the beginning: the genetic risk of a couple aiming to conceive.
      ). Thus, the application of genotyping tests is associated with a higher reproductive risk, although this can be reduced by performing a complete study of the CFTR gene by NGS.
      The lower detection rate of CF carriers with panels I, H, and SG is mainly due to the fact that none of them studies the 5T-TG variants as a possible cause of CF.
      The pathogenic variants most frequently detected in our study were c.1210-34TG (
      • Sánchez-Pozo M.C.
      • Mendiola J.
      • Serrano M.
      • Mozas J.
      • Björndahl L.
      • Menkveld R.
      • et al.
      Proposal of guidelines for the appraisal of SEMen QUAlity studies (SEMQUA).
      )T(5), c.1210-34TG (
      • Grody W.W.
      • Cutting G.R.
      • Klinger K.W.
      • Richards C.S.
      • Watson M.S.
      • Desnick R.J.
      Laboratory standards and guidelines for population-based cystic fibrosis carrier screening.
      )T(5), c.1210-34TG(13)T(5), R75Q, L997F, R668C, G756A, and V754M. None of them were detected by any of the 4 genotyping tests analyzed, with the exception of the Poly 5T variants, which only the Elucigene test detected as a cause of CF. It should be noted that the Preconception Focus test includes a study of 5T-TG and of the L997F variant of the CFTR gene; however, it does not report them as causative variants of CF, but as a different clinical entity that only provokes the congenital bilateral absence of the vas deferens (CBAVD). Both variants have been associated with other CF-related clinical characteristics (
      • Delgado I.
      • Pérez E.
      • Álvarez A.I.
      • Macías Y.
      • Carrasco L.
      • et al.
      Results of the Andalusian cystic fibrosis neonatal screening program, five years after implementation.
      ,
      • Salinas D.B.
      • Sosnay P.R.
      • Azen C.
      • Young S.
      • Raraigh K.S.
      • Keens T.G.
      • et al.
      Benign outcome among positive cystic fibrosis newborn screen children with non-CF-causing variants.
      ,
      • Gomez M.
      • Patuzzo C.
      • Castellani C.
      • Bovo P.
      • Cavallini G.
      • Mastella G.
      • et al.
      CFTR and cationic trypsinogen mutations in idiopathic pancreatitis and neonatal hypertrypsinemia.
      ,
      • Groman J.D.
      • Hefferon T.W.
      • Casals T.
      • Bassas L.
      • Estivill X.
      • Des Georges M.
      • et al.
      Variation in a repeat sequence determines whether a common variant of the cystic fibrosis transmembrane conductance regulator gene is pathogenic or benign.
      ); thus, limiting the presence of these variants to CBAVD, as panel SG does, would not be correct.
      Although the L997F variant has been known since 1992, its functional role and impact on pathogenicity remain unclear. Initially it was described as a polymorphism, owing to its high population frequency and because, according to CFTR2, it does not cause CF (
      • Lucarelli M.
      • Narzi L.
      • Pierandrei S.
      • Bruno S.M.
      • Stamato A.
      • d’Avanzo M.
      • et al.
      A new complex allele of the CFTR gene partially explains the variable phenotype of the L997F mutation.
      ). This variant has been described in a patient with genotype p.Leu997Phe/c.2909-92G>A, who showed a typical clinical presentation of CF (
      • Delgado I.
      • Pérez E.
      • Álvarez A.I.
      • Macías Y.
      • Carrasco L.
      • et al.
      Results of the Andalusian cystic fibrosis neonatal screening program, five years after implementation.
      ). An in vitro functional study has shown that the presence of this variant reduces the conductivity of the Cl− ion with respect to the reference protein. In that same study, conducted in newborns with a positive sweat test result, the variant was identified in 4% of patients. However, it is not known how many subsequently experienced other respiratory or gastrointestinal disorders or manifestations of infertility associated with CF (
      • Salinas D.B.
      • Sosnay P.R.
      • Azen C.
      • Young S.
      • Raraigh K.S.
      • Keens T.G.
      • et al.
      Benign outcome among positive cystic fibrosis newborn screen children with non-CF-causing variants.
      ). In addition, the presence of this variant has been associated with pancreatic insufficiency (
      • Gomez M.
      • Patuzzo C.
      • Castellani C.
      • Bovo P.
      • Cavallini G.
      • Mastella G.
      • et al.
      CFTR and cationic trypsinogen mutations in idiopathic pancreatitis and neonatal hypertrypsinemia.
      ,
      • Lucarelli M.
      • Narzi L.
      • Pierandrei S.
      • Bruno S.M.
      • Stamato A.
      • d’Avanzo M.
      • et al.
      A new complex allele of the CFTR gene partially explains the variable phenotype of the L997F mutation.
      ), lung disease (
      • Strom C.M.
      • Redman J.B.
      • Peng M.
      The dangers of including nonclassical cystic fibrosis variants in population-based screening panels: p.L997F, further genotype/phenotype correlation data.
      ,
      • Lucarelli M.
      • Narzi L.
      • Pierandrei S.
      • Bruno S.M.
      • Stamato A.
      • d’Avanzo M.
      • et al.
      A new complex allele of the CFTR gene partially explains the variable phenotype of the L997F mutation.
      ), disseminated bronchiectasis, normal sweat test result with neonatal hypertripsinemia, and congenital absence of the vas deferens (
      • Lucarelli M.
      • Narzi L.
      • Pierandrei S.
      • Bruno S.M.
      • Stamato A.
      • d’Avanzo M.
      • et al.
      A new complex allele of the CFTR gene partially explains the variable phenotype of the L997F mutation.
      ).
      The 5T allele in intron 8 (IVS8) causes abnormal splicing in the CFTR gene and is considered a mutation of incomplete penetrance (
      • Groman J.D.
      • Hefferon T.W.
      • Casals T.
      • Bassas L.
      • Estivill X.
      • Des Georges M.
      • et al.
      Variation in a repeat sequence determines whether a common variant of the cystic fibrosis transmembrane conductance regulator gene is pathogenic or benign.
