Clinical Evaluation of the CF(12)m Cystic Fibrosis DNA Diagnostic Kit

¹ Department of Clinical Biochemistry, Hôpital Armand Trousseau, 75012 Paris, France, and
² Department of Biochemistry and Molecular Genetics, Hôpital Henri Mondor, 94010 Creteil, France;
^a author for correspondence: Fax 33 01 44 73 66 65, e-mail dpt-mb{at}infobiogen.fr

Cystic fibrosis (CF) is the most common lethal autosomal recessive disease in the Caucasian population. CF has a carrier frequency of about 1 in 25 and an incidence of 1 in 2000 to 3000 births, depending on the population group (1). Over 650 different mutations of the CFTR gene (2) have been described to date. Rapid and reliable methods are needed for the extensive investigations routinely performed in many laboratories. The CF(12)m kit uses multiplexed amplification refractory mutation system (ARMS^(TM)) technology (3), which allows the simultaneous identification of the more prevalent CFTR gene mutations (4) in 1 working day.

Forty DNA samples from CF patients or carriers and an unaffected individual were typed by two genetic testing laboratories. The reference method for DNA typing was the analysis of the 27 exons and the intron-exon boundaries of the CFTR gene as described previously (5). Six additional samples bearing the I507, Rl17C, R347H, D1152H, and Rl17P mutations and one bearing the 1540AG polymorphism, not detectable with the CF(12)m kit, were also tested to evaluate cross-reactivity.

DNA was extracted from thawed blood and analyzed by using the CF(12)m kit as recommended by the manufacturer (Zeneca Diagnostics). The CF(12)m test consists of two tubes. The A tube contains ARMS primers specific for the 1717–1GA, G542X, W1282X, N1303K, F508, and 384910kbCT mutations. The B tube contains ARMS primers specific for the 6211GT, R553X, G551D, R117H, R1162X, and R334W mutations. The B tube also contains an ARMS primer specific for the unaffected F508 allele. There are also two control reactions in each tube. After DNA amplification, the reaction mixtures were separated by agarose gel electrophoresis to reveal the diagnostic and amplification control DNA fragments. The presence of the control products and a specific ARMS product, defined by electrophoretic mobility, is diagnostic of the presence of the respective mutant allele and/or unaffected F508 allele (6).

The extraction of 10 blood samples could be completed in half a day. All samples were analyzed in both laboratories. In one laboratory, one sample required a repeat analysis because of the absence of a control DNA fragment. One sample, different in each laboratory, was not interpreted, even after reamplification. In laboratory 1, a compound heterozygous sample (1717–1GA/F508) had the allele carrying the 1717-lGA mutation, which was amplified only at a low concentration. In laboratory 2, one sample showed no amplification control band and no F508 ARMS products.

As expected, the mutations 27895GA, 2176insC, 1078delT, and S1235R and the polymorphisms 87540A/G and 2694A/C identified by the reference method were not detected by the kit, neither were they erroneously typed. This was also found for the I507, Rl17C, R347H, D1152H, Rl17P, and 1540AG samples (data not shown). None of the CF mutations detectable by the kit were incorrectly identified as other mutations.

In all cases (with the exception above) where one or more of the 12 mutations detected by the kit were present in the test set, i.e., 1717–1GA, G542X, W1282X, N1303K, F508, 6211GT, R553X, and G551D, the kit accurately identified them. When compared with the reference method, the kit detected the same mutations in 34 of the 40 samples (85%). Four samples carried a rarer mutation that is not detected by the kit. Two other samples were from patients homozygous for the W1282X and G542X mutations. Fig. 1 shows a result typical of those produced using the CF(12)m kit.

