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.

Cystic fibrosis testing 8 years on: lessons learned from carrier screening and sequencing analysis.

PURPOSE: This study reviews data from our cystic fibrosis testing program to evaluate the performance of population-based carrier screening and compare observed detection rates with predicted results of the American College of Medical Genetics/American College of Obstetricians and Gynecologists recommended panel of 23 mutations. METHODS: We queried our proprietary databases containing approximately 3 million cystic fibrosis screening tests, 1300 prenatal diagnostic tests, and 2400 cystic fibrosis sequencing analyses. RESULTS: We observed an overall cystic fibrosis carrier frequency of 1:37.6 individuals in the pan-ethnic tested population. This represents a detection rate of 77%, given an estimated US pan-ethnic carrier frequency of 1:29. For patients self-identified as white or Ashkenazi Jewish, a carrier frequency of 1:29 and 1:27 were observed, respectively. A combined frequency of 1:28, representing close to 90% of carriers, was identified in these two highest risk populations. In total, 119 affected fetuses were identified by prenatal diagnoses, a ratio of 1 affected fetus per 25,000 carrier screens. Of 62 newborns with positive immunoreactive trypsinogen and positive sweat tests, almost all of whom had been tested using the American College of Medical Genetics/American College of Obstetricians and Gynecologists panel, only two individuals would have been identified using an expanded mutation panel. CONCLUSION: The American College of Medical Genetics/American College of Obstetricians and Gynecologists panel of 23 mutations is performing as predicted in detecting cystic fibrosis carriers in the United States among all ethnic groups. No recurrent mutations have been detected in sufficient numbers to justify including any additional mutations to the existing panel. An expanded American College of Medical Genetics/American College of Obstetricians and Gynecologists panel would have a minimal impact on the prevention of births of children affected with cystic fibrosis.

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.

Development of a web-based query tool for quality assurance of clinical molecular genetic test results.

The College of American Pathologists molecular pathology checklist item (MOL.20550) calls for periodic review of molecular genetic statistics, including percentages of normal and abnormal findings and allele frequencies. A web-based query tool application for clinical molecular genetic test results was developed to plot dynamically and display genotype and/or allele frequencies for any time period. This tool is used to produce plots of all high-volume molecular genetic assays (>50 samples per month). A single web page contains pull-down menus, enabling the user to select the type of chart to be generated (genotype or allele frequency), the molecular genetic assays to chart (from one to all), the ending date for data in the chart (month and year), and the duration of the time period to plot (1 to 12 months). The rendered graphical and textual frequency data can then be viewed or printed. This tool can be used by any laboratory and interfaced with a standard laboratory information system. Monthly quality control charts and tables are now generated in minutes compared with the hours it took using manual charting applications. This simplified process enables timely compliance with a College of American Pathologists checklist item.

Technical validation of a TM Biosciences Luminex-based multiplex assay for detecting the American College of Medical Genetics recommended cystic fibrosis mutation panel.

The American College of Medical Genetics (ACMG) and the American College of Obstetrics and Gynecology have recommended population-based carrier screening for cystic fibrosis to include 23 mutations and 5 polymorphisms in the cystic fibrosis transmembrane regulator gene(CFTR). We estimate 20% of all pregnant women are being tested for their CF carrier status. We assessed two commercially available analyte-specific reagents (ASRs) capable of testing all 25 mutations of the original ACMG-recommended panel, Tag-It CFTR40 + 4 Luminex-based reagent from Tm Biosciences, and our current assay platform, CF Genotyper V. 3.0 from Abbott/Celera. Blinded testing using genomic controls containing known CFTRmutations demonstrated that the Tag-It platform detected all mutations on the ACMG-recommended panel. We next performed a platform comparison with 1,029 consecutive patient samples. There were no discrepant results in 1,029 consecutive analyses between the two platforms, yielding an impressive figure of >25,000 individual genotypes without error for both platforms. In conclusion, both the Abbott/Celera ASR reagent and the Luminex-based Tag-It CF ASR reagent are appropriate for use in the clinical laboratory.

Molecular testing for Fragile X Syndrome: lessons learned from 119,232 tests performed in a clinical laboratory.

