The dangers of including nonclassical cystic fibrosis variants in population-based screening panels: p.L997F, further genotype/phenotype correlation data.

PURPOSE: : Recently, a major CLIA-certified commercial laboratory began offering an extended cystic fibrosis (CF) carrier screening panel containing 103 variants including p.L997F. Our laboratory has already received two invasive prenatal diagnostic samples where one parent carries a classic CF mutation and the other carries p.L997F. One fetus inherited both variants. METHODS: : We queried our databases containing >2500 CF sequencing analyses to find all individuals with the p.L997F variant. For all compound heterozygous patients, clinical information was obtained by a genetic counselor telephoning the medical provider. RESULTS: : There were four compound heterozygous patients carrying the p.L997F variant and a second pathogenic CF allele. Three patients were discovered by newborn screening and were asymptomatic at ages 28, 40, and 60 months, respectively. The fourth individual is currently aged 10 years and has the diagnosis of atypical CF with recurrent pancreatitis, sinusitis with nasal polyps, and mild lung disease. His length and weight are in the 90th and 75th centile, respectively. The fifth patient was a compound heterozygote for p.F508del and a complex allele containing p.L997F and a deletion of exons 2-9. This patient has the diagnosis of classical CF. CONCLUSION: : The p.L997F variant is not a classical CF mutation, and its inclusion in population-based carrier screening panels is a disservice to couples who may make poorly informed reproductive decisions based on incorrect assumptions.

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.

Improving accuracy of Tay Sachs carrier screening of the non-Jewish population: analysis of 34 carriers and six late-onset patients with HEXA enzyme and DNA sequence analysis.

The purpose of this study was to determine whether combining different testing modalities namely beta-hexosaminidase A (HEXA) enzyme analysis, HEXA DNA common mutation assay, and HEXA gene sequencing could improve the sensitivity for carrier detection in non-Ashkenazi (AJ) individuals. We performed a HEXA gene sequencing assay, a HEXA DNA common mutation assay, and a HEXA enzyme assay on 34 self-reported Tay-Sachs disease (TSD) carriers, six late-onset patients with TSD, and one pseudodeficiency allele carrier. Sensitivity of TSD carrier detection was 91% for gene sequencing compared with 91% for the enzyme assay and 52% for the DNA mutation assay. Gene sequencing combined with enzyme testing had the highest sensitivity (100%) for carrier detection. Gene sequencing detected four novel mutations, three of which are predicted to be disease causing [118.delT, 965A-->T (D322V), and 775A-->G (T259A)]. Gene sequencing is useful in identifying rare mutations in patients with TSD and their families, in evaluating spouses of known carriers for TSD who have indeterminate enzyme analysis and negative for common mutation analysis, and in resolving ambiguous enzyme testing results.

Apparent homozygosity of a novel frame shift mutation in the CFTR gene because of a large deletion.

Patients develop cystic fibrosis because of a variety of homozygous recessive mutations, including single nucleotide polymorphisms, insertions, and deletions, in the cystic fibrosis transmembrane regulator (CFTR) gene, or because of compound heterozygosity for two mutations in the CFTR gene. A false determination of homozygosity for a particular CFTR mutation could negatively affect both carrier screens for a patient's family as well as researchers' ability to study the physiological implications of a particular mutation. We argued previously that homozygosity for rare or novel mutations in the CFTR gene could result from a mutation on one allele and the presence of a large deletion encompassing the same sequence region on the second allele. We present here a patient with classic cystic fibrosis who has a novel microdeletion in exon 7 on one allele and a large deletion encompassing exon 7 on the second allele. These data highlight the need to prevent misdiagnosis of homozygous mutations, which can lead to misinterpretation of mutation penetrance and its effects on protein function.

Identification of cystic fibrosis variants by polymerase chain reaction/oligonucleotide ligation assay.

The purpose of this work is to define rare variants of cystic fibrosis (CF) that are potential sources of error and can confound molecular genetic testing methods. We performed routine, clinical CF mutation screening using a laboratory-developed test and the oligonucleotide ligation assay reagents from Abbott/Celera. In this report, we describe 11 unique allele drop outs [3849 + 10kb C>T (NM_000492.2:c.3718-2477C>T), V520F (c.1558G>T), 1078delT (c.948delT), A455E (c.1364C>A), R347P (c.1040G>C), 2184delA (c.2052delA), W1282X (c.3846G>A), R117H (c.350G>A), G85E (c.254G>A), 621 + 1G>T (c.489 + 1G>T), and 2789 + 5G>A (c.2657 + 5G>A)] observed with this platform. The allele drop outs account for less than 0.01% of all results reported in our laboratory. Both the recognition and enumeration of such variants along with clinical information in CF testing is valuable in avoiding false-positive and false-negative results.