View larger version (69K): [in this window] [in a new window]   Figure 1. Nusieve(TM) 3:1 agarose gel electrophoresis of six CF(12) analyses. Each sample is amplified in two tubes (A and B). The identification of specific mutations are determined by presence of PCR products of the appropriate sizes in the relevant reactions. Lanes 1, 3, 6, 8, 10, 13, and 15, A tube; lanes 2, 4, 7, 9, 11, 14, and 16, B tube; lanes 5 and 12, molecular weight marker. Upper and lower PCR products: amplification controls. Genotypes detected, from left to right: lanes 1 and 2 (sample 1), F508 homozygous; lanes 3 and 4 (sample 2), G551D heterozygous; lanes 6 and 7 (sample 3), F508 homozygous; lanes 8 and 9 (sample 4), F508 homozygous; lanes 10 and 11 (sample 5), F508/G542X; lanes 13 and 14 (control), no mutation detected; lanes 15 and 16 (blank), no amplification.

Routine determination of disease-causing mutations requires accurate, rapid, reliable, and low-cost methods, permitting the simultaneous detection of multiple mutations. Various screening methods have been proposed for this purpose, such as multiplex denaturing gradient gel electrophoresis analysis (7), PCR-allele-specific oligonucleotide (8), ARMS (9), or DNA enzyme immunoassay (10). The CF(12)m kit uses multiplex ARMS (11), which is extremely rapid and allows the screening of at least 10 samples for 12 CFTR mutations in a work day. A major feature of the kit, therefore, is the speed with which the results are delivered. We found the kit to be reliable and accurate after evaluating it in a blind study in two independent laboratories, both of which are experienced in CF molecular diagnostics. The results obtained were in accordance with those described by the manufacturer and demonstrated a first-time amplification success rate of 97%.

To achieve consistently reliable results, we found that strict adherence to the protocols was required. The control DNA fragments should be visible routinely to avoid the risk of getting a false-negative result. The absence of the upper control fragment in conjunction with no mutant allele amplicons indicates that a repeat test is required.

The 12 mutations that the CF(12)m kit can detect are among the most frequent observed in European populations (1). The diagnostic power of the test, depending on the population under study, has been calculated (6). In the French population generally, 78.8% of CF chromosomes should be detected using the CF(12)m kit; in our hands, 85% were detected in both laboratories. This seems satisfactory because screening for very rare mutations does not markedly increase the detection frequency of CF chromosomes.

A potential limitation of the CF(12)m kit is that it does not distinguish between homozygotes and heterozygotes for mutations other than F508. Although homozygotes for the rarer mutations are usually encountered infrequently, this characteristic of the kit must be considered when interpreting the test result. This was demonstrated in this investigation when the genotype of the W1282X and G542X homozygous patients could not be determined because the kit does not differentiate between one or two mutated alleles, except for the F508 mutation.

The identification of mutations in the CFTR gene is required primarily in patients suspected of suffering from CF and is important for genetic counseling in the patient family. The CF(12)m kit is useful in this case for first-line screening and can be complemented by other methods if two mutations are not detected initially. The detection rate of the kit allows the calculation by Bayesian analysis of the posterior risk of an "at risk" individual with a negative result. Similar assessments of carrier risk estimation can be calculated for individuals with a family history of CF where affected or obligate heterozygous individuals are not available for mutation typing.

The CF(12)m kit may prove useful if CF carrier screening programs are envisaged (12), and it could be of particular use in CF neonatal screening in association with immunoreactive trypsin (IRT) (13) or pancreatitis-associated protein (14).

Acknowledgments

The CF(12)m-PCR test kit has been developed by ZENECA Diagnostics. The authors gratefully acknowledge ZENECA Diagnostics for making the kit available for this study before the kit launch. We are grateful to Susie Howes for helpful discussions during the course of the study and to Michel Bahuau for his generous cooperation. The PCR process is covered by patents held by Hoffmann-La Roche Inc. ARMS is a trademark of ZENECA Limited. The ARMS technology is the subject of European Patent No. 0 332 435, US Patent No. 5595890, and corresponding world-wide patent property (All ZENECA Limited).

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