      ). The genotype-phenotype correlation is not clear; thus, it has been reported that when the 5T variant is found in trans with a severe CFTR mutation it can lead to male infertility, nonclassic CF, or a normal phenotype (
      • Groman J.D.
      • Hefferon T.W.
      • Casals T.
      • Bassas L.
      • Estivill X.
      • Des Georges M.
      • et al.
      Variation in a repeat sequence determines whether a common variant of the cystic fibrosis transmembrane conductance regulator gene is pathogenic or benign.
      ,
      • Chiang H.S.
      • Lu J.F.
      • Liu C.H.
      • Wu Y.N.
      • Wu C.C.
      CFTR (TG)m(T)n polymorphism in patients with CBAVD in a population expressing low incidence of cystic fibrosis.
      ); that the number of TG repeats adjacent to 5T is correlated with the phenotype and disease penetrance; that the number of 11TG repeats is associated with the absence of the vas deferens or with a normal phenotype; and that 12TG and 13TG are more frequent in affected individuals, whether producing the absence of the vas deferens or a nonclassic CF phenotype (
      • Groman J.D.
      • Hefferon T.W.
      • Casals T.
      • Bassas L.
      • Estivill X.
      • Des Georges M.
      • et al.
      Variation in a repeat sequence determines whether a common variant of the cystic fibrosis transmembrane conductance regulator gene is pathogenic or benign.
      ).
      Although the 5T variant is found in 10% of the general population (
      • Groman J.D.
      • Hefferon T.W.
      • Casals T.
      • Bassas L.
      • Estivill X.
      • Des Georges M.
      • et al.
      Variation in a repeat sequence determines whether a common variant of the cystic fibrosis transmembrane conductance regulator gene is pathogenic or benign.
      ), in our study its frequency was approximately 7%. This discrepancy may be due to the fact that the variant has been associated with obstructive azoospermia and oligozoospermia (
      • Chiang H.S.
      • Lu J.F.
      • Liu C.H.
      • Wu Y.N.
      • Wu C.C.
      CFTR (TG)m(T)n polymorphism in patients with CBAVD in a population expressing low incidence of cystic fibrosis.
      ,
      • Mak V.
      • Zielenski J.
      • Tsui L.C.
      • Durie P.
      • Zini A.
      • Martin S.
      • et al.
      Cystic fibrosis gene mutations and infertile men with primary testicular failure.
      ,
      • Chen H.
      • Ruan Y.C.
      • Xu W.M.
      • Chen J.
      • Chan H.C.
      Regulation of male fertility by CFTR and implications in male infertility.
      ), and donor candidates with these sperm characteristics are not included in the donation program. Moreover, donor candidates who reported a family history of CF were excluded from the donation program. In consequence, our population is biased in this respect.
      Among the donors predefined as individuals with good sperm quality, there were no statistically significant differences in sperm quality between carriers and noncarriers of CFTR variants. Studies in which a relationship has been found between CF carrier status and sperm quality have been conducted among the general population, but not in a study limited to individuals with high sperm quality (
      • Lewis-Jones D.I.
      • Gazvani M.R.
      • Mountford R.
      Cystic fibrosis in infertility: screening before assisted reproduction: opinion.
      ). However, we would expect to find a higher rate of CF carriers among oocyte donors, inasmuch as heterozygous carrier status has not been associated with any marker analyzed in the screening of oocyte donors. At first, R75Q and R668C/G756A (isolated or in allelic complex in cis) variants were classified as benign polymorphisms. However, their frequent occurrence in individuals with a CF-related phenotype suggests that a pathogenic role may also be played. In addition, their presence is associated with a decrease of 30% to 50% in mRNA levels of CFTR and a decrease of 17% to 26% in mature CFTR protein (
      • Bergougnoux A.
      • Viart V.
      • Miro J.
      • Bommart S.
      • Molinari N.
      • des Georges M.
      • et al.
      Should diffuse bronchiectasis still be considered a CFTR-related disorder?.
      ). When the R75Q variant is in trans with another pathogenic variant, it is frequently detected in patients with asthma, CBAVD, disseminated bronchiectasis, obstructive pulmonary disease, or pancreatitis (
      • Tzetis M.
      • Efthymiadou A.
      • Strofalis S.
      • Psychou P.
      • Dimakou A.
      • Pouliou E.
      • et al.
      CFTR gene mutations–including three novel nucleotide substitutions–and haplotype background in patients with asthma, disseminated bronchiectasis and chronic obstructive pulmonary disease.
      ,
      • Martinez B.
      • Heller M.
      • Gaitch N.
      • Hubert D.
      • Burgel P.R.
      • Levy P.
      • et al.
      p.Arg75Gln, a CFTR variant involved in the risk of CFTR-related disorders?.
      ,
      • Divac A.
      • Nikolic A.
      • Mitic-Milikic M.
      • Nagorni-Obradovic L.
      • Petrovic-Stanojevic N.
      • Dopudja-Pantic V.
      • et al.
      High frequency of the R75Q CFTR variation in patients with chronic obstructive pulmonary disease.
      ). Variants G576A and R668C are associated with abnormal splicing (
      • Bergougnoux A.
      • Viart V.
      • Miro J.
      • Bommart S.
      • Molinari N.
      • des Georges M.
      • et al.
      Should diffuse bronchiectasis still be considered a CFTR-related disorder?.
      ). Both variants are associated with a reduction in normal transcript quantity, of 57% and 37% respectively, with respect to the wild-type gene. The G576A/R668C haplotype in cis has been found in patients with disseminated and idiopathic bronchiectasis (
      • Tzetis M.
      • Efthymiadou A.
      • Strofalis S.
      • Psychou P.
      • Dimakou A.
      • Pouliou E.
      • et al.
      CFTR gene mutations–including three novel nucleotide substitutions–and haplotype background in patients with asthma, disseminated bronchiectasis and chronic obstructive pulmonary disease.
      ,
      • Martinez B.
      • Heller M.
      • Gaitch N.
      • Hubert D.
      • Burgel P.R.
      • Levy P.
      • et al.
      p.Arg75Gln, a CFTR variant involved in the risk of CFTR-related disorders?.
      ) and with CF-related phenotypes such as cholestasis, nasal polyps, and idiopathic pancreatitis (
      • El-Seedy A.
      • Girodon E.
      • Norez C.
      • Pajaud J.
      • Pasquet M.C.
      • de Becdelièvre A.
      • et al.
      CFTR mutation combinations producing frequent complex alleles with different clinical and functional outcomes.
      ).
      The R668C variant has been associated with CBAVD and azoospermia (
      • Tzetis M.
      • Efthymiadou A.
      • Strofalis S.
      • Psychou P.
      • Dimakou A.
      • Pouliou E.
      • et al.
      CFTR gene mutations–including three novel nucleotide substitutions–and haplotype background in patients with asthma, disseminated bronchiectasis and chronic obstructive pulmonary disease.
      ,
      • El-Seedy A.
      • Girodon E.
      • Norez C.
      • Pajaud J.
      • Pasquet M.C.
      • de Becdelièvre A.
      • et al.
      CFTR mutation combinations producing frequent complex alleles with different clinical and functional outcomes.
      ). On the other hand, studies have associated the presence of the G576A variant with individuals presenting a classic CF phenotype, and it has been detected in patients with CF-related symptoms such as idiopathic pancreatitis, CBAVD, and lung disease (
      • Pagani F.
      • Stuani C.
      • Tzetis M.
      • Kanavakis E.
      • Efthymiadou A.
      • Doudounakis S.
      • et al.
      New type of disease causing mutations: the example of the composite exonic regulatory elements of splicing in CFTR exon 12.
      ).
      The individuals who presented >1 variant were healthy. The variants detected in these individuals (p.[D443Y; G576A; R668C] and p.[R74W; V201M; D1270N]) have been reported as variants in cis on the same allele (
      • Bareil C.
      • Guittard C.
      • Altieri J.P.
      • Templin C.
      • Claustres M.
      des Georges M. Comprehensive and rapid genotyping of mutations and haplotypes in congenital bilateral absence of the vas deferens and other cystic fibrosis transmembrane conductance regulator-related disorders.
      ,
      • Grangeia A.
      • Sá R.
      • Carvalho F.
      • Martin J.
      • Girodon E.
      • Silva J.
      • et al.
      Molecular characterization of the cystic fibrosis transmembrane conductance regulator gene in congenital absence of the vas deferens.
      ,
      • Ratbi I.
      • Legendre M.
      • Niel F.
      • Martin J.
      • Soufir J.C.
      • Izard V.
      • et al.
      Detection of cystic fibrosis transmembrane conductance regulator (CFTR) gene rearrangements enriches the mutation spectrum in congenital bilateral absence of the vas deferens and impacts on genetic counselling.
      ), which could explain the fact that these individuals did not present symptoms related to CF.
      The presence of the V754M variant is also associated with a 30% to 50% decrease in mRNA levels of the CFTR gene and with a 39% decrease in mature CFTR protein levels (
      • Bergougnoux A.
      • Viart V.
      • Miro J.
      • Bommart S.
      • Molinari N.
      • des Georges M.
      • et al.
      Should diffuse bronchiectasis still be considered a CFTR-related disorder?.
      ). Moreover, it has been described in a patient with atypical CF, in whom a second mutation was not detected, and in patients with bronchiectasis and CBAVD (
      • Bergougnoux A.
      • Viart V.
      • Miro J.
      • Bommart S.
      • Molinari N.
      • des Georges M.
      • et al.
      Should diffuse bronchiectasis still be considered a CFTR-related disorder?.
      ,
      • Grangeia A.
      • Sá R.
      • Carvalho F.
      • Martin J.
      • Girodon E.
      • Silva J.
      • et al.
      Molecular characterization of the cystic fibrosis transmembrane conductance regulator gene in congenital absence of the vas deferens.
      ,
      • Dal'Maso V.B.
      • Mallmann L.
      • Siebert M.
      • Simon L.
      • Saraiva-Pereira M.L.
      Dalcin P de T. Diagnostic contribution of molecular analysis of the cystic fibrosis transmembrane conductance regulator gene in patients suspected of having mild or atypical cystic fibrosis.
      ).
      The great clinical variability associated with the variants found in the CFTR gene is in accordance with the previously described genetic and clinical heterogeneity of the disease (
      • Castellani C.
      • Cuppens H.
      • Macek M.
      • Cassiman J.J.
      • Kerem E.
      • Durie P.
      • et al.
      Consensus on the use and interpretation of cystic fibrosis mutation analysis in clinical practice.
      ). Our findings suggest that CF could be influenced by multiple additive effects such as mutations in other alternative or modifier genes, epigenetic factors, or environmental influence (
      • Gallati S.
      Disease-modifying genes and monogenic disorders: experience in cystic fibrosis.
      ,
      • Magalhães M.
      • Rivals I.
      • Claustres M.
      • Varilh J.
      • Thomasset M.
      • Bergougnoux A.
      • et al.
      DNA methylation at modifier genes of lung disease severity is altered in cystic fibrosis.
      ,
      • Magalhães M.
      • Tost J.
      • Pineau F.
      • Rivals I.
      • Busato F.
      • Alary N.
      • et al.
      Dynamic changes of DNA methylation and lung disease in cystic fibrosis: lessons from a monogenic disease.
      ).
      In conclusion, in view of the evident clinical heterogeneity associated with the variants of the CFTR gene, population studies are needed of patients with CF to clarify the genotype-phenotype relationship in CF, thus facilitating genetic counseling for the individuals concerned and improving decision making in assisted reproduction treatment based on gamete donation. Despite the higher cost of studying the CFTR gene by NGS with nontargeted analysis of the variants compared with genotyping, the higher detection rate of healthy individuals who are carriers of CF helps reduce the number of children born with CF, which ultimately produces a cost-effectiveness benefit (
      • Azimi M.
      • Schmaus K.
      • Greger V.
      • Neitzel D.
      • Rochelle R.
      • Dinh T.
      Carrier screening by next-generation sequencing: health benefits and cost effectiveness.
      ).
      Although our study was not based on the general population, we recommend that complete sequencing of the CFTR gene be carried out using NGS to screen for CF in sperm donors, because genotype testing alone may result in many mutations being missed. The low rate of detection of CF carriers in the targeted panels and the differences between the results obtained highlight the need to seek a consensus on the basic variants that should be included in a study of CF carriers. In this regard, we emphasize the importance of identifying the risks involved and of informing patients of the existence of a statistically significantly high reproductive risk when targeted panels are used.

      Acknowledgement

      This article is related to the Ph.D. doctoral thesis of M. Molina.

      Supplementary data

      References

        • Rowntree R.K.
        • Harris A.
        The phenotypic consequences of CFTR mutations.
        Ann Hum Genet. 2003; 67: 471-485
        • Rommens J.M.
        • Iannuzzi M.C.
        • Kerem B.
        • Drumm M.L.
        • Melmer G.
        • Dean M.
        • et al.
        Identification of the cystic fibrosis gene: chromosome walking and jumping.
        Science. 1989; 245: 1059-1065
        • Drumm M.L.
        • Ziady A.G.
        • Davis P.B.
        Genetic variation and clinical heterogeneity in cystic fibrosis.
        Annu Rev Pathol. 2012; 7: 267-282
        • Kammesheidt A.
        • Kharrazi M.
        • Graham S.
        • Young S.
        • Pearl M.
        • Duncop C.
        • et al.
        Comprehensive genetic analysis of the cystic fibrosis transmembrane conductance regulator from dried blood specimens: implications for newborn screening.
        Genet Med. 2006; 8: 557-562
        • Strom C.M.
        • Redman J.B.
        • Peng M.
        The dangers of including nonclassical cystic fibrosis variants in population-based screening panels: p.L997F, further genotype/phenotype correlation data.
        Genet Med. 2011; 13: 1042-1044
        • Lucarelli M.
        • Bruno S.M.
        • Pierandrei S.
        • Ferraguti G.
        • Testino G.
        • Truglio G.
        • et al.
        The impact on genetic testing of mutational patterns of CFTR gene in different clinical macrocategories of cystic fibrosis.
        J Mol Diagn. 2016; 18: 554-565
        • Sims C.A.
        • Callum P.
        • Ray M.
        • Iger J.
        • Falk R.E.
        Genetic testing of sperm donors: survey of current practices.
        Fertil Steril. 2010; 94: 126-129
      1. WHO laboratory manual for the examination and processing of human semen. Fifth edition. World Health Organization.
        (Available at:)
        • Richards S.
        • Aziz N.
        • Bale S.
        • Bick D.
        • Das S.
        • Gastier-Foster J.
        • et al.
        Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology.
        Genet Med. 2015; 17: 405-424
        • Castellani C.
        • Cuppens H.
        • Macek M.
        • Cassiman J.J.
        • Kerem E.
        • Durie P.
        • et al.
        Consensus on the use and interpretation of cystic fibrosis mutation analysis in clinical practice.
        J Cyst Fibros. 2008; 7: 179-196
        • Sánchez-Pozo M.C.
        • Mendiola J.
        • Serrano M.
        • Mozas J.
        • Björndahl L.
        • Menkveld R.
        • et al.
        Proposal of guidelines for the appraisal of SEMen QUAlity studies (SEMQUA).
        Hum Reprod. 2013; 28: 10-21
        • Grody W.W.
        • Cutting G.R.
        • Klinger K.W.
        • Richards C.S.
        • Watson M.S.
        • Desnick R.J.
        Laboratory standards and guidelines for population-based cystic fibrosis carrier screening.
        Genet Med. 2001; 3: 149-154
        • Silver A.J.
        • Larson J.L.
        • Silver M.J.
        • Lim R.M.
        • Borroto C.
        • Spurrier B.
        • et al.
        Carrier screening is a deficient strategy for determining sperm donor eligibility and reducing risk of disease in recipient children.
        Genet Test Mol Biomarkers. 2016; 20: 276-284
        • Landaburu I.
        • Gonzalvo M.C.
        • Clavero A.
        • Ramírez J.P.
        • Zamora S.
        • Martinez L.
        • et al.
        Genetic testing of sperm donors for cystic fibrosis and spinal muscular atrophy: evaluation of clinical utility.
        Eur J Obstet Gynecol Reprod Biol. 2013; 170: 183-187
        • Urbano A.
        • Montoya E.
        • Ochando I.
        • Sánchez M.
        • Rueda J.
        Ventajas del cribado de portadores en donantes de gametos con un panel de 15 genes mediante tecnología Next-Generation Sequencing.
        Genética Médica y Genómica. 2018; 2: 21-29
        • Martin J.
        • Asan Yi Y.
        • Alberola T.
        • Rodríguez-Iglesias B.
        • Jimenez-Almazán J.
        • et al.
        Comprehensive carrier genetic test using next-generation deoxyribonucleic acid sequencing in infertile couples wishing to conceive through assisted reproductive technology.
        Fertil Steril. 2015; 104: 1286-1293
        • Abulí A.
        • Latre L.
        • Boada M.
        • Palacios-Verdú G.
        • Clua E.
        • Rodríguez-Santiago B.
        • et al.
        Cribado ampliado de portadores en un programa de donación de ovocitos: Implementación de un nuevo test y resultados tras dos años de experiencia.
        MEDRE. 2017; : 4113-4121
        • Cribado Genético en Donación de Gametos
        Recomendaciones sobre el cribado genético en donación de gametos de la Sociedad Española de Fertilidad.
        (Available at:)
        • Pfeifer S.
        • Goldberg J.
        • McClure R.
        • Lobo R.
        • Thomas M.
        • Widra E.
        • et al.
        Recommendations for gamete and embryo donation: a committee opinion.
        Fertil Steril. 2013; 99: 47-62
        • Zenke U.
        • Chetkowski R.J.
        Inclusion of heterozygotes for cystic fibrosis in the egg donor pool.
