Variation in a Repeat Sequence Determines Whether a Common Variant of the Cystic Fibrosis Transmembrane Conductance Regulator Gene Is Pathogenic or Benign
2004; Elsevier BV; Volume: 74; Issue: 1 Linguagem: Inglês
10.1086/381001
ISSN1537-6605
AutoresJoshua D. Groman, Timothy Hefferon, Teresa Casals, L. Bassas, Xavier Estivill, Marie des Georges, Caroline Guittard, Monika Koudová, M. Daniele Fallin, Krisztína Németh, György Fekete, L Kádasi, K.J. Friedman, Martin Schwarz, Cristina Bombieri, Pier Franco Pignatti, Emmanuel Kanavakis, Maria Tzetis, Marianne Schwartz, Giuseppe Novelli, Maria Rosaria D’Apice, Agnieszka Sobczyńska‐Tomaszewska, Jerzy Bal, Manfred Stuhrmann, Milan Maçek, Mireille Claustres, Garry R. Cutting,
Tópico(s)Tracheal and airway disorders
ResumoAn abbreviated tract of five thymidines (5T) in intron 8 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene is found in ∼10% of individuals in the general population. When found in trans with a severe CFTR mutation, 5T can result in male infertility, nonclassic cystic fibrosis, or a normal phenotype. To test whether the number of TG repeats adjacent to 5T influences disease penetrance, we determined TG repeat number in 98 patients with male infertility due to congenital absence of the vas deferens, 9 patients with nonclassic CF, and 27 unaffected individuals (fertile men). Each of the individuals in this study had a severe CFTR mutation on one CFTR gene and 5T on the other. Of the unaffected individuals, 78% (21 of 27) had 5T adjacent to 11 TG repeats, compared with 9% (10 of 107) of affected individuals. Conversely, 91% (97 of 107) of affected individuals had 12 or 13 TG repeats, versus only 22% (6 of 27) of unaffected individuals (P<.00001). Those individuals with 5T adjacent to either 12 or 13 TG repeats were substantially more likely to exhibit an abnormal phenotype than those with 5T adjacent to 11 TG repeats (odds ratio 34.0, 95% CI 11.1–103.7, P<.00001). Thus, determination of TG repeat number will allow for more accurate prediction of benign versus pathogenic 5T alleles. An abbreviated tract of five thymidines (5T) in intron 8 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene is found in ∼10% of individuals in the general population. When found in trans with a severe CFTR mutation, 5T can result in male infertility, nonclassic cystic fibrosis, or a normal phenotype. To test whether the number of TG repeats adjacent to 5T influences disease penetrance, we determined TG repeat number in 98 patients with male infertility due to congenital absence of the vas deferens, 9 patients with nonclassic CF, and 27 unaffected individuals (fertile men). Each of the individuals in this study had a severe CFTR mutation on one CFTR gene and 5T on the other. Of the unaffected individuals, 78% (21 of 27) had 5T adjacent to 11 TG repeats, compared with 9% (10 of 107) of affected individuals. Conversely, 91% (97 of 107) of affected individuals had 12 or 13 TG repeats, versus only 22% (6 of 27) of unaffected individuals (P<.00001). Those individuals with 5T adjacent to either 12 or 13 TG repeats were substantially more likely to exhibit an abnormal phenotype than those with 5T adjacent to 11 TG repeats (odds ratio 34.0, 95% CI 11.1–103.7, P<.00001). Thus, determination of TG repeat number will allow for more accurate prediction of benign versus pathogenic 5T alleles. Genes that are responsible for inherited disorders can have mutations that cause disease phenotypes in some but not all individuals. These incompletely penetrant mutations become problematic if they are incorporated into clinical testing prior to an understanding of the factors affecting penetrance. One variant that presents a problem in the interpretation of genetic testing of the cystic fibrosis transmembrane conductance regulator (CFTR) gene is an abbreviated tract of five thymidines (5T) in intron 8 (Noone et al. Noone et al., 2000Noone PG Pue CA Zhou Z Friedman KJ Wakeling EL Ganeshananthan M Simon RH Silverman LM Knowles MR Lung disease associated with the IVS8 5T allele of the CFTR gene.Am J Respir Crit Care