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

Strom, Charles M.; Huang, Donghui; Chen, Christina; Buller, Arlene; Peng, Mei; Quan, Franklin; Redman, Joy B.; Sun, Weimin

doi:10.1097/00125817-200301000-00002

Cited by 56 publications

(42 citation statements)

References 12 publications

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“…This is demonstrated in Figure 2A. To confirm this putative arrangement, we performed PCR using an exon 10 forward primer and an exon 6B reverse primer 19 and successfully amplified a fragment of ϳ3.8 kb from the patient sample DNA but not from samples not harboring the duplication ( Figure 2B). The amplification of this fragment provides a second confirmation for the presence of this large duplication in the patient's DNA.…”

Section: Amplification Of Duplication Junctionmentioning

confidence: 77%

Characterization of a Recurrent Novel Large Duplication in the Cystic Fibrosis Transmembrane Conductance Regulator Gene

Hantash

Redman

Goos

et al. 2007

The Journal of Molecular Diagnostics

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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, singlestrand 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 ⌬F508. 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 ⌬F508 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. (J Mol Diagn

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Section: Amplification Of Duplication Junctionmentioning

confidence: 77%

Characterization of a Recurrent Novel Large Duplication in the Cystic Fibrosis Transmembrane Conductance Regulator Gene

Hantash

Redman

Goos

et al. 2007

The Journal of Molecular Diagnostics

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“…In two assays 25,26 the CFTR gene was studied in 32 amplicons, and each PCR primer contained an M13 linker sequence ensuring a single PCR condition and the use of universal priming in cycle sequencing. All PCR primers had to be redesigned because of the presence of the M13 linker sequence.…”

Section: Discussionmentioning

confidence: 99%

Comprehensive and Rapid Genotyping of Mutations and Haplotypes in Congenital Bilateral Absence of the Vas Deferens and Other Cystic Fibrosis Transmembrane Conductance Regulator-Related Disorders

Bareil

Guittard

Altiéri

et al. 2007

The Journal of Molecular Diagnostics

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Available commercial kits only screen for the most common cystic fibrosis transmembrane conductance regulator (CFTR) mutations causing classic cystic fibrosis and for the Tn variant in IVS8. However, full scanning of CFTR is needed for the diagnosis of patients with cystic fibrosis or CFTR-related disorders (including congenital bilateral absence of the vas deferens) bearing rare mutations. Standard strategies for detecting point mutations rely on extensive scanning of the gene by denaturing gradient gel electrophoresis or denaturing high performance liquid chromatography, which are time-consuming. Moreover, the haplotyping of IVS8-(TG)m and Tn tracts is still challenging despite several recent improvements. We have optimized both the detection of mutations and the haplotyping of IVS8 polyvariants in developing two methods: i) a rapid and robust direct sequence analysis of all exons/flanking introns of the CFTR gene based on single condition touchdown amplification/sequencing in 96-well plates, and ii) a fluorescent assay that allows haplotyping of IVS8-(TG)mTn even without family linkage study. Combined with search for rare large rearrangements, this strategy detected 87.9% of CFTR defects in congenital bilateral absence of the vas deferens patients, a proportion considerably higher than those usually reported. These highly efficient tests, scanning each sample in a few days, greatly improve the genotyping of patients with CFTR-related symptoms and may be particularly important in emergency situations such as fetus with hyperechogenic bowel suggestive of cystic fibrosis. (J Mol Diagn 2007, 9:582-588;

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“…With the California CF NBS algorithm, hypertrypsinogenemic newborns (top 1.6%) are considered screen positive either when two CFTR mutations are detected by a panel of 40 CF-causing mutations (CF40 mut ) or 1 by the panel and ‡1 by subsequent DNA sