Testing for variants in CYP2C19: population frequencies and testing experience in a clinical laboratory.

PURPOSE: We sought to determine the genotype frequencies for cytochrome p450 enzyme 2C19 variant alleles both in the US pan-ethnic population and various US ethnic groups and to establish the frequency of clinically actionable genotypes. METHODS: Analytical results were obtained from 1,396 consecutive samples submitted for cytochrome p450 enzyme 2C19 genotyping tests and stored in a proprietary database. This database was queried and genotypes and predicted phenotypes established. Anonymized samples were obtained from specimens submitted for cystic fibrosis genotyping that contained ethnicity information. Samples from 357, 149, and 346 individuals self-identified as white, African American, and Hispanic, respectively, were analyzed. In addition, 342 anonymized samples submitted for Ashkenazi Jewish panel testing were analyzed. RESULTS: Significant ethnic differences were observed in the frequencies of the *17 ultrarapid allele among the various groups studied. In the pan-ethnic population, 3.8% of tested patients were classified as ultrarapid metabolizers, 24% as extensive metabolizers heterozygous for a *17 ultrarapid allele, 27% as intermediate metabolizers, and 3.5% as poor metabolizers. Using stringent criteria, 7.3% of individuals would have clinically actionable genotypes. In addition, we detected two individuals with a haplotype of *2/*17 and a single individual with a haplotype of *4/*17 indicating that the *17 hypermetabolic allele can occur on a *1, *2, or *4 background.

FMR1 premutation carrier frequency in patients undergoing routine population-based carrier screening: insights into the prevalence of fragile X syndrome, fragile X-associated tremor/ataxia syndrome, and fragile X-associated primary ovarian insufficiency in the United States.

PURPOSE: Fragile X syndrome is caused by expansion and methylation of a CGG tract in the 5' untranslated region of the FMR1 gene. The estimated frequency of expanded alleles (≥55 repeats) in the United States is 1:257-1:382, but these estimates were not calculated from unbiased populations. We sought to determine the frequency of fragile X syndrome premutation (55-200 repeats) and full mutation (>200 repeats) alleles in nonselected, unbiased populations undergoing routine carrier screening for other diseases. METHODS: A previously validated laboratory-developed test using triplet-primed polymerase chain reaction was used to detect premutation and full mutation alleles in an unselected series of 11,759 consecutive cystic fibrosis carrier screening samples and 2011 samples submitted for screening for genetic diseases prevalent among the Ashkenazi Jewish population. RESULTS: Premutations were identified in 48 cystic fibrosis screening samples (1:245) and 15 samples (1:134) from the Ashkenazi Jewish population. Adjusted for the ethnic mix of the US population and self-reported ethnicity in our screening population, the estimated female premutation carrier frequency in the United States was 1:178. The calculated frequency of full mutation alleles was 1:3335 overall, and the calculated premutation frequency in males was 1:400. Based on frequency of larger, ≥70 repeat alleles, and reported penetrance, the calculated fragile X-associated tremor and ataxia syndrome, and fragile X-associated primary ovarian insufficiency frequencies is 1:4848 and 1:3560, respectively. CONCLUSION: Our calculated fragile X syndrome carrier rate is higher than previous estimates for the US population and warrants further consideration of population-based carrier screening.

Qualitative assessment of FMR1 (CGG)n triplet repeat status in normal, intermediate, premutation, full mutation, and mosaic carriers in both sexes: implications for fragile X syndrome carrier and newborn screening.

PURPOSE: Fragile X syndrome is caused by expansion and subsequent methylation of a CGG trinucleotide repeat in the FMR1 5'-untranslated region. Southern blot analysis is typically required to determine expansion size for triplet repeat lengths >200. We describe a triplet-primed polymerase chain reaction-based method using automated capillary electrophoresis detection for qualitative assessment of expanded CGG repeats. METHODS: The assay uses triplet-primed polymerase chain reaction in combination with GC-melting reagents and substitution of 7-deaza-2-deoxyGTP for dGTP. Amplicons are resolved by capillary electrophoresis. RESULTS: A distinctive pattern of tapering or "stutter" polymerase chain reaction amplification was evident on capillary electrophoresis in male and female patients harboring all expanded allele lengths examined (up to 2000 CGG repeats) and could be used to differentiate normal, intermediate, premutation, and full mutation alleles. Full mutation alleles exhibited an additional late-migrating amplicon on capillary electrophoresis. Mixing experiments demonstrated sensitivity as low as 1% for detection of the full mutation allele. In a 1275-sample concordance study against our existing polymerase chain reaction platform (with Southern blot analysis for repeat lengths ≥55), the triplet-primed polymerase chain reaction method exhibited 100% concordance for normal, intermediate, expanded, and full mutation alleles. This method also detected the full mutation alleles in DNA isolated from blood spots. CONCLUSION: This assay provides an accurate assessment of FMR1 repeat status and holds promise for use in carrier and newborn screening. The method distinguishes normal homozygous females from full mutation carrying females. Although the method is not useful for accurate sizing, it supplements the classic polymerase chain reaction method and results in significant reduction in the number of Southern blot analyses required to be performed in the laboratory to accurately assess the FMR1 genotype in all individuals.

Characterization of a recurrent novel large duplication in the cystic fibrosis transmembrane conductance regulator gene.

Recently, DNA rearrangements in the cystic fibrosis transmembrane conductance regulator (CFTR) gene have been described with increasing frequency. These large DNA rearrangements are not detected using conventional methods of DNA sequencing, single-strand conformational polymorphism, or denaturing high-performance liquid chromatography. We and others have described methods to detect such rearrangements in the CFTR gene. With one exception, all rearrangements reported thus far are single or multiple exon deletions, whereas only one report has described a large duplication. We describe here the detection and characterization of a novel large duplication in the CFTR gene. This duplication, referred to as gIVS6a + 415_IVS10 + 2987Dup26817bp, was detected in a classic CF female patient whose other mutation was DeltaF508. The duplication was inherited paternally. The duplication encompassed exons 6b to 10 and occurred on the IVS8-11TG/IVS8-7T/G1540 haplotype. This large duplication is predicted to result in the production of a truncated CFTR protein lacking the terminal part of NBD1 domain and beyond and thus can be considered a null allele. The combination of the DeltaF508 and gIVS6a + 415_IVS10 + 2987Dup26817bp mutation probably causes the severe CF phenotype in this patient. We designed a simple polymerase chain reaction test to detect the duplication, and we further detected the same duplication from another independent laboratory. The duplication breakpoint is identical in all three patients, suggesting a likely founder mutation.