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Technical Briefs |
1 Department of Pathology, Duke University Medical Center, Durham, NC; 2 Coriell Cell Repositories, Coriell Institute for Medical Research, Camden, NJ; 3 Department of Human Genetics, Emory University School of Medicine, Atlanta, GA; 4 Center for Genetic Testing at Saint Francis, Tulsa, OK; 5 Department of Pathology, State University of New York Upstate Medical University, Syracuse, NY; 6 DNA Technologies Group, National Institute of Standards and Technology, Gaithersburg, MD; 7 ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT;
aaddress correspondence to this author at: Abbott Molecular, 1300 E. Touhy Ave., Des Plaines, IL 60016; fax 224-361-7054, e-mail timothy.stenzel{at}abbott.com
Clinical genetic testing laboratories offer tests for >800 conditions (1) and need both positive and negative genetic control materials for proficiency evaluation, quality control, and test development/validation. Few such materials are available(2)(3)(4)(5). Genetic material, generally DNA, can be purified from Epstein–Barr virus (EBV)-transformed lymphocyte cell lines. The resulting preparations closely resemble DNA purified from patient samples and often are appropriate controls for clinical genetic testing applications. The National Institute of General Medical Sciences (NIGMS) Human Genetic Cell Repository(6) and the National Institute on Aging (NIA) Aging Cell Repository(7) house thousands of EBV-transformed cell lines, but mutations have been described in only 
600 of these.
We screened 13 cell lines from the NIGMS Repository for medically important mutations in 11 different genes: CFTR, F5, F2, MTHFR, HFE, GJB2 (connexin 26), FMR1 (fragile X), HBA1/HBA2 (
-thalassemia), FGFR3, HD, and HbS/HbC. Six of the cell lines had previously been shown to carry 1 or more mutations in at least one of these genes, and 7 were derived from apparently healthy individuals with no known mutations. Some cell lines were subsequently developed for use as clinically validated positive controls for molecular genetic testing or were used as negative control materials [see accompanying article by Bernacki et al (8), in this issue].
DNA was extracted according to the Puregene protocol (Gentra Systems, Inc.). Selected mutations were analyzed by standard molecular diagnostic techniques at the Duke Molecular Diagnostics Laboratory and/or at other CLIA-certified clinical testing facilities where indicated. The 11 diseases characterized, the specific mutations analyzed, and the general methods used are listed below. The OMIM numbers (9) are listed in Table 1
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Cystic Fibrosis (CFTR; ABCC7).
Four tests were used to analyze the CFTR gene: the Roche Linear Array CF-31 (Roche Molecular Diagnostics); ABI CF Assay (Applied Biosystems); an in-house assay using SNaPshot technology from Applied Biosystems for detection of I148T (10), which was not detected by the commercial reagent sets used when this study was undertaken; and direct DNA sequence analysis for detection of S1235R. The combination of assays detects all 25 mutations in the 2001 American College of Medical Genetics (ACMG)-recommended screening panel(11), including T allele status, plus S1235R, 405 + 3A>C, G480C, A559T, 230insA, Q493X, V520F, S549R, S549N, 3849 + 4A>G, 3905insT, Y122X, R347P, and R347H. For one cell line (GM13591), long-range allele-specific PCR was used with oligonucleotide ligation analysis (ABI CF assay) to determine the cis/trans status of the IVS-8 polyT allele and the R117H mutation [see below; ARUP Laboratories(12)]. Four cell lines, GM07441, GM13591, GM03469, and GM00130, were tested at additional laboratories. One laboratory (Ambry Genetics) scanned all exons and significant splice sites in the CFTR gene using modified temporal temperature gradient electrophoresis(13). Suspect regions were sequenced.
Factor V Leiden thrombophilia (FVL; F5), prothrombin thrombophilia (F2), and methylenetetrahydrofolate reductase (MTHFR) deficiency.
