Issues in implementing prenatal screening for cystic fibrosis: results of a working conference.

PURPOSE: To summarize a conference convened to examine how cystic fibrosis screening might appropriately be introduced into routine prenatal practice. METHODS: Participants included experts from various relevant disciplines. Systematic reviews and data from individual trials were presented; issues were identified and discussed. RESULTS: Judged by published criteria, prenatal cystic fibrosis screening is suitable for introduction. Screening can be performed cost-effectively by identifying racial/ethnic groups at sufficient risk and then using either of two models for delivering laboratory services. Validated educational materials exist. Ethical issues are not unique. CONCLUSIONS: Once adequate facilities for patient and provider education, testing, counseling, quality control, and monitoring are in place, individual programs can begin prenatal screening for cystic fibrosis.

Hereditary haemochromatosis mutation frequencies in the general population.

OBJECTIVES: This study aims to expand our knowledge of the general population frequency of two mutations, C282Y and H63D, identified in the candidate gene for hereditary haemochromatosis, and to determine whether the testing can be performed using routinely obtained cheek-brush (buccal) samples. SETTING: Banked buccal lysate samples, randomised and coded for anonymity, from a cohort of couples who underwent prenatal cystic fibrosis screening in Maine. METHODS: A multiplex ARMS test was performed on buccal cell lysates to identify the two mutations. RESULTS: Genotype frequencies found among the 1001 subjects studied (502 women, 499 men) were: seven C282Y homozygotes, 22 C282Y/H63D compound heterozygotes, 97 C282Y heterozygotes, 17 H63D homozygotes, 246 H63D heterozygotes, and 612 individuals with no detectable mutation. The allele frequencies for C282Y and H63D were 0.066 and 0.151, respectively. CONCLUSIONS: Observed genotype frequencies in Maine are consistent with expectations and with consensus data from five smaller studies. Combined mutational analysis data indicate that homozygosity for C282Y (the genotype found in about 85% of subjects with diagnosed hereditary haemochromatosis) occurs in 51 per 10,000 white subjects of northern European heritage; the corresponding total hereditary haemochromatosis prevalence of about 60 per 10,000 is consistent with previous estimates. The study also confirms that H63D would not be useful in general population screening for hereditary haemochromatosis.

A multiplex ARMS test for 10 cystic fibrosis (CF) mutations: evaluation in a prenatal CF screening program.

In Maine, prenatal screening for cystic fibrosis (CF) is offered through primary care providers. Cheekbrush (buccal) samples are routinely tested for eight mutations by multiplex PCR amplification of five exons, followed by dot-blot hybridization with pooled allele-specific oligonucleotides (ASO). The ASO methodology is widely used and effective, but somewhat time and labor intensive when applied to CF carrier testing or couple-based prenatal screening in the general pregnant population. Amplification Refractory Mutation System (ARMS) is an improvement of the PCR that allows rapid detection of mutations involving single base changes or small deletions/insertions. In this study, two multiplex ARMS reactions are used to test for 10 common CF mutations. Clinical evaluation of the ARMS test includes a retrospective study of 140 banked samples (54 cell line, proficiency testing, and buccal controls; 86 clinical buccal samples) with known CF genotype (57 with CF mutations, 83 no mutation), followed by a prospective trial in which 309 buccal samples are analyzed con-currently using both methods. The success rate of the ARMS test in buccal lysates is comparable to the ASO method; all CF mutations are successfully identified. For testing nonsterile buccal lysates with low DNA concentrations, optimized performance in the ARMS method is obtained using Amplitaq Gold polymerase. The ARMS method developed is easy, rapid (1 day), and avoids the need for ASO probe labeling, dot-blotting and autoradiography. This study provides further evidence that ARMS methodology is suitable for clinical CF mutation analysis.

A homogeneous method for genotyping with fluorescence polarization.

We combined the amplification refractory mutation system (ARMS) and fluorescence polarization (FP) to give a homogeneous genomic DNA genotype analysis method. Oligonucleotide probes labeled with the fluorescein dyes fluorescein isothiocyanate and 5-([4,6-dichlorotriazin-2-yl]amino)fluorescein and the rhodamine dye 6-carboxyrhodamine were included in amplification mixes and were annealed to PCR products after amplification. Hybridization was accompanied by an increase in the FP of the probe. We demonstrated homogeneous genotyping by analyzing human DNA samples for delta F508 mutation status of the cystic fibrosis transmembrane conductance regulator gene. The genotypes determined with the method described herein were in full agreement with those obtained by the conventional application of ARMS. We also demonstrated the simultaneous detection of two PCR products in a single reaction. The assay method described is homogeneous and so obviates the necessity to open reaction vessels after amplification. This therefore eliminates PCR carryover contamination.

Development, multiplexing, and application of ARMS tests for common mutations in the CFTR gene.

The amplification refractory mutation system (ARMS) is a simple, rapid and reliable method for the detection of any mutation involving single base changes or small deletions. We have applied ARMS methodology to the detection of mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Single ARMS tests have been developed for 11 CFTR mutations found in the northwest of England. ARMS reactions for the most common mutations have been multiplexed to give a test which will detect the presence of the delta F508, G551D, G542X, and 621 + 1G----T mutations in a DNA sample. The multiplex test has been validated by the analysis of over 500 previously genotyped samples and has been found to be completely accurate. The rapid detection of the most common mutations has enabled early molecular confirmation of suspected cystic fibrosis in neonates, rapid typing of cystic fibrosis patients and their relatives, and testing of sperm and egg donors.