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Fig. 1. Specificity of the Sangtec 100 IRMA. Human recombinant S100 proteins (20 g/L) were added to the Sangtec 100 in vitro assay to test its specificity for S100B.
pendent hydrophobic interaction, size-exclusion, and/or exchange chromatography (1, 3 ). Purified dimeric S100 proteins were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and their metal-binding and immunologic properties. The exact masses of S100 proteins were determined by electrospray ionization mass spectrometry (SCIEX API 365; Perkin-Elmer). Native proteins (in contrast to the recombinant proteins) were found to be mostly acetylated with no other posttranslational modifications. Human S100A8 and S100A9 were purchased from Bachem AG. The protocol of the supplier was followed except that the Sangtec 100 IRMA calibrators (containing lyophilized partly purified bovine S100 peptides) were replaced by the same concentration (0.5– 60 g/L) of human recombinant S100 proteins. The reagent blank (negative control) contained no protein calibrators. As illustrated in Fig. 1, the assay recognized the human S100B protein equally well and in the same concentration range as the bovine S100B calibrator. The Sangtec IRMA was linear over a range of 0 –20 g/L; the same linearity was obtained with identical concentrations of the recombinant human S100B. To test the specificity of this assay, we replaced the bovine S100B calibrator with the various human recombinant S100 proteins in concentrations of 10 –30 g/L. There was no interference/cross-reactivity by the other S100 proteins tested, including S100A6 and S100A4, which are highly expressed in brain tissue. According to these results this diagnostic assay is specific and reliable for measurement of S100B in human body fluids.
I thank C. Acklin for technical assistance, Dr. A. Rowlerson for critical reading, and D. Are´ valo for assistance with the preparation of the manuscript. References 1. Heizmann CW, Fritz G, Scha¨ fer B. W. S100 proteins: structure, functions and pathology [Review]. Front Biosci 2002;7:d1356 – 68.
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2. Michetti F, Gazzolo D. S100B protein in biological fluids: a tool for perinatal medicine [Review]. Clin Chem 2002;48:2097–104. 3. Fritz G, Heizmann CW. 3D-structures of the Ca2⫹- and Zn2⫹-binding S100 proteins. In: Bode W, Messerschmidt A, Cygler M, eds. Handbook of metalloproteins, Vol. 3. Chichester, NY: John Wiley & Sons, 2004:in press. 4. Wolf R, Mirmohammadsadegh A, Walz M, Lysa B, Tartler U, Remus R, et al. Molecular cloning and characterization of alternatively spliced mRNA isoforms from psoriatic skin encoding a novel member of the S100 family. FASEB J 2003;17:1969 –71. 5. Heizmann CW, Cox JA. New perspectives on S100 proteins: a multifunctional Ca2⫹-, Zn2⫹- and Cu2⫹-binding protein family [Review]. Biometals 1998;11:383–97. 6. Tiu SC, Chan WY, Heizmann CW, Scha¨ fer BW, Shu SY, Yew DT. Differential expression of S100B and S100A6 in the human fetal and aged cerebral cortex. Develop Brain Res 2000;119:159 – 68. 7. Chan WY, Xia CL, Dong DC, Heizmann CW, Yew DT. Differential expression of S100 proteins in the developing human hippocampus and temporal cortex. Microsc Res Tech 2003;60:600 –13. 8. Vives V, Alonso G, Solai AC, Joubert D, Legraverend C. Visualization of S100B-positive neurons and glia in the central nervous system of EGFP transgenic mice. J Comp Neurol 2003;457:404 –19. 9. Hofmann MA, Drury S, Fu C, Qu W, Taguchi A, Lu Y, et al. RAGE mediates a novel proinflammatory axis: a central cell surface receptor for S100/ calgranulin polypeptides. Cell 1999;97:889 –901. 10. Schmidt AM, Yan SD, Yan SF, Stern D. The multiligand receptor RAGE as a progression factor amplifying immune and inflammatory responses. J Clin Invest 2001;108:949 –55. 11. Huttunen HJ, Kuja-Panulat J, Sorci G, Angeletti AL, Donato R, Rauvala H. Coregulation of neurite outgrowth and cell survival by amphoterin and S100 proteins through receptor for advanced glycation end products (RAGE) activation. J Biol Chem 2000;275:40096 –105. 12. Kanner AA, Marchi N, Fazio V, Mayberg MR, Koltz MT, Siomin V, et al. Serum S100. A noninvasive marker of blood-brain barrier function and brain lesions. Cancer 2003;97:2806 –13. 13. Garbe C, Leiter U, Ellwanger U, Blaheta H-J, Meier F, Rassner G, et al. Diagnostic value and prognostic significance of protein S-100, melanomainhibitory activity, and tyrosinase/MART-1 reverse transcription-polymerase chain reaction in the follow-up of high-risk melanoma patients. Cancer 2003;97:1737– 45. 14. Ba´ nfalvi T, Udvarhelyi N, Orosz Z, Gergye M, Gilde K, Tima´ r J. Heterogeneous S-100B protein expression patterns in malignant melanoma and association with serum protein levels. Oncology 2003;64:374 –9. 15. Hauschild A, Engel G, Brenner W, Ga¨ swer R, Mo¨ nig H, Henze E, et al. Predictive value of serum S100B for monitoring patients with metastatic melanoma during chemotherapy and/or immunotherapy. Br J Dermatol 1999;140:1065–71. 16. Stigbrand T, Nyberg L, Ulle´ n A, Haglid K, Sandstro¨ m E, Brundell J. A new specific method for measuring S-100B in serum. Int J Biol Markers 2000;15:33– 40.
