Charles M. Strom

Cystic fibrosis population carrier screening: 2004 revision of American College of Medical Genetics mutation panel

Cystic fibrosis population carrier screening: 2004 revision of American College of Medical Genetics mutation panel

Mutations in TMEM216 perturb ciliogenesis and cause Joubert, Meckel and related syndromes

Joubert syndrome (JBTS), related disorders (JSRDs) and Meckel syndrome (MKS) are ciliopathies. We now report that MKS2 and CORS2 (JBTS2) loci are allelic and caused by mutations in TMEM216, which encodes an uncharacterized tetraspan transmembrane protein. Individuals with CORS2 frequently had nephronophthisis and polydactyly, and two affected individuals conformed to the oro-facio-digital type VI phenotype, whereas skeletal dysplasia was common in fetuses affected by MKS. A single G218T mutation (R73L in the protein) was identified in all cases of Ashkenazi Jewish descent (n = 10). TMEM216 localized to the base of primary cilia, and loss of TMEM216 in mutant fibroblasts or after knockdown caused defective ciliogenesis and centrosomal docking, with concomitant hyperactivation of RhoA and Dishevelled. TMEM216 formed a complex with Meckelin, which is encoded by a gene also mutated in JSRDs and MKS. Disruption of tmem216 expression in zebrafish caused gastrulation defects similar to those in other ciliary morphants. These data implicate a new family of proteins in the ciliopathies and further support allelism between ciliopathy disorders.

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.

Cystic fibrosis screening using the College panel: Platform comparison and lessons learned from the first 20,000 samples

Purpose: To determine the accuracy of two commercially available kits for cystic fibrosis (CF) genotyping and determine allele frequencies for the ACMG/ACOG recommended mutations.Methods: A total of 1,040 consecutive analyses using Roche CF Gold Strips and the ABI CF Genotyper were performed. Subsequently we performed analyses of 20,103 samples.Results: Both kits accurately determined CF genotypes. The I148T mutation was found >100 times more frequently in carrier screening than in CF patients. Asymptomatic patients were identified who are compound heterozygotes for delta F508 and I148T. Four of 13 patients heterozygous for delta F508 and the IVS8-5T polymorphism had some symptoms of CF.Conclusion: Accurate and timely analysis can be performed for the ACMG CF panel. I148T is a low penetrance CF allele.

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 CFTR 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 CFTR gene provides important information for CF patients and is a valuable adjunct to a carrier screening program to resolve ambiguities in panel testing.

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.

Development of a novel, accurate, automated, rapid, high-throughput technique suitable for population-based carrier screening for Fragile X syndrome.

Purpose: To develop a high-throughput, automated, accurate method suitable for population-based carrier detection of fragile X syndrome.Methods: We developed a new method called capillary Southern analysis that allows automated high-throughput screening for expanded fragile X mental retardation 1 (FMR1) alleles. Initially samples are analyzed by a multiplex polymerase chain reaction that contains an internal control to establish gender. All females heterozygous for two normal alleles are reported as normal without further analysis. All females homozygous at the FMR1 locus (24% of all analysis) are then analyzed by capillary Southern analysis. Theoretically this method can detect expansion as high as 2000 CGG repeats, although in our series the largest nonmosaic FMR1 present was 950 CGG repeats. After assay development, we performed capillary Southern analysis on 995 female and 557 male samples submitted for fragile X syndrome testing by polymerase chain reaction and Southern blot.Results: The polymerase chain reaction/capillary Southern analysis technique identified 100% of six female premutation carriers, seven full mutation carrier females, one premutation male, and five affected males. There was only one discrepancy between analysis by polymerase chain reaction/Southern blot and analysis by polymerase chain reaction/capillary Southern analysis. A single female sample appeared to be heterozygous for a premutation allele by polymerase chain reaction/capillary Southern analysis but was negative by Southern blot. It is possible this patient is a mosaic for the premutation allele, but because samples were deidentified, we were unable to determine whether this was a true false positive.Conclusion: We have developed an automated, high-throughput technique capable of detecting carriers of fragile X syndrome with 100% sensitivity and at least 99.5% specificity. This should allow population-based carrier detection for the most commonly inherited form of mental retardation.

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.