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Multicenter characterization and validation of the intron-8 poly(T) tract (IVS8-T) status in 25 coriell cell repository cystic fibrosis reference cell lines for cystic fibrosis transmembrane conductance regulator (CFTR) gene mutation assays.

Cystic fibrosis (CF) is the most common life-limiting recessive genetic disorder in Caucasians, with a carrier frequency of ~1 in 25 and incidence of ~1 in 2500-3300 live births (1). CF is caused by mutations affecting the transmembrane conductance regulator (CFTR) gene localized on the long arm of chromosome 7 (7831.2). CFTR contains 27 exons and encodes a protein of 1480 amino acids that functions as a CAMP-regulated chloride channel in the apical membrane of epithelial cells (2, 3). Mutations in the CFTR gene lead to dysfunction of the lungs, sweat glands, testes, ovaries, intestines, and pancreas. More than 1000 mutations in this gene have been identified to date (4). The clinical manifestations of the disease are variable, ranging from severe pulmonary disease with pancreatic insufficiency to mild pulmonary disease and pancreatic sufficiency (1). Moreover, mutations in the CFTR gene have also been found in patients who have normal lung function but show other clinical signs, such as congenital bilateral absence of the vas deferens (CBAVD), nasal polyposis, bronchiectasis, and bronchopulmonary allergic aspergillosis (5, 6).

Some of the variability in the CF phenotype has been attributed to the influence of the 5T allele at a polymorphic poly(T) tract in intron 8 (IVS8-T) of the CFTR gene. Genotype-phenotype correlations have shown that there is a strong association of the 5T allele with male infertility caused by congenital CBAVD and with other monosymptomatic forms of CF, such as bronchiectasis and chronic idiopathic pancreatitis (5-7). At the IVS8-T locus, which functions as a splice acceptor site, three variants designated 5T, 7T, and 9T have been identified. The 5T variant is a poor splice acceptor site and gives rise to skipping of exon 9 in a high percentage of CFTR mRNA transcripts (7-11). CFTR mRNA missing exon 9 does not produce a functional protein. A CFTR gene with the 5T allele produces only 5% of the normal concentration of normal mRNA, and when coupled with a CF mutation, this can have a clinical effect (9).

There are three main molecular scenarios that are clinically relevant: (a) When 5T is present with a severe CF mutation on the opposite chromosome (in trans), individuals may be asymptomatic, may have mild symptoms of CF, or if male, may have CBAVD (5,6). (b) The 5T allele modifies the penetrance of the mild CF mutation R117H. When R117H is on the same allele (in cis) with 5T and another CF mutation is present on the other chromosome, the outcome is usually mild CF with pancreatic sufficiency, although some cases of classic pancreatic-insufficient CF have been seen (12,13). (c) In males, R117H in trans with 5T (without the presence of another CF mutation) is associated with CBAVD (5,13).

The 7T variant also plays a clinical role. When R117H is in cis with 7T and another CF mutation is present on the other chromosome (in males), CBAVD may result with or without late onset of mild lung disease (6,12). The American College of Medical Genetics has recommended reflex testing for the 5T/7T/9T variant when the R117H mutation is found (1,13,14). If 5T is present, further testing of parents or offspring is recommended to determine whether the 5T is in cis or trans with R117H. This increased diagnostic relevance of CFTR IVS8-T status in fully evaluating genotype-phenotype correlation in CF and CBAVD prompted us to devise methods to determine poly(T) tract status in CFTR.