Molecular testing: improving patient care through partnering with laboratory genetic counselors.

The utility of molecular diagnostics in clinical practice has been steadily increasing and is expected to continue to do so as the applications of genomic medicine increase. The goal of this article was to describe the roles and responsibilities of genetic counselors who work in the customer service area of molecular diagnostics laboratories. In this role, genetic counselors provide recommendations to clinicians on issues that are specific to DNA-based testing. This article will address some issues that are specifically relevant to disease genetic tests. Many molecular diagnostic laboratories employ genetic counselors, who have extensive training in how to communicate genetic information, to provide information in the preanalytic, analytic, and postanalytic stages of testing. To maximize the quality of the service, it is important to establish an understanding of what can be expected of both the practitioner and the laboratory genetic counselor. Although some complications in the laboratory cannot be anticipated, discussing the case with the laboratory genetic counselors beforehand may avert certain problems. This article discusses real cases from laboratory genetic counselors to illustrate issues that arise due to technical difficulties and the inherent limitations of molecular testing. The summary describes practical ways in which clinicians and laboratory personnel can work together to either avoid or, when unavoidable, better manage problems and delays. The responsibilities of genetic counselors working in molecular diagnostics are discussed.

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.

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.

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.

Validation and clinical application of a locus-specific polymerase chain reaction- and minisequencing-based assay for congenital adrenal hyperplasia (21-hydroxylase deficiency).

Congenital adrenal hyperplasia is an autosomal recessive disorder caused by defective adrenal steroid biosynthesis, resulting in reduced glucocorticoid and increased androgen production. The majority of cases are due to inactivation of the 21-hydroxylase gene (CYP21A2), most commonly caused by genomic rearrangements with the adjacent, highly homologous pseudogene CYP21A. The most common deletions and gene conversion events have been defined and are typically detected by Southern hybridization detection of CYP21 rearrangements and/or polymerase chain reaction (PCR). However, Southern hybridization is laborious, and allele-specific PCR results may be difficult to interpret. We have therefore developed a locus-specific, PCR-based, minisequencing method for detecting the 12 most common CYP21A2 mutations. We validated the assay using a panel of 20 previously genotyped samples obtained from individuals who collectively have a broad spectrum of mutations causing 21-hydroxylase deficiency. We also used 19 control samples having no CYP21 mutations. All validation samples were correctly typed, and we identified haplotypes that may be useful for clinical diagnosis. Results from 102 clinical samples demonstrate that this assay is a rapid, reliable, and robust method for locus-specific identification of mutations and is suitable for routine clinical use and prenatal diagnosis.

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.

Frequency of the cystic fibrosis 3199del6 mutation in individuals heterozygous for I148T.

PURPOSE: To determine the carrier frequency of the 3199del6 cystic fibrosis (CF) mutation in individuals heterozygous for I148T in a large-scale CF testing population. METHODS: DNA samples from 439 consecutive I148T-heterozygous individuals were screened for the 3199del6 mutation using a laboratory-developed test. RESULTS: Genotyping revealed four samples heterozygous for the 3199del6 mutation (0.9%). The four samples positive for 3199del6 had an IVS 8 genotype of 7T/9T. The 3199del6 mutation was not observed after genotyping of 348 random, anonymous samples. CONCLUSION: The 3199del6 mutation occurs in 0.9% of individuals positive for the I148T mutation and <0.07% of chromosomes that are wild type for the ACMG panel mutations.

Usher syndrome clinical types I and II: could ocular symptoms and signs differentiate between the two types?

PURPOSE: Usher syndrome types I and II are clinical syndromes with substantial genetic and clinical heterogeneity. We undertook the current study in order to identify ocular symptoms and signs that could differentiate between the two types. METHODS: Sixty-seven patients with Usher syndrome were evaluated. Based on audiologic and vestibular findings, patients were classified as either Usher type I or II. The severity of the ocular signs and symptoms present in each type were compared. RESULTS: Visual acuity, visual field area, electroretinographic amplitude, incidence of cataract and macular lesions were not significantly different between Usher types I and II. However, the ages when night blindness was perceived and retinitis pigmentosa was diagnosed differed significantly between the two types. CONCLUSIONS: There seems to be some overlap between types I and II of Usher syndrome in regard to the ophthalmologic findings. However, night blindness appears earlier in Usher type I (although the difference in age of appearance appears to be less dramatic than previously assumed). Molecular elucidation of Usher syndrome may serve as a key to understanding these differences and, perhaps, provide a better tool for use in clinical diagnosis, prognosis and genetic counseling.