PURPOSE: To examine the data from over 119,000 Fragile X Syndrome tests and 307 prenatal tests to detect unsuspected findings and obtain clinical data when indicated to optimize genetic counseling. METHODS: A proprietary database containing 119,232 consecutive postnatal and 307 prenatal FXS tests performed between November 2, 1992 and June 1, 2006 was queried. RESULTS: The distribution of normal FMR1 alleles was a bimodal distribution with a major peak at 30 repeats and a minor peak at 21 repeats. Of 59,707 tests performed for males, 1.4% had a fully expanded and methylated FMR1 allele. Of 59,525 tests performed for females, 0.61% had an affected FMR1 allele, and 1.7% had a premutation FMR1 allele for a total carrier frequency of 1.3%. When fetuses inherited an expanded maternal allele, the risk of expansion to a full affected allele was 0%, 5%, 30% and 100% for allele sizes of <50, 50-75, 76-100 and >100 repeats, respectively. CONCLUSIONS: These figures can be used for genetic counseling of patients presenting for carrier detection and prenatal diagnosis for Fragile X Syndrome.

Novel and recurrent rearrangements in the CFTR gene: clinical and laboratory implications for cystic fibrosis screening.

Because standard techniques used to detect mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene do not detect single or multiple exonic rearrangements, the importance of such rearrangements may be underestimated. Using an in-house developed, single-tube, semi-quantitative fluorescent PCR (SQF PCR) assay, we analyzed 36 DNA samples submitted for extensive CFTR sequencing and identified ten samples with rearrangements. Of 36 patients with classic CF, 10 (28%) harbored various deletions in the CFTR gene, accounting for 14% of CF chromosomes. A deletion encompassing the CFTR promoter and exons 1 and 2 was detected in a sample from one proband, and in the maternal DNA as well. In another family, a deletion of the promoter and exon 1 was detected in three siblings. In both of these cases, the families were African American and the 3120+1G > A splice site mutation was also identified. These promoter deletions have not been previously described. In a third case, a deletion of exons 17a, 17b, and 18 was identified in a Caucasian female and the same mutation was detected in the paternal DNA. In the other seven cases, we identified the following deletions: exons 2 and 3 (n = 2); exons 4, 5, and 6a; exons 17a and 17b; exons 22 and 23; and exons 22, 23, and 24 (n = 2). In our series, the frequency of CFTR rearrangements in classic CF patients, when only one mutation was identified by extensive DNA sequencing, was >60% (10/16). Screening for exon deletions and duplications in the CFTR gene would be beneficial in classic CF cases, especially when only one mutation is identified by standard methodologies.

CFTR 5T variant has a low penetrance in females that is partially attributable to its haplotype.

PURPOSE: The study's purpose was to understand the molecular basis for different clinical phenotypes of the 5T variant, a tract of 5 thymidines in intron 8 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which disrupts processing of CFTR mRNA and reduces synthesis from the corresponding CFTR alleles. METHOD: We analyzed the polymorphic TG dinucleotide repeat adjacent to the 5T variant in intron 8 and the codon 470 in exon 10. Patients selected for this study were positive for both the 5T variant and the major cystic fibrosis mutation, Delta F508. Almost all Delta F508 mutation alleles occur in a 10TG-9T-470M haplotype. Therefore, it is possible to determine the haplotype of the 5T variant in trans. RESULTS: Of the 74 samples analyzed, 41 (55%) were 11TG-5T-470M, 31 (42%) were 12TG-5T-470V, and 2 (3%) were 13TG-5T-470M. Of the 49 cases for which we had clinical information, 17.6% of females (6/34) and 66.7% of males (10/15) showed symptoms resembling atypical cystic fibrosis. The haplotype with the highest penetrance in females (42% or 5/12) and more than 80% (5/6) in males is 12TG-5T-470V. We also evaluated 12 males affected with congenital bilateral absence of vas deferens and positive for the 5T variant; 10 of 12 had the 12TG-5T-470V haplotype. CONCLUSION: Overall, the 5T variant has a milder clinical consequence than previously estimated in females. The clinical presentations of the 5T variant are associated with the 5T-12TG-470M haplotype.

Contrasting phenotypes in three patients with novel mutations in mitochondrial tRNA genes.