The present study was undertaken to characterize IVS8-T status in the cell lines of the CFTR mutation panel (Order No. MUTCF) provided by the Coriell Cell Repository. The panel contains 21 of the 25 alleles recommended by the American College of Medical Genetics for routine diagnostic and carrier testing and is widely used for procedure validation and positive control samples in CF testing. However, IVS8-T status of CFTR in this mutation panel has not been reported. Additionally, we validated these results with eight other molecular diagnostic laboratories that routinely conduct CF testing to establish the potential utility of these CFTR cell lines as test controls in determining CFTR IVS8-T tract variant status. We determined IVS8-T allele status in all 21 cell lines included in the Coriell CFTR mutation panel (MUTCF) and in 4 additional Coriell CF cell lines (NA11290, NA13032, NA13033, and NA07464). We tested the cell lines with the INNO-LiPA CFTR 17+Tn (Innogenetics), a line probe assay system based on the reverse hybridization principle ( To verify IVS8-T results for samples NA11275 and NA11280, which showed discrepant test results at one participating laboratory, IVS8-T status was additionally tested by use of the ELUCIGENE[TM] CF-PolyT ASR system (Orchid Biosciences), which uses the ARMS[TM] technology ( UK.pdf). In all cases, instructions provided by the manufacturers were followed with modifications. For the INNO-LiPA system, instead of following the manufacturer's instructions to process each strip (CFTR16 and CFTR170) in separate troughs, we routinely processed both strips in the same trough by placing one strip facing down and other facing up. In the case of the INNO-LiPA system, the amount of genomic DNA used in the PCR was in the range of ~1000-3000 ng, and for the ELUCIGENE assay, it was 50 ng. To further confirm the T-allele status in samples NA11275 and NA11280, we performed nucleotide sequencing using the Big Dye Terminator v.1.1 Cycle sequencing reagent set (Applied Biosystems).

The data were compared with the IVS8-T allele status found by eight other well-established molecular diagnostic laboratories that routinely conduct CF assays. Laboratories 2, 3, 4, and 6 used LINEAR ARRAY CF Gold 1.0 supplied by Roche Diagnostics Corporation (http://www.; laboratories 5, 8, and 9 used the INNO-LiPA system (Innogenetics); and laboratory 7 used an in-house-developed assay procedure (15).

The results of IVS8-T allele status in 25 DNA samples containing well-characterized CFTR mutations are depicted in Table 1. The results agree in all but two samples, NA11275 and NA11280, for which the results reported by laboratory 4 differed from the results reported by the others. Nucleotide sequencing confirmed the consensus T-allele status (shown in the "Consensus" column).

In conclusion, our study determined and validated the IVS-8 T allele status in all 25 cell lines routinely used as test controls in CF assays. The commercial availability of the test samples described in this study provides easily accessible routine controls to monitor and evaluate respective CF test results. The data may also be useful for validating analytic performance of CF testing methods. In addition, we believe that the multicenter-validated CFTR T-allele status in MUTCF samples could facilitate interlaboratory standardization and proficiency testing for these clinically relevant CFTR mutations.

This work was supported in part by the CDC (Grant 200-2000-10050). We thank Drs. Jeanne Beck of Coriell Cell Repository (Camden, NJ), Ana Stankovic, and Laurina Williams of the CDC (Atlanta, GA) for assistance, and Dr. Susan Bernacki for critical review of the manuscript. The contents of this publication do not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the US Government. We received as a gift one 50-assay ELUCIGENE CF-PolyT ASR system from Orchid Biosciences (Oxfordshire, UK).


(1.) Richards CS, Bradley LA, Amos J, Allitto B, Grody WW, Maddalena A, et al. Standards and guidelines for CFTR mutation testing. Genet Med 2002;4: 379-91.

(2.) Kerem B, Rommens JM, Buchanan JA, Markiewicz D, Cox TK, Chakravarti A, et al. Identification of the cystic fibrosis gene: genetic analysis. Science 1989;245:1073-80.

(3.) Rommens JM, lannuzzi MC, Kerem B, Drumm ML, Melmer G, Dean M, et al. Identification of the cystic fibrosis gene: chromosome walking and jumping. Science 1989;245:1059-65.

(4.) Cystic Fibrosis Consortium. Cystic fibrosis mutations database. http:// (Accessed June 2003).

(5.) Chillon M, Casals T, Mercier B, Bassas L, Lissens W, Silber S, et al. Mutations in the cystic fibrosis gene in patients with congenital absence of the vas deferens. N Engl J Med 1995;332:1475-80.

(6.) Kiesewetter S, Macek M, Davis C, Curristin SM, Chu CS, Graham C, et al. A mutation in CFTR produces different phenotypes depending on chromosomal background. Nat Genet 1993;5:274-8.

(7.) Pignatti PE, Bombieri C, Benetazzo M, Casartelli A, Trabetti E, Gile LS, et al. CFTR gene variant IVS8-5T in disseminated bronchiectasis. Am J Hum Genet 1996;58:889-92.

(8.) Chu 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;3:151-6.

(9.) Cuppens H, Lin W, Jaspers M, Costes B, Teng H, Vankeerberghen A, et al. Polyvariant mutant cystic fibrosis transmembrane conductance regulator genes. The polymorphic (Tg)m locus explains the partial penetrance of the T5 polymorphism as a disease mutation. J Clin Invest 1998;101:487-96.