DOI: 10.1373/clinchem.2003.027367
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, 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. Stenzel1* (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
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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
[email protected]) 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 (7q31.2). 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 (http://www.innogenetics.com/site/diagnostics.html). 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 ELUCIGENETM CF-PolyT ASR system (Orchid Biosciences), which uses the ARMSTM technology (http://www.elucigene.co.uk/pdf/PolyT_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. roche-applied-science.com/pack-insert/3253660A.pdf); lab-
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Table 1. IVS8-T variant status in 25 CF cell lines used as test controls for CFTR gene mutation assays. Laboratoryb Samplea
Cell line
1
2
3
4
5
6
7
8
9
Consensus
Reported allelic variants
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
NA01531 NA07441 NA07552 NA08338 NA11275 NA11277 NA11280 NA11281 NA11282 NA11283 NA11284 NA11472 NA11496 NA11723 NA11859 NA11860 NA12444 NA12585 NA12785 NA12960 NA13591 NA11290 NA13032 NA13033 NA07464
9T/9T 7T/9T 7T/9T 7T/9T 7T/9T 7T/7T 7T/9T 9T/9T 7T/9T 9T/9T 7T/9T 7T/9T 9T/9T 5T/7T 7T/7T 7T/7T 7T/7T 7T/7T 7T/7T 7T/7T 5T/9T 9T/9T 5T/7T 7T/7T 7T/7T
9T/9T 7T/9T 7T/9T 7T/9T 7T/9T 7T/7T 7T/9T 9T/9T 7T/9T 9T/9T 7T/9T 7T/9T 9T/9T 5T/7T 7T/7T 7T/7T 7T/7T 7T/7T 7T/7T 7T/7T 5T/9T ND ND ND ND
NDc ND ND ND 7T/9T 7T/7T 7T/9T ND 7T/9T ND 7T/9T 7T/9T 9T/9T 5T/7T 7T/7T 7T/7T 7T/7T 7T/7T 7T/7T 7T/7T 5T/9T 9T/9T 5T/7T 7T/7T 7T/7T
9T/9T 7T/9T 7T/9T 7T/9T 7T/7Te 7T/7T 7T/7Te 9T/9T 7T/9T 9T/9T 7T/9T 7T/9T 9T/9T 5T/7T 7T/7T 7T/7T 7T/7T 7T/7T 7T/7T 7T/7T 5T/9T ND ND ND ND
9T/9T 7T/9T 7T/9T 7T/9T 7T/9T 7T/7T 7T/9T 9T/9T 7T/9T 9T/9T 7T/9T 7T/9T 9T/9T 5T/7T 7T/7T 7T/7T 7T/7T 7T/7T 7T/7T 7T/7T 5T/9T ND ND ND ND
ND ND ND ND 7T/9T 7T/7T 7T/9T ND 7T/9T 9T/9T 7T/9T 7T/9T 9T/9T 5T/7T 7T/7T 7T/7T 7T/7T 7T/7T 7T/7T 7T/7T 5T/9T 9T/9T 5T/7T 7T/7T ND
9T/9T 7T/9T 7T/9T 7T/9T 7T/9T 7T/7T 7T/9T 9T/9T 7T/9T 9T/9T 7T/9T 7T/9T 9T/9T 5T/7T 7T/7T 7T/7T 7T/7T 7T/7T 7T/7T 7T/7T 5T/9T 9T/9T 5T/7T 7T/7T ND
9T/9T 7T/9T 7T/9T 7T/9T 7T/9T 7T/7T 7T/9T 9T/9T 7T/9T 9T/9T 7T/9T 7T/9T 9T/9T 5T/7T 7T/7T 7T/7T 7T/7T 7T/7T 7T/7T 7T/7T 5T/9T ND 5T/7T 7T/7T ND
ND 7T/9T ND ND ND ND ND ND 7T/9T ND ND ND ND ND 7T/7T ND ND 7T/7T ND ND ND ND ND ND ND
9T/9T 7T/9T 7T/9T 7T/9T 7T/9T 7T/7T 7T/9T 9T/9T 7T/9T 9T/9T 7T/9T 7T/9T 9T/9T 5T/7T 7T/7T 7T/7T 7T/7T 7T/7T 7T/7T 7T/7T 5T/9T 9T/9T 5T/7T 7T/7T 7T/7T
⌬F508; ⌬F508d 3120 ⫹ 1G⬎A; 621 ⫹ 1G⬎Td R553X; ⌬F508d G551D 3659delC; ⌬F508d ⌬I507 711 ⫹ 1G⬎T(T); 621 ⫹ 1G⬎Td 621 ⫹ 1G⬎T; ⌬F508d G85E;621 ⫹ 1G⬎Td A455E; ⌬F508d R560T; ⌬F508d N1303K; G1349Dd G542X; G542Xd W1282X 2789 ⫹ 5G⬎A; 2789 ⫹ 5G⬎Ad 3849 ⫹ 10C⬎T; 3849 ⫹ 10C⬎Td 1717-1G⬎T(A) R1162X R347P; G551Dd R334W/?d R117H; F508d A455E; 621 ⫹ 1G⬎Td I506V F508C 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.
oratories 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). References 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, Iannuzzi 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.
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