Med. 2000; 162: 1919-1924Crossref PubMed Scopus (94) Google Scholar; Strom et al. Strom et al., 2002Strom CM Huang D Buller A Redman J Crossley B Anderson B Entwistle T Sun W Cystic fibrosis screening using the College panel: platform comparison and lessons learned from the first 20,000 samples.Genet Med. 2002; 4: 289-296Crossref PubMed Scopus (50) Google Scholar). Some individuals bearing 5T in trans with a severe cystic fibrosis–causing mutation may have nonclassic cystic fibrosis (CF [MIM 219700]); others may have male infertility due to congenital bilateral absence of the vas deferens (CBAVD [MIM 277180]); and ∼40% may be healthy and fertile as a consequence of incomplete penetrance (Chillón et al. Chillón et al., 1995Chillón M Casals T Mercier B Bassas L Lissens W Silber S Romey MC et al.Mutations in the cystic fibrosis gene in patients with congenital absence of the vas deferens.N Engl J Med. 1995; 332: 1475-1480Crossref PubMed Scopus (770) Google Scholar; Zielenski et al. Zielenski et al., 1995Zielenski J Patrizio P Corey M Handelin B Markiewicz D Asch R Tsui LC CFTR gene variant for patients with congenital absence of vas deferens.Am J Hum Genet. 1995; 57: 958-960PubMed Google Scholar). Thus, 5T frequently creates an interpretative challenge for clinicians due to the commonness of the variant (found in 1 in 10 individuals) and the large number of CFTR genetic tests performed each year. The 5T variant is a stretch of five contiguous thymidines at the 3′ end of intron 8 that exacerbates skipping of exon 9, thereby resulting in reduced levels of functional CFTR protein (Chu et al. Chu et al., 1993Chu CS Trapnell BC Curristin S Cutting GR Crystal RG Genetic basis of variable exon 9 skipping in cystic fibrosis transmembrane conductance regulator mRNA.Nat Genet. 1993; 3: 151-156Crossref PubMed Scopus (423) Google Scholar). Prior studies have demonstrated that the number of TG repeats immediately adjacent to 5T correlates with exon 9 skipping (Cuppens et al. Cuppens et al., 1998Cuppens H Lin W Jaspers M Costes B Teng H Vankeerberghen A Jorissen M Droogmans G Reynaert I Goossens M Nilius B Cassiman JJ Polyvariant mutant cystic fibrosis transmembrane conductance regulator genes: the polymorphic (TG)m locus explains the partial penetrance of the 5T polymorphism as a disease mutation.J Clin Invest. 1998; 101: 487-496Crossref PubMed Scopus (343) Google Scholar; Niksic et al. Niksic et al., 1999Niksic M Romano M Buratti E Pagani F Baralle FE Functional analysis of cis-acting elements regulating the alternative splicing of human CFTR exon 9.Hum Mol Genet. 1999; 8: 2339-2349Crossref PubMed Scopus (127) Google Scholar). Cuppens et al. (Cuppens et al., 1998Cuppens H Lin W Jaspers M Costes B Teng H Vankeerberghen A Jorissen M Droogmans G Reynaert I Goossens M Nilius B Cassiman JJ Polyvariant mutant cystic fibrosis transmembrane conductance regulator genes: the polymorphic (TG)m locus explains the partial penetrance of the 5T polymorphism as a disease mutation.J Clin Invest. 1998; 101: 487-496Crossref PubMed Scopus (343) Google Scholar) have also suggested that increased exon 9 skipping from transcripts bearing more TG repeats increases the penetrance of 5T. However, an adequate calculation of the effect of TG repeat number on 5T penetrance has been hampered by small sample sizes. The aim of this collaborative study was to evaluate whether TG repeat number significantly aids in prediction of benign versus pathogenic 5T alleles, thereby warranting its use in clinical situations. Identifying information was removed from all DNA samples prior to submission to this study. The relative frequencies of 5T and TG-T combinations were estimated by screening 1,269 DNA samples from the general populations of five countries (United States, Czech Republic, Poland, Italy, and Germany). To assess the effect of TG repeat variation on penetrance of 5T, we analyzed DNA samples from 27 unaffected fertile men, 98 patients with CBAVD, and 9 patients with nonclassic CF. Each of the 134 individuals in this study had a severe CF mutation on one CFTR gene and 5T on the