We studied three patients, each harboring a novel mutation at a highly conserved position in a different mitochondrial tRNA gene. The mutation in patient 1 (T5543C) was associated with isolated mitochondrial myopathy, and occurred in the anticodon loop of tRNA(Trp). In patient 2, with mitochondrial myopathy and marked retinopathy, the mutation (G14710A) resulted in an anticodon swap (Glu to Lys) in tRNA(Glu). Patient 3, who manifested mitochondrial encephalomyopathy and moderate retinal dysfunction, harbored a mutation (C3287A) in the TpsiC loop of tRNA(Leu(UUR)). The mutations were heteroplasmic in muscle in all cases, and sporadic in two cases. PCR-RFLP analysis in all patients showed much higher amounts of mutated mtDNA in affected tissue (muscle) than unaffected tissue (blood), and significantly higher levels of mutated mtDNA in cytochrome c oxidase (COX)-negative muscle fibers than in COX-positive fibers, confirming the pathogenicity of these mutations. The mutation was also detected in single hair roots from all three patients, indicating that each mutation must have arisen early in embryonic development or in maternal germ cells. This suggests that individual hair root analyses may reflect a wider tissue distribution of mutated mtDNA than is clinically apparent, and might be useful in predicting prognosis and, perhaps, the risk of transmitting the mutation to offspring. Our data suggest a correlation between clinical phenotype and distribution of mutated mtDNA in muscle versus hair roots. Furthermore, the high threshold for phenotypic expression in single muscle fibers (92-96%) suggests that therapies may only need to increase the percentage of wild-type mtDNA by a small amount to be beneficial.

Technical validation of a multiplex platform to detect thirty mutations in eight genetic diseases prevalent in individuals of Ashkenazi Jewish descent.

PURPOSE: This study determines the analytic accuracy of a Luminex bead-based commercial analyte-specific reagent for the simultaneous analysis of 30 mutations prevalent in Ashkenazi Jews at eight genetic disease loci. METHODS: DNA from 20 samples with known abnormal genotypes were run a total of 109 times. DNA from 820 patients with unknown genotypes submitted for Ashkenazi Jewish testing panels were analyzed using our current laboratory techniques. The 820 samples were then stripped of identifiers, coded, and reanalyzed using the Tm Biosciences (Toronto, Canada) Ashkenazi Jewish panel analyte-specific reagent in a blinded fashion. For the controls, comparisons were made with their known genotypes. For the patient samples, the results of the Tm assay were compared with the results of our current assay. For 24 of the 30 mutations, we had genomic DNA controls or detected patients' samples heterozygous for these mutations. RESULTS: There were no discrepant results in the control or patient samples. In the patient samples, 19,680 genotyping reactions were performed without error in both our laboratory-developed single-disease assays and the Tm multiplex assay. Including the controls, 22,296 genotypes were determined without error. CONCLUSION: The Tm Biosciences Ashkenazi Jewish analyte-specific reagent is capable of performing accurate analyses of 24 different mutations in eight different genes in a single multiplex reaction and can be used with confidence in the clinical molecular genetics laboratory.

Extensive sequencing of the CFTR gene: lessons learned from the first 157 patient samples.

Cystic fibrosis (CF) is one of the most common monogenic diseases affecting Caucasians and has an incidence of approximately 1:3,300 births. Currently recommended screening panels for mutations in the responsible gene (CF transmembrane regulator gene, CFTR) do not detect all disease-associated mutations. Our laboratory offers extensive sequencing of the CFTR (ABCC7) gene (including the promoter, all exons and splice junction sites, and regions of selected introns) as a clinical test to detect mutations which are not found with conventional screening. The objective of this report is to summarize the findings of extensive CFTR sequencing from our first 157 consecutive patient samples. In most patients with classic CF symptoms (18/24, 75%), extensive CFTR sequencing confirmed the diagnosis by finding two disease-associated mutations. In contrast, only 5 of 75 (7%) patients with atypical CF had been identified with two CFTR mutations. A diagnosis of CF was confirmed in 10 of 17 (58%) newborns with either positive sweat chloride readings or positive immunoreactive trypsinogen (IRT) screen results. We ascertained ten novel sequence variants that are potentially disease-associated: two deletions (c.1641AG>T, c.2949_2853delTACTC), seven missense mutations (p.S158T, p.G451V, p.K481E, p.C491S, p.H949L, p.T1036N, p.F1099L), and one complex allele ([p.356_A357del; p.358I]). We ascertained three other apparently novel complex alleles. Finally, several patients were found to carry partial CFTR gene deletions. In summary, extensive CFTR gene sequencing can detect rare mutations which are not found with other screening and diagnostic tests, and can thus establish a definitive diagnosis in symptomatic patients with previously negative results. This enables carrier detection and prenatal diagnosis in additional family members.

Detection of 677CT/1298AC "double variant" chromosomes: implications for interpretation of MTHFR genotyping results.