        Fertil Steril. 2002; 78: 557-561
        • Simpson J.L.
        • Rechitsky S.
        • Kuliev A.
        Before the beginning: the genetic risk of a couple aiming to conceive.
        Fertil Steril. 2019; 112: 622-630
        • Kanavakis E.
        • Efthymiadou A.
        • Strofalis S.
        • Duodounakis S.
        • Traeger-Synodinos J.
        • Tzetis M.
        Cystic fibrosis in Greece: molecular diagnosis, haplotypes, prenatal diagnosis and carrier identification amongst high-risk individuals.
        Clin Genet. 2003; 63: 400-409
        • Delgado I.
        • Pérez E.
        • Álvarez A.I.
        • Macías Y.
        • Carrasco L.
        • et al.
        Results of the Andalusian cystic fibrosis neonatal screening program, five years after implementation.
        Arch Bronconeumol. 2018; 54: 551-558
        • Salinas D.B.
        • Sosnay P.R.
        • Azen C.
        • Young S.
        • Raraigh K.S.
        • Keens T.G.
        • et al.
        Benign outcome among positive cystic fibrosis newborn screen children with non-CF-causing variants.
        J Cyst Fibros. 2015; 14: 714-719
        • Gomez M.
        • Patuzzo C.
        • Castellani C.
        • Bovo P.
        • Cavallini G.
        • Mastella G.
        • et al.
        CFTR and cationic trypsinogen mutations in idiopathic pancreatitis and neonatal hypertrypsinemia.
        Pancreatology. 2001; 1: 538-542
        • Groman J.D.
        • Hefferon T.W.
        • Casals T.
        • Bassas L.
        • Estivill X.
        • Des Georges M.
        • et al.
        Variation in a repeat sequence determines whether a common variant of the cystic fibrosis transmembrane conductance regulator gene is pathogenic or benign.
        Am J Hum Genet. 2004; 74: 176-179
        • Lucarelli M.
        • Narzi L.
        • Pierandrei S.
        • Bruno S.M.
        • Stamato A.
        • d’Avanzo M.
        • et al.
        A new complex allele of the CFTR gene partially explains the variable phenotype of the L997F mutation.
        Genet Med. 2010; 12: 548-555
        • Chiang H.S.
        • Lu J.F.
        • Liu C.H.
        • Wu Y.N.
        • Wu C.C.
        CFTR (TG)m(T)n polymorphism in patients with CBAVD in a population expressing low incidence of cystic fibrosis.
        Clin Genet. 2009; 76: 282-286
        • Mak V.
        • Zielenski J.
        • Tsui L.C.
        • Durie P.
        • Zini A.
        • Martin S.
        • et al.
        Cystic fibrosis gene mutations and infertile men with primary testicular failure.
        Hum Reprod. 2000; 15: 436-439
        • Chen 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: 703-713
        • Lewis-Jones D.I.
        • Gazvani M.R.
        • Mountford R.
        Cystic fibrosis in infertility: screening before assisted reproduction: opinion.
        Hum Reprod. 2000; 15: 2415-2417
        • Bergougnoux A.
        • Viart V.
        • Miro J.
        • Bommart S.
        • Molinari N.
        • des Georges M.
        • et al.
        Should diffuse bronchiectasis still be considered a CFTR-related disorder?.
        J Cyst Fibros. 2015; 14: 646-653
        • Tzetis M.
        • Efthymiadou A.
        • Strofalis S.
        • Psychou P.
        • Dimakou A.
        • Pouliou E.
        • et al.
        CFTR gene mutations–including three novel nucleotide substitutions–and haplotype background in patients with asthma, disseminated bronchiectasis and chronic obstructive pulmonary disease.
        Hum Genet. 2001; 108: 216-221
        • Martinez B.
        • Heller M.
        • Gaitch N.
        • Hubert D.
        • Burgel P.R.
        • Levy P.
        • et al.
        p.Arg75Gln, a CFTR variant involved in the risk of CFTR-related disorders?.
        J Hum Genet. 2014; 59: 206-210
        • Divac A.
        • Nikolic A.
        • Mitic-Milikic M.
        • Nagorni-Obradovic L.
        • Petrovic-Stanojevic N.
        • Dopudja-Pantic V.
        • et al.
        High frequency of the R75Q CFTR variation in patients with chronic obstructive pulmonary disease.
        J Cyst Fibros. 2004; 3: 189-191
        • El-Seedy A.
        • Girodon E.
        • Norez C.
        • Pajaud J.
        • Pasquet M.C.
        • de Becdelièvre A.
        • et al.
        CFTR mutation combinations producing frequent complex alleles with different clinical and functional outcomes.
        Hum Mutat. 2012; 33: 1557-1565
        • Pagani F.
        • Stuani C.
        • Tzetis M.
        • Kanavakis E.
        • Efthymiadou A.
        • Doudounakis S.
        • et al.
        New type of disease causing mutations: the example of the composite exonic regulatory elements of splicing in CFTR exon 12.
        Hum Mol Genet. 2003; 12: 1111-1120
        • Bareil C.
        • Guittard C.
        • Altieri J.P.
        • Templin C.
        • Claustres M.
        des Georges M. Comprehensive and rapid genotyping of mutations and haplotypes in congenital bilateral absence of the vas deferens and other cystic fibrosis transmembrane conductance regulator-related disorders.
        J Mol Diagno. 2007; 9: 582-588
        • Grangeia A.
        • Sá R.
        • Carvalho F.
        • Martin J.
        • Girodon E.
        • Silva J.
        • et al.
        Molecular characterization of the cystic fibrosis transmembrane conductance regulator gene in congenital absence of the vas deferens.
        Genet Med. 2007; 9: 163-172
        • Ratbi I.
        • Legendre M.
        • Niel F.
        • Martin J.
        • Soufir J.C.
        • Izard V.
        • et al.
        Detection of cystic fibrosis transmembrane conductance regulator (CFTR) gene rearrangements enriches the mutation spectrum in congenital bilateral absence of the vas deferens and impacts on genetic counselling.