(10.) Rave-Harel N, Kerem E, Nissim-Rafinia M, Madjar I, Goshen R, Augarten A, et al. The molecular basis of partial penetrance of splicing mutations in cystic fibrosis. Am J Hum Genet 1997;60:87-94.

(11.) Niksic 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;8:2339-49.

(12.) Massie RJH, Poplawski N, Wilcken B, Goldblatt J, Byrnes C, Robertson C. Intron-8 polythymidine sequence in Australasian individuals with CF mutations R117H and R117C. Eur Respir J 2001;17:1195-200.

(13.) Grody WW, Cutting GR, Klinger KW, Richards CS, Watson MS, Desnick RJ. Laboratory standards and guidelines for population-based cystic fibrosis carrier screening. Genet Med 2001;3:149-54.

(14.) Watson MS, Desnick RJ, Grody WW, Mennuti MT, Popovich BV, Richards CS. Cystic fibrosis carrier screening: issues in implementation. Genet Med 2002;4:407-9.

(15.) Millson A, Spangler F, Lyon E. Comparison of the linear array CF-31 (Roche) and the cystic fibrosis assay (ABI) to detect cystic fibrosis mutations [Abstract]. J Mol Diagn 2001;3:194.

DOI: 10.1373/clinchem.2003.028068

Siby Sebastian, [1] Silvia G. Spitzer, [2] Leonard E. Grosso, [3] Jean Amos, [4] Frederick V. Schaefer, [5] Elaine Lyon, [6] Daynna J. Wolff, [7] Atieh Hajianpour, [8] Annette K. Taylor, [9] Alison Millson, [6] and Timothy T. Stenzel [1] * ([1] Department of Pathology, Molecular Diagnostics Laboratory, Duke University Medical Center, Durham, NC; [2] Molecular Genetics Laboratory of SUNY at Stony Brook, Stony Brook, NY; [3] Department of Pathology, St. Louis University School of Medicine, St. Louis, MO; [4] Specialty Laboratories Inc., Santa Monica, CA; [5] Chapman Institute of Medical Genetics, Tulsa, OK; [6] ARUP Laboratories, University of Utah, Salt Lake City, UT; [7] Department of Pathology and Laboratory Medicine, Medical University of South Carolina, Charleston, SC; [8] Molecular Genetics Laboratory, Alfigen, The Genetics Institute, Pasadena, CA; [9] Kimball Genetics Inc., Denver, CO; * address correspondence to this author at: Vysis, Inc., an Abbott Laboratories Company, 3100 Woodcreek Dr., Downers Grove, IL 60615-5400; e-mail
Table 1. IVS8-T variant status in 25 CF cell lines used as test
controls for CFTR gene mutation assays.

 Laboratory (b)

Sample (a) Cell line 1 2 3 4 5

 1 NA01531 9T/9T 9T/9T ND (c) 9T/9T 9T/9T
 2 NA07441 7T/9T 7T/9T ND 7T/9T 7T/9T
 3 NA07552 7T/9T 7T/9T ND 7T/9T 7T/9T
 4 NA08338 7T/9T 7T/9T ND 7T/9T 7T/9T
 5 NA11275 7T/9T 7T/9T 7T/9T 7T/7T (e) 7T/9T
 6 NA11277 7T/7T 7T/7T 7T/7T 7T/7T 7T/7T
 7 NA11280 7T/9T 7T/9T 7T/9T 7T/7T (e) 7T/9T
 8 NA11281 9T/9T 9T/9T ND 9T/9T 9T/9T
 9 NA11282 7T/9T 7T/9T 7T/9T 7T/9T 7T/9T
 10 NA11283 9T/9T 9T/9T ND 9T/9T 9T/9T
 11 NA11284 7T/9T 7T/9T 7T/9T 7T/9T 7T/9T
 12 NA11472 7T/9T 7T/9T 7T/9T 7T/9T 7T/9T
 13 NA11496 9T/9T 9T/9T 9T/9T 9T/9T 9T/9T
 14 NA11723 5T/7T 5T/7T 5T/7T 5T/7T 5T/7T
 15 NA11859 7T/7T 7T/7T 7T/7T 7T/7T 7T/7T
 16 NA11860 7T/7T 7T/7T 7T/7T 7T/7T 7T/7T
 17 NA12444 7T/7T 7T/7T 7T/7T 7T/7T 7T/7T
 18 NA12585 7T/7T 7T/7T 7T/7T 7T/7T 7T/7T
 19 NA12785 7T/7T 7T/7T 7T/7T 7T/7T 7T/7T
 20 NA12960 7T/7T 7T/7T 7T/7T 7T/7T 7T/7T
 21 NA13591 5T/9T 5T/9T 5T/9T 5T/9T 5T/9T
 22 NA11290 9T/9T ND 9T/9T ND ND
 23 NA13032 5T/7T ND 5T/7T ND ND
 24 NA13033 7T/7T ND 7T/7T ND ND
 25 NA07464 7T/7T ND 7T/7T ND ND