Testing for FVL (R506Q), prothrombin (20210G>A), and MTHFR 677C>T mutations was performed with multiplex PCR amplification with 3 sets of disease-specific primers followed by restriction enzyme analysis of amplified fragments.
Hereditary hemochromatosis (HFE).
HFE mutations H63D, S65C, and C282Y were detected by PCR amplification followed by restriction enzyme analysis.
Nonsyndromic hereditary hearing loss/connexin 26 (GJB2).
The 35delG mutation was detected by PCR amplification followed by direct DNA sequence analysis.
Fragile X.
FMR1 gene analysis was performed at the NIST, by specifically designed PCR and sequencing methods (14). Because preferential amplification may occur when 2 alleles are present, results are reported only for cell lines derived from males.
-Thalassemia.
Deletions in the -globin gene region (HBA1 and HBA2) were detected by Southern blot analysis of restriction enzyme digests with probes for both - and 
-globin. Some -thalassemia testing was performed at the Emory Molecular Genetics Laboratory with use of both Southern blot and PCR analysis.
Nonsyndromic craniosynostosis (Muenke syndrome; FGFR3)
The FGFR3 749C>G mutation was detected by PCR amplification followed by direct DNA sequence analysis.
Huntington disease (HD).
HD testing was performed at the H.A. Chapman Institute of Medical Genetic. The CAG repeat region was analyzed by PCR.
Sickle cell/hemoglobin C disease (HbS/HbC).
HbS and HbC mutations were detected by PCR amplification of the appropriate region, followed by identification of allele type by allele-specific oligonucleotide analysis of PCR fragments immobilized on filters.
We confirmed the presence of all mutations reported previously by NIGMS and detected 16 previously unreported mutations and sequence variants (Table 1
), including a novel CFTR single-base deletion. CFTR IVS-8 polyT allele status was established for all cell lines.
GM16028 had been reported previously to carry mutations in the F2, F5, and MTHFR genes and was considered particularly useful as a multiplex positive control. We found that this cell line is heterozygous for S65C (15) in the HFE gene as well as heterozygous for type 2 -thalassemia. GM16000, which was previously reported to have mutations in the HFE and F2 genes, also carried MTHFR 677C>T.
The donor patient for GM07441 was described as having symptoms of cystic fibrosis and was known to carry the deleterious CFTR mutation 3120 + 1G>A. We detected 1 copy of 621 + 1G>T. Both of these mutations are included in the 2001 and 2004 screening panels recommended by the ACMG (11)(16); therefore, this cell line represents an important compound heterozygous genotype. GM07441 was clinically validated as a positive control for both CFTR mutations in the context of the CDC project. GM07441 was also found to be heterozygous for MTHFR 677C>T and homozygous for type 2 -thalassemia.
CFTR IVS-8 polyT analysis of GM13591, reported by NIGMS to carry the CFTR mutations 
F508 and R117H, indicated a genotype of 5T/9T. Additional analysis showed that the 5T allele was in cis with R117H, which can produce a more severe phenotype (17).
Previously unreported MTHFR 677C>T mutations were detected in 5 of the 13 cell lines, reflecting the estimated allelic frequency of up to 38% (18)(19). Similarly, previously unreported mutations in the HFE gene were found in 4 cell lines. Overall mutation carrier frequency for HFE has been estimated at 1 in 10 individuals of northern European descent(20). The HFE S65C mutation, present in GM16028, may be associated with mild disease(15), and clinical testing is performed for this mutation. S65C also has the potential to interfere with testing for HFE H63D because the 2 sites are so close together. S65C has not been reported previously in a publicly available cell line.