        Hum Reprod. 2007; 22: 1285-1291
        • Dal'Maso V.B.
        • Mallmann L.
        • Siebert M.
        • Simon L.
        • Saraiva-Pereira M.L.
        Dalcin P de T. Diagnostic contribution of molecular analysis of the cystic fibrosis transmembrane conductance regulator gene in patients suspected of having mild or atypical cystic fibrosis.
        J Bras Pneumol. 2013; 39: 181-189
        • Gallati S.
        Disease-modifying genes and monogenic disorders: experience in cystic fibrosis.
        Appl Clin Genet. 2014; 7: 133-146
        • Magalhães M.
        • Rivals I.
        • Claustres M.
        • Varilh J.
        • Thomasset M.
        • Bergougnoux A.
        • et al.
        DNA methylation at modifier genes of lung disease severity is altered in cystic fibrosis.
        Clin Epigenet. 2017; 9: 19
        • Magalhães M.
        • Tost J.
        • Pineau F.
        • Rivals I.
        • Busato F.
        • Alary N.
        • et al.
        Dynamic changes of DNA methylation and lung disease in cystic fibrosis: lessons from a monogenic disease.
        Epigenomics. 2018; 10: 1131-1145
        • Azimi M.
        • Schmaus K.
        • Greger V.
        • Neitzel D.
        • Rochelle R.
        • Dinh T.
        Carrier screening by next-generation sequencing: health benefits and cost effectiveness.
        Mol Genet Genomic Med. 2016; 4: 292-302
        • LaRusch J.
        • Jung J.
        • General I.J.
        • Lewis M.D.
        • Woo Park H.
        • Brand R.E.
        • et al.
        Mechanisms of CFTR functional variants that impair regulated bicarbonate permeation and increase risk for pancreatitis but not for cystic fibrosis.
        PLoS Genet. 2014; 10e1004376
        • Havasi V.
        • Rowe S.M.
        • Kolettis P.N.
        • Dayangac D.
        • Sxahin A.
        • Grangeia A.
        • et al.
        Association of cystic fibrosis genetic modifiers with congenital bilateral absence of the vas deferens.
        Fertil Steril. 2010; 94: 2122-2127
        • Behar D.M.
        • Inbar O.
        • Shteinberg M.
        • Gur M.
        • Mussaffi H.
        • Shoseyov D.
        • et al.
        Nationwide genetic analysis for molecularly unresolved cystic fibrosis patients in a multiethnic society: implications for preconception carrier screening.
        Mol Genet Genomic Med. 2017; 5: 223-226
        • Masson E.
        • Chen J.M.
        • Audrézet M.P.
        • Cooper D.N.
        • Férec C.
        A conservative assessment of the major genetic causes of idiopathic chronic pancreatitis: data from a comprehensive analysis of PRSS1, SPINK1, CTRC and CFTR genes in 253 young French patients.
        PLoS One. 2013; 8e73522
        • Audrézet M.P.
        • Novelli G.
        • Mercier B.
        • Sangiuolo F.
        • Maceratesi P.
        • Férec C.
        • et al.
        Identification of three novel cystic fibrosis mutations in a sample of Italian cystic fibrosis patients.
        Hum Hered. 1993; 43: 295-300
        • Scotet V.
        • De Braekeleer M.
        • Audrézet M.P.
        • Lodé L.
        • Verlingue C.
        • Quéré I.
        • et al.
        Prevalence of CFTR mutations in hypertrypsinaemia detected through neonatal screening for cystic fibrosis.
        Clin Genet. 2001; 59: 42-47
        • Dörk T.
        • Dworniczak B.
        • Aulehla-Scholz C.
        • Wieczorek D.
        • Böhm I.
        • Mayerova A.
        • et al.
        Distinct spectrum of CFTR gene mutations in congenital absence of vas deferens.
        Hum Genet. 1997; 100: 365-377
        • Schrijver I.
        • Ramalingam S.
        • Sankaran R.
        • Swanson S.
        • Dunlop C.L.
        • Keiles S.
        • et al.
        Diagnostic testing by CFTR gene mutation analysis in a large group of Hispanics: novel mutations and assessment of a population-specific mutation spectrum.
        J Mol Diagn. 2005; 7: 289-299
        • Casals T.
        • De-Gracia J.
        • Gallego M.
        • Dorca J.
        • Rodríguez-Sanchón B.
        • Ramos M.D.
        • et al.
        Bronchiectasis in adult patients: an expression of heterozygosity for CFTR gene mutations?.
        Clin Genet. 2004; 65: 490-495
        • Kolesár P.
        • Minárik G.
        • Baldovic M.
        • Ficek A.
        • Kovács L.
        • Kádasi L.
        Mutation analysis of the CFTR gene in Slovak cystic fibrosis patients by DHPLC and subsequent sequencing: identification of four novel mutations.
        Gen Physiol Biophys. 2008; 27: 299-305
        • Ziętkiewicz E.
        • Rutkiewicz E.
        • Pogorzelski A.
        • Klimek B.
        • Voelkel K.
        • Witt M.
        CFTR mutations spectrum and the efficiency of molecular diagnostics in Polish cystic fibrosis patients.
        PLoS One. 2014; 9e89094
        • de Cid R.
        • Ramos M.D.
        • Aparisi L.
        • García C.
        • Mora J.
        • Estivill X.
        • et al.
        Independent contribution of common CFTR variants to chronic pancreatitis.
        Pancreas. 2010; 39: 209-215
        • Bombieri C.
        • Giorgi S.
        • Carles S.
        • de Cid R.
        • Belpinati F.
        • Tandoi C.
        • et al.
        A new approach for identifying non-pathogenic mutations: an analysis of the cystic fibrosis transmembrane regulator gene in normal individuals.
        Hum Genet. 2000; 106: 172-178
        • Padoan R.
        • Genoni S.
        • Moretti E.
        • Seia M.
        • Giunta A.
        • Corbetta C.
        Genetic and clinical features of false-negative infants in a neonatal screening programme for cystic fibrosis.