other. Either the 5T variant was confirmed—by studying allele transmission in the family—to be in trans with a severe CF mutation, or it was assumed to be in trans if the severe CF mutation was a common mutation previously shown not to be in linkage disequilibrium with 5T. To identify unaffected individuals with this genotype, we screened for 5T in 1,685 men who fathered at least one child with classic CF (fertile, obligate carriers). Since >98% of men with CF are infertile, it is unlikely that fathers of patients with CF have an unrecognized form of the disease, making them an appropriate control population for this study. Each of the 27 fathers with 5T had one of the following mutations: ΔF508 (18), G542X (2), 1812-1G→A (2), ΔI507 (1), 936delTA (1), N1303K (1), 3600+2insT (1), or 1717-1 G→A (1). 5T was inferred to be in trans with ΔF508 and ΔI507, since these mutations have never been shown to be in linkage disequilibrium with 5T. The 5T variant was confirmed by use of pedigree analysis to be in trans with the other mutations. Samples from fathers of patients with CF were referred from CF care centers from the following countries (number of samples screened and number with the CF mutation/5T genotype in parentheses): Czech Republic (202, 5), Denmark (172, 2), France (109, 2), Germany (114, 1), Greece (210, 1), Hungary (30, 2), Italy (230, 2), Poland (100, 5), Slovakia (46, 0), Spain (384, 5), and the United States (88, 2). Patients with CBAVD were diagnosed by palpation, analysis of sperm, and/or rectal ultrasound. Each of the 98 patients with CBAVD had 5T with one of the following mutations: ΔF508 (78), G542X (6), N1303K (3), 711+1G→T (2), R1066C (2), R1162X (2), R764X (1), Y563X (1), H609R (1), L206W (1), or R334W (1). 5T was confirmed by use of pedigree analysis to be in trans with R764X, Y563X, and H609X and was inferred as in trans for the remaining mutations, since these mutations have never been found to be in linkage disequilibrium with 5T in previous population sampling. Patients with CBAVD were referred from the following countries (number with the CF mutation/5T genotype): Denmark (6), France (35), Germany (8), Greece (1), Italy (4), Poland (6), Spain (29), the United Kingdom (6), and the United States (3). Nine patients with nonclassic CF were referred from CF care centers in the United States and were confirmed to have a 5T in trans with one of the following CFTR mutations: ΔF508 (6), 2814insA (1), G463D (1), or F693L (1). For each patient, we scanned the entire coding region of the CFTR gene to rule out the presence of rare mutations in addition to 5T (DNA sequencing n=6, denaturating gradient gel electrophoresis [DGGE] n=3). Each patient met current diagnostic criteria for CF by demonstrating both CFTR dysfunction, and at least one clinical feature of CF (Rosenstein and Cutting Rosenstein and Cutting, 1998Rosenstein BJ Cutting GR The diagnosis of cystic fibrosis: a consensus statement. Cystic Fibrosis Foundation Consensus Panel.J Pediatr. 1998; 132: 589-595Abstract Full Text Full Text PDF PubMed Scopus (869) Google Scholar). Specifically, six of the nine patients had respiratory disease consistent with CF, as well as CFTR dysfunction, as indicated by elevated mean sweat Cl− concentration (58–70 mmol/L). Of the remaining three patients, each had evidence of CFTR dysfunction, as indicated by borderline sweat Cl− concentration (43–49 mmol/L), and abnormally reduced low Cl− buffer/isoproterenol response, as demonstrated by the nasal potential difference (NPD) test. Of these three patients, one had mild respiratory disease, one had elevated serum trypsinogen in the newborn period, and one had elevated quantitative fecal fat levels consistent with pancreatic insufficiency. To detect samples positive for 5T, PCR products were fixed on Hybond N+ nylon membranes (Amersham Biosciences) and were probed with the 32P end-labeled probe 5′-TGTGTGTGTTTTTAACAG. Samples that were positive for 5T were sequenced bidirectionally and analyzed by automated capillary electrophoresis (Applied Biosystems) to determine TG repeat number and phase of TG-T repeat combinations. Samples from France, Italy, Greece, and Spain were screened using DGGE or restriction enzyme digestion. Allele frequencies for 5T were similar among the five general population groups and were consistent with previous reports (table 1). 