 Laboratory (b)

Sample (a) 6 7 8 9 Consensus

 1 ND 9T/9T 9T/9T ND 9T/9T
 2 ND 7T/9T 7T/9T 7T/9T 7T/9T
 3 ND 7T/9T 7T/9T ND 7T/9T
 4 ND 7T/9T 7T/9T ND 7T/9T
 5 7T/9T 7T/9T 7T/9T ND 7T/9T
 6 7T/7T 7T/7T 7T/7T ND 7T/7T
 7 7T/9T 7T/9T 7T/9T ND 7T/9T
 8 ND 9T/9T 9T/9T ND 9T/9T
 9 7T/9T 7T/9T 7T/9T 7T/9T 7T/9T
 10 9T/9T 9T/9T 9T/9T ND 9T/9T
 11 7T/9T 7T/9T 7T/9T ND 7T/9T
 12 7T/9T 7T/9T 7T/9T ND 7T/9T
 13 9T/9T 9T/9T 9T/9T ND 9T/9T
 14 5T/7T 5T/7T 5T/7T ND 5T/7T
 15 7T/7T 7T/7T 7T/7T 7T/7T 7T/7T
 16 7T/7T 7T/7T 7T/7T ND 7T/7T
 17 7T/7T 7T/7T 7T/7T ND 7T/7T
 18 7T/7T 7T/7T 7T/7T 7T/7T 7T/7T
 19 7T/7T 7T/7T 7T/7T ND 7T/7T
 20 7T/7T 7T/7T 7T/7T ND 7T/7T
 21 5T/9T 5T/9T 5T/9T ND 5T/9T
 22 9T/9T 9T/9T ND ND 9T/9T
 23 5T/7T 5T/7T 5T/7T ND 5T/7T
 24 7T/7T 7T/7T 7T/7T ND 7T/7T
 25 ND ND ND ND 7T/7T

Sample (a) Reported allelic variants

 1 [DELTA] F508; [DELTA] F508 (d)
 2 3120 + 1G > A; 621 + 1G > T (d)
 3 R553X; [DELTA] F508d
 4 G551D
 5 3656delC; [DELTA] F508 (d)
 6 [DELTA] I507
 7 711 + 1G > T(T); 621 + 1G T (d)
 8 621 + 1G > T; [DELTA] F508 (d)
 9 G85E;621 + 1G > T (d)
 10 A455E; [DELTA] F508 (d)
 11 R560T; [DELTA] F508 (d)
 12 N1303K; G1349D (d)
 13 G542X; G542X (d)
 14 W1282X
 15 2789 + 5G > A; 2789 + 5G > A (d)
 16 3849 + 10C > T; 3849 + 10C T (d)
 17 1717-1G > T(A)
 18 R1162X
 19 R347P; G551D (d)
 20 R334W/? (d)
 21 R117H; F508 (d)
 22 A455E; 621 + 1G > T (d)
 23 I506V
 24 F508C
 25 R553X

(a) Samples 1-21 are from the Coriell MUTCF panel, and samples
22-25 are cell lines with characterized CFTR mutations available
from Coriell as test controls for CF DNA assays.

(b) Laboratory numbers are arbitrary and do not correspond to
the order of authors.

(c) ND, not determined.

(d) Clinically affected.

(e) Varied results.
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Title Annotation:Technical Briefs
Author:Sebastian, Siby; Spitzer, Silvia G.; Grosso, Leonard E.; Amos, Jean; Schaefer, Frederick V.; Lyon, E
Publication:Clinical Chemistry
Date:Jan 1, 2004
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