PURPOSE: Genotyping 37,026 individuals as part of a thrombophilia evaluation, we determined and analyzed the genotypic frequencies of the 677CT and 1298AC mutations in the methylenetetrahydrofolate reductase (MTHFR) gene. METHODS: The 677CT and 1298AC mutations in the MTHFR gene were determined by either a laboratory-developed test involving PCR amplification and restriction digestion utilizing the ABI 3100 capillary electrophoresis apparatus (Applied Biosystems Inc) or by using an Analyte Specific Reagent (ASR) supplied by Third Wave Technologies. The genotype for three specimens with triple variant MTHFR mutations were confirmed by DNA sequencing on the ABI 3100 capillary electrophoresis apparatus. RESULTS: The MTHFR frequencies of the 677CT/1298AA, 677CC/1298AC, 677CT/1298AC, 677CC/1298AA, 677TT/1298AA, 677CC/1298CC, 677TT/1298AC, and 677CT/1298CC genotypes were 0.228, 0.208, 0.198, 0.153, 0.122, 0.088, 0.0005, and 0.0003, respectively. CONCLUSIONS: Individuals containing double variant MTHFR mutations on one allele (cis) cannot be distinguished between compound heterozygotes (trans) for 677CT and 1298AC mutations in routine clinical testing, a genotype associated with thrombophilia. Such patients could be inappropriately counseled for being at high risk for thrombotic episodes. Until information regarding prevalence and the clinical consequences of this double variant (cis) allele becomes available, caution should be used in interpreting the genotyping results of compound heterozygosity for 677CT and 1298AC.

Extensive sequencing of the cystic fibrosis transmembrane regulator gene: assay validation and unexpected benefits of developing a comprehensive test.

PURPOSE: To develop a sequencing assay for the gene to identify mutations in patients with cystic fibrosis (CF). METHODS: An automated assay format was developed to sequence all exons and splice junctional sequences, the promotor region, and parts of introns 11 and 19. RESULTS: After validating the assay using 20 known samples, DNA of seven patients, four of whom were heterozygous for a known CF mutation, was sequenced. Known CF mutations were detected in seven of the eight chromosomes, and a novel missense mutation was detected in the eighth. In addition, this assay allowed 14 ambiguous results obtained using the Roche CF gold strips to be resolved. Three false-positive diagnoses were prevented; a different mutation at the same codon was identified in two patients and confirmation was provided in the remaining nine cases. CONCLUSIONS: Sequencing of the gene provides important information for CF patients and is a valuable adjunct to a carrier screening program to resolve ambiguities in panel testing.

Molecular screening for diseases frequent in Ashkenazi Jews: lessons learned from more than 100,000 tests performed in a commercial laboratory.

PURPOSE: To determine the frequency of carriers of Ashkenazi Jewish (AJ) genetic diseases in the US population and compare these numbers with previously published frequencies reported in smaller more isolated cohorts. METHODS: A database containing more than 100,000 genotyping assays was queried. Assays for 10 separate AJ genetic diseases where comparisons were made with published data. RESULTS: As expected, we observed lower carrier frequencies in a general, US population than those reported in literature. In 2427 patients tested for a panel of 8 AJ diseases, 20 (1:121) were carriers of two diseases and 331 (1:7) were carriers of a single disease. Fifty-three of 7184 (1:306) individuals tested for Gaucher disease had 2 Gaucher Disease mutations indicating a potentially affected phenotype. CONCLUSIONS: As the number of AJ diseases increases, progressively more individuals will be identified as carriers of at least one disease.

Cystic fibrosis screening: lessons learned from the first 320,000 patients.

PURPOSE: To examine the data from > 335,000 Cystic fibrosis (CF) tests to detect unsuspected findings and obtain clinical data when indicated to optimize genetic counseling. METHODS: A proprietary database containing 335,204 consecutive CF DNA tests and 445 CF prenatal diagnostic tests was queried. Clinical information was obtained for prenatal and selected nonprenatal cases by telephone contact with physician offices. RESULTS: The mutation 1078delT was found in much lower frequency than expected with rates of only 1:55,867 tests and 0.06% of CF mutations. This level is below the threshold set by the American College of Medical Genetics. Homozygosity was observed for 2789+5G>A in a 29-year-old women and compound heterozygosity with delta F408 in a 40-year-old woman with isolated chronic sinusitis. Many patients elected prenatal diagnosis when not at a 1:4 risk due to echogenic bowel or IVS-8 5T issues. CONCLUSIONS: With the exception of 1078delT, all CF mutations in the ACMG panel were detected with a frequency of > 0.1% of CF chromosomes. When ACMG guidelines are strictly adhered to, population-based CF carrier screening will accurately identify couples at risk for having children with CF.