5T was found in cis with three different TG repeats (TG11-5T, TG12-5T, TG13-5T). Although variation in the frequencies of these combinations was observed among populations, TG11-5T was by far the most common in all five populations, followed by TG12-5T and TG13-5T (table 1).Table 1Frequency of the 5T Allele and TG-5T Combinations in the General Population5T Combinations (Proportion of 5T Alleles)Country of OriginDNA Samples Screened5T Allele FrequencyTG11-5TTG12-5TTG13-5TUnited States5170.04836 (0.72)13 (0.26)1 (0.02)Czech Republic2510.03014 (0.93)1 (0.07)0 (0.00)Poland1320.05313 (0.93)1 (0.07)0 (0.00)Italy1730.0389 (0.70)4 (0.30)0 (0.00)Germany1960.04111 (0.69)4 (0.25)1 (0.06)Total12690.04383 (0.77)23 (0.21)2 (0.02) Open table in a new tab TG12-5T and TG13-5T were more frequent in the affected patients than controls across each of the populations sampled (table 2), and the frequencies of the different TG-T combinations differed significantly between the unaffected and affected groups (table 3). The most frequent combination, TG11-5T, was found in 78% of unaffected individuals, versus 9% of affected individuals (table 3), suggesting that TG11-5T is generally benign. TG12-5T was the most common disease- associated combination, and was found in 76% of affected individuals versus 22% of unaffected individuals. It is interesting to note that TG13-5T was found exclusively in affected individuals (table 3). These findings demonstrate that, when 5T is found in trans with a severe CF mutation, the odds of pathogenicity are 28 and 34 times greater for TG12-5T and TG13-5T, respectively, than for TG11-5T (table 3).Table 2Distribution of the TG-5T Combinations in Unaffected and Affected Individuals, by Country of OriginNo. of Occurrences inUnaffected IndividualsAffected IndividualsCountry of OriginTG11-5TTG12-5TTG13-5TTG11-5TTG12-5TTG13-5TFrance2……2285Spain41…425…United States11……84Poland5……141Germany…1…143Denmark2……222Italy2………4…United Kingdom…………51Czech Republic32…………Greece1………1…Hungary11………… Open table in a new tab Table 3Frequency of the TG-5T Combinations in Unaffected and Affected Individuals with a CF Mutation/5T GenotypeNo. of IndividualsStatusAll SamplesTG11-5TTG12-5TaOdds ratio 28.4, 95% CI 9.3–86.9, P<.00001.TG13-5TbOdds ratio 34.0, 95% CI 4.0–289.8, P<.0001.Unaffected: Fertile men272160Affected: CBAVD98107612 Nonclassic CF9054 Total affected107108116Note.—Odds ratios were calculated by comparing the frequency of the TG12-T5 or TG13-T5 combination to the frequency of the putatively benign TG11-T5 combination in affected versus unaffected individuals.a Odds ratio 28.4, 95% CI 9.3–86.9, P<.00001.b Odds ratio 34.0, 95% CI 4.0–289.8, P<.0001. Open table in a new tab Note.— Odds ratios were calculated by comparing the frequency of the TG12-T5 or TG13-T5 combination to the frequency of the putatively benign TG11-T5 combination in affected versus unaffected individuals. Previous studies have suggested that another common variant in CFTR, a methionine or valine at codon 470, also influences the penetrance of 5T (Cuppens et al. Cuppens et al., 1998Cuppens H Lin W Jaspers M Costes B Teng H Vankeerberghen A Jorissen M Droogmans G Reynaert I Goossens M Nilius B Cassiman JJ Polyvariant mutant cystic fibrosis transmembrane conductance regulator genes: the polymorphic (TG)m locus explains the partial penetrance of the 5T polymorphism as a disease mutation.J Clin Invest. 1998; 101: 487-496Crossref PubMed Scopus (343) Google Scholar; de Meeus et al. de Meeus et al., 1998de Meeus A Guittard C Desgeorges M Carles S Demaille J Claustres M Linkage disequilibrium between the M470V variant and the IVS8 polyT alleles of the CFTR gene in CBAVD.J Med Genet. 