        Acta Paediatr. 2002; 91: 82-87
        • Casals T.
        • Ramos M.D.
        • Giménez J.
        • Larriba S.
        • Nunes V.
        • Estivill X.
        High heterogeneity for cystic fibrosis in Spanish families: 75 mutations account for 90% of chromosomes.
        Hum Genet. 1997; 101: 365-370
        • Bombieri 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: 180-193
        • Hamoir C.
        • Pepermans X.
        • Piessevaux H.
        • Jouret-Mourin A.
        • Weynand B.
        • Habyalimana J.B.
        • et al.
        Clinical and morphological characteristics of sporadic genetically determined pancreatitis as compared to idiopathic pancreatitis: higher risk of pancreatic cancer in CFTR variants.
        Digestion. 2013; 87: 229-239
        • Kilinç M.O.
        • Ninis V.N.
        • Dağli E.
        • Demirkol M.
        • Ozkinay F.
        • Arikan Z.
        • et al.
        Highest heterogeneity for cystic fibrosis: 36 mutations account for 75% of all CF chromosomes in Turkish patients.
        Am J Med Genet. 2002; 113: 250-257
        • Kahnoski K.
        • Khoo S.K.
        • Nassif N.T.
        • Chen J.
        • Lobo G.P.
        • Segelov E.
        • et al.
        Alterations of the Birt-Hogg-Dubé gene (BHD) in sporadic colorectal tumours.
        J Med Genet. 2003; 40: 511-515
        • de Prada A.
        • Bütschi F.N.
        • Bouchardy I.
        • Beckmann J.S.
        • Morris M.A.
        • Hafen G.M.
        • Fellmann F.
        [R74W;R1070W;D1270N]: a new complex allele responsible for cystic fibrosis.
        J Cyst Fibros. 2010; 9: 447-449
        • de Gracia J.
        • Mata F.
        • Alvarez A.
        • Casals T.
        • Gatner S.
        • Vendrell M.
        • et al.
        Genotype-phenotype correlation for pulmonary function in cystic fibrosis.
        Thorax. 2005; 60: 558-563
        • Borowitz D.
        CFTR, bicarbonate, and the pathophysiology of cystic fibrosis.
        Pediatr Pulmonol. 2015; 50: S24-S30
        • Schneider A.
        • Larusch J.
        • Sun X.
        • Aloe A.
        • Lamb J.
        • Hawes R.
        • et al.
        Combined bicarbonate conductance-impairing variants in CFTR and SPINK1 variants are associated with chronic pancreatitis in patients without cystic fibrosis.
        Gastroenterology. 2011; 140: 162-171
        • Mak V.
        • Zielenski J.
        • Tsui L.C.
        • Durie P.
        • Zini A.
        • Martin S.
        • et al.
        Proportion of cystic fibrosis gene mutations not detected by routine testing in men with obstructive azoospermia.
        J Am Med Assoc. 1999; 281 (JM: Ref 69 changed away from JAMA per style manual, even though JAMA has been universally used for years. Your opinion?): 2217-2224
        • Desgeorges M.
        • Mégarbané A.
        • Guittard C.
        • Carles S.
        • Loiselet J.
        • Demaille J.
        • et al.
        Cystic fibrosis in Lebanon: distribution of CFTR mutations among Arab communities.
        Hum Genet. 1997; 100: 279-283
        • Gomez M.
        • Benetazzo M.G.
        • Marzari M.G.
        • Bombieri C.
        • Belpinati F.
        • Castellani C.
        • et al.
        High frequency of cystic fibrosis transmembrane regulator mutation L997F in patients with recurrent idiopathic pancreatitis and in newborns with hypertrypsinemia.
        Am J Hum Genet. 2000; 66: 2013-2014
        • Keiles S.
        • Kammesheidt A.
        Identification of CFTR, PRSS1 and SPINK1 mutations in 381 patients with pancreatitis.
        Pancreas. 2006; 33: 221-227
        • Alonso M.J.
        • Heine-Suñer D.
        • Calvo M.
        • Rosell J.
        • Giménez J.
        • Ramos M.D.
        • et al.
        Spectrum of mutations in the CFTR gene in cystic fibrosis patients of Spanish ancestry.
        Ann Hum Genet. 2007; 71: 194-201
        • Casals T.
        • Bassas L.
        • Egozcue S.
        • Ramos M.D.
        • Giménez J.
        • Segura A.
        • et al.
        Heterogeneity for mutations in the CFTR gene and clinical correlations in patients with congenital absence of the vas deferens.
        Hum Reprod. 2000; 15: 1476-1483
        • Van Goor F.
        • Yu H.
        • Burton B.
        • Hoffman B.J.
        Effect of ivacaftor on CFTR forms with missense mutations associated with defects in protein processing or function.
        J Cyst Fibros. 2014; 13: 29-36
        • Puéchal X.
        • Bienvenu T.
        • Génin E.
        • Berthelot J.M.
        • Sibilia J.
        • Gaudin P.
        • et al.
        Mutations of the cystic fibrosis gene in patients with bronchiectasis associated with rheumatoid arthritis.
        Ann Rheum Dis. 2011; 70: 653-659
        • De Braekeleer M.
        • Allard C.
        • Leblanc J.P.
        • Simard F.
        • Aubin G.
        Phenotypic variability in five cystic fibrosis patients compound heterozygous for the Y1092X mutation.
        Hum Hered. 1998; 48: 158-162
        • Schrijver I.
        • Oitmaa E.
        • Metspalu A.
        • Gardner P.
        Genotyping microarray for the detection of more than 200 CFTR mutations in ethnically diverse populations.
        J Mol Diagn. 2005; 7: 375-387
        • Terlizzi V.
        • Carnovale V.
        • Castaldo G.
        • Castellani C.
        • Cirilli N.
        • Colombo C.
        • et al.
        Clinical expression of patients with the D1152H CFTR mutation.
        J Cyst Fibros. 2015; 14: 447-452
        • Peleg L.
        • Karpati M.
        • Bronstein S.
        • Berkenstadt M.