Seven cell lines with no previously reported mutations were evaluated. Four were negative for all mutations included in the initial analysis, and 2 cell lines, GM03469 and GM00130, were selected for use as negative controls for the CDC clinical validation project mentioned above. The mutational analysis performed by the laboratories participating in the clinical validation project was in some cases more comprehensive than the testing performed for this report. Ambry Genetics (Costa Mesa, CA) scanned all exons and significant splice sites in the CFTR gene and detected mutations or sequence variants in both "negative" cell lines. All mutations and variants were confirmed by direct DNA sequence analysis. GM03469 was found to carry R170H, a known deleterious mutation associated with congenital bilateral absence of the vas deferens (21). R170H was also detected in this cell line by sequence analysis at Laboratory Prof Seelig and Colleagues (Karlsruhe, Germany). GM00130 was found to be homozygous for M470V, which has been associated with disease in particular haplotype backgrounds(17)(22). A novel CFTR variant, 4375-36delT, was also detected in GM00130 and independently confirmed by sequence analysis at the State University of New York (Syracuse, NY). It is not known whether this variant is associated with disease, although the position of the deletion (36 bp 5' to exon 24) suggests it would most likely be benign. One copy of M470V was also detected in GM13591, which carries 5T in cis with R117H and 9T in cis with F508. These results illustrate the importance of precisely defining the scope of mutational analyses performed for allelic characterization.
GM03469 was apparently normal for all other mutations tested, but 1 of the 5 laboratories testing for -thalassemia used a PCR-based method (23) that reproducibly gave a type 2 heterozygous genotype for this cell line. The same laboratory detected no deletions by Southern analysis. The other 4 laboratories reported no deletions.
GM06160 also gave anomalous results in testing for -thalassemia. Southern blot analysis showed an unusual banding pattern with a -globin gene probe, with additional smaller bands in BamHI and BglII digests (an EcoRI digest appeared normal). The -globin probe gave a normal pattern for all digests. The reason for the unusual pattern was not evident; we do not recommend that this cell line be used as a control for -globin analysis.
All cell lines tested negative/normal for Huntington disease, Muenke syndrome, and nonsyndromic hereditary hearing loss. All cell lines derived from males also tested negative for mutations of the FMR1 gene, although GM16266, with 44 CGG repeats, is approaching the "gray zone" (24) for CGG expansion. This cell line may be an important sizing calibrator or control for clinical assays.
These cell lines should be a useful resource. Existing cell repositories provide rich resources that have been greatly underutilized. We recommend that funding be made available for further exploration of the potential of these cell lines to provide clinically useful material.
Acknowledgments
This work was supported by Contract 200-2000-10050 from the Centers for Disease Control and Prevention (Atlanta, GA). The content of this publication does 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 thank Drs. Ana Stankovic, Laurina Williams, and Joe Boone of the Centers for Disease Control and Prevention, Public Health Practices Program Office, Division of Laboratory Services, for their contributions to this study. T.T.S. received research funding from Abbott Laboratories during the course of this work for unrelated projects, and he now works for Abbott Laboratories. Abbott Laboratories currently markets a subsequent version of the ABI CF Assay used in this work.
Footnotes
1 current affiliation: Department of Biomedical Engineering, North Carolina State University, Raleigh, NC; 
References
A and I148T mutations in cystic fibrosis. Clin Chem 2001;47:2053-2055.T mutation of the methylenetetrahydrofolate reductase gene in different ethnic groups. Thromb Haemost 1998;79:119-121.[Web of Science][Medline]
[Order article via Infotrieve]
]-thalassemia genotypes by polymerase chain reaction. J Lab Clin Med 2001;137:290-295.[CrossRef][Web of Science][Medline]
[Order article via Infotrieve]
The following articles in journals at HighWire Press have cited this article:
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  S. H. Bernacki, J. C. Beck, A. K. Stankovic, L. O. Williams, J. Amos, K. Snow-Bailey, D. H. Farkas, M. J. Friez, F. M. Hantash, K. J. Matteson, et al. Genetically Characterized Positive Control Cell Lines Derived from Residual Clinical Blood Samples Clin. Chem., November 1, 2005; 51(11): 2013 - 2024. [Abstract] [Full Text] [PDF]
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