1998; 35: 594-596Crossref PubMed Scopus (45) Google Scholar). To test whether this variant affects the penetrance of 5T, we established phase between TG-T combinations and this variant in 22 unaffected and 92 affected individuals. As reported elsewhere, TG11-5T was always found with M470, and, in all but five cases, TG12-5T was found with V470 (data not shown). The highly penetrant TG13-5T was found to occur only with M470 in all of the cases in which phase could be established (n=15). The correlation between increased penetrance and TG repeat number, lack of correlation with variation at codon 470, and experimental evidence of increased exon 9 skipping with more TG repeats indicates that TG repeat number, rather than M470V status, is the major determinant of penetrance for 5T. Our analysis demonstrates that knowledge of TG repeat number in individuals with 5T is of diagnostic value. When TG repeat number is determined, the sensitivity for identifying affected individuals is 91%, whereas the specificity for identifying those who are unaffected is 78%. After adjusting for differences in sample sizes of the affected and unaffected groups, we used the proportion of each TG-T combination in affected individuals to estimate that the disease risk for males with TG11-5T is 0.10, TG12-5T is 0.78, and TG13-5T is 1.0. Since CBAVD accounts for a large component of the disease risk in this study, the risk of pathogenicity will likely be lower for females. Nonetheless, a female carrying TG12-5T or TG13-5T in trans with a CF mutation will be at higher risk of developing nonclassic CF than if she carried the TG11-5T allele. The association between TG repeat number and disease penetrance demonstrated in this study is consistent with molecular studies correlating TG repeat number with aberrant CFTR splicing (Cuppens et al. Cuppens et al., 1998Cuppens H Lin W Jaspers M Costes B Teng H Vankeerberghen A Jorissen M Droogmans G Reynaert I Goossens M Nilius B Cassiman JJ Polyvariant mutant cystic fibrosis transmembrane conductance regulator genes: the polymorphic (TG)m locus explains the partial penetrance of the 5T polymorphism as a disease mutation.J Clin Invest. 1998; 101: 487-496Crossref PubMed Scopus (343) Google Scholar; Niksic et al. Niksic et al., 1999Niksic M Romano M Buratti E Pagani F Baralle FE Functional analysis of cis-acting elements regulating the alternative splicing of human CFTR exon 9.Hum Mol Genet. 1999; 8: 2339-2349Crossref PubMed Scopus (127) Google Scholar; Hefferon et al., Hefferon et al., in pressHefferon TW, Groman JD, Yurk CE, Cutting GR. A variable dinucleotide repeat in the CFTR gene contributes to phenotype diversity by forming RNA secondary structures that alter splicing. Proc Natl Acad Sci USA (in press)Google Scholar) and validates clinical use of TG repeat testing in counseling individuals with 5T. This information will have important clinical relevance, since ∼25 million individuals in the United States are predicted to carry at least one 5T allele. Although TG repeat number proves to be a reliable predictor of penetrance for 5T, further studies are required to elucidate the factors that determine whether an individual will develop multisymptomatic (nonclassic CF) or monosymptomatic (CBAVD) disease. We wish to thank M. Boyle, S. Hsu, and T. Holtzman, for review of the manuscript, and L. Ramos, J. Gimenez, C. Yurk, X. Wang, D. Arking, C. Castellani, M. De Gironcoli, F. Belpinati, M. Macek, Sr., A. Krebsova, and V. Vavrova, for technical and logistical assistance. This study was supported by grants from the Italian MIUR and Ministry of Health “Genetic Tests” project DL229/99 9 and MURST-Italy (to P.F.P. and C.B.); the Italian Ministry of Health and Ministry of Education (to G.N.); IGA MZCR-00000064203/6463–3, MSMT-111300003/LN00A079, EC-CFChip, and CRMGEN (to M.M.Jr.); F. La Marato TV3 (980410) and ISCiii (PI020099) and ICS (to T.C., L.C., and X.E.); and the Cystic Fibrosis Foundation (CUTTIN98AO) and the National Institutes of Health (DK44003 and HL68927) (to G.R.C.). The URL for data presented herein is as follows: Online Mendelian Inheritance in Man (OMIM), http://www.ncbi.nlm.nih.gov/Omim/ (for CF and CBAVD)
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