        • Frydman M.
        • Yonath H.
        • et al.
        The D1152H cystic fibrosis mutation in prenatal carrier screening, patients and prenatal diagnosis.
        J Med Screen. 2011; 18: 169-172
        • Feldmann D.
        • Couderc R.
        • Audrezet M.P.
        • Ferec C.
        • Bienvenu T.
        • Desgeorges M.
        • et al.
        CFTR genotypes in patients with normal or borderline sweat chloride levels.
        Hum Mutat. 2003; 22: 340
        • Augarten A.
        • Kerem B.S.
        • Yahav Y.
        • Noiman S.
        • Rivlin Y.
        • Tal A.
        • et al.
        Mild cystic fibrosis and normal or borderline sweat test in patients with the 3849 + 10 kb C-->T mutation.
        Lancet. 1993; 342: 25-26
        • Duguépéroux I.
        • De Braekeleer M.
        The CFTR 3849+10kbC->T and 2789+5G->A alleles are associated with a mild CF phenotype.
        Eur Respir J. 2005; 25: 468-473
        • Ataseven F.
        • Ozer S.
        • Yilmaz R.
        • Senayli A.
        Bilateral spontaneous pneumothorax in a newborn with N1303K mutation of cystic fibrosis (CFTR) gene.
        Tuberk Toraks. 2010; 58: 181-183
        • Osborne L.
        • Santis G.
        • Schwarz M.
        • Klinger K.
        • Dörk T.
        • McIntosh I.
        • et al.
        Incidence and expression of the N1303K mutation of the cystic fibrosis (CFTR) gene.
        Hum Genet. 1992; 89: 653-658
        • Chávez-Saldaña M.
        • Yokoyama E.
        • Lezana J.L.
        • Carnevale A.
        • Macías M.
        • Vigueras R.M.
        • et al.
        CFTR allelic heterogeneity in Mexican patients with cystic fibrosis: implications for molecular screening.
        Rev Invest Clin. 2010; 62: 546-552
        • Steiner B.
        • Rosendahl J.
        • Witt H.
        • Teich N.
        • Keim V.
        • Schulz H.U.
        • et al.
        Common CFTR haplotypes and susceptibility to chronic pancreatitis and congenital bilateral absence of the vas deferens.
        Hum Mutat. 2011; 32: 912-920
        • Claustres M.
        • Guittard C.
        • Bozon D.
        • Chevalier F.
        • Verlingue C.
        • Ferec C.
        • et al.
        Spectrum of CFTR mutations in cystic fibrosis and in congenital absence of the vas deferens in France.
        Hum Mutat. 2000; 16: 143-156
        • Gené G.G.
        • Llobet A.
        • Larriba S.
        • de Semir D.
        • Martínez I.
        • Escalada A.
        • et al.
        N-terminal CFTR missense variants severely affect the behavior of the CFTR chloride channel.
        Hum Mutat. 2008; 29: 738-749
        • Bernardino A.L.
        • Ferri A.
        • Passos-Bueno M.R.
        • Kim C.E.
        • Nakaie C.M.
        • Gomes C.E.
        • et al.
        Molecular analysis in Brazilian cystic fibrosis patients reveals five novel mutations.
        Genet Test. 2000; 4: 69-74
        • Boudaya M.
        • Fredj S.H.
        • Haj R.B.
        • Khrouf M.
        • Bouker A.
        • Halouani L.
        • et al.
        Cystic fibrosis transmembrane conductance regulator mutations and polymorphisms associated with congenital bilateral absence of vas deferens in a restricted group of patients from North Africa.
        Ann Hum Biol. 2012; 39: 76-79
        • Giusti R.
        • Badgwell A.
        • Iglesias A.D.
        New York State cystic fibrosis consortium: the first 2.5 years of experience with cystic fibrosis newborn screening in an ethnically diverse population.
        Pediatrics. 2007; 119: e460-e467
        • Rozen R.
        • Ferreira-Rajabi L.
        • Robb L.
        • Colman N.
        L206W mutation of the cystic fibrosis gene, relatively frequent in French Canadians, is associated with atypical presentations of cystic fibrosis.
        Am J Med Genet. 1995; 57: 437-439
        • Clain J.
        • Lehmann-Che J.
        • Duguépéroux I.
        • Arous N.
        • Girodon E.
        • Legendre M.
        • et al.
        Misprocessing of the CFTR protein leads to mild cystic fibrosis phenotype.
        Hum Mutat. 2005; 25: 360-371
        • De Wachter E.
        • Thomas M.
        • Wanyama S.S.
        • Seneca S.
        • Malfroot A.
        What can the CF registry tell us about rare CFTR-mutations? A Belgian study.
        Orphanet J Rare Dis. 2017; 12: 142
        • Masvidal L.
        • Giménez J.
        • Ramos M.D.
        • Domingo C.
        • Farré A.
        • Bassas L.
        • et al.
        The p.Arg258Gly mutation in intracellular loop 2 of CFTR is associated with CFTR-related disorders.
        Genet Test Mol Biomarkers. 2009; 13: 765-768
        • Nakano E.
        • Masamune A.
        • Niihori T.
        • Kume K.
        • Hamada S.
        • Aoki Y.
        • et al.
        Targeted next-generation sequencing effectively analyzed the cystic fibrosis transmembrane conductance regulator gene in pancreatitis.
        Dig Dis Sci. 2015; 60: 1297-1307
        • Jurkuvenaite A.
        • Varga K.
        • Nowotarski K.
        • Kirk K.L.
        • Sorscher E.J.
        • Li Y.
        • et al.
        Mutations in the amino terminus of the cystic fibrosis transmembrane conductance regulator enhance endocytosis.
        J Biol Chem. 2006; 281: 3329-3334
        • Pelletier A.L.
        • Bienvenu T.
        • Rebours V.
        • O'Toole D.
        • Hentic O.
        • Maire F.
        • et al.
        CFTR gene mutation in patients with apparently idiopathic pancreatitis: lack of phenotype-genotype correlation.
        Pancreatology. 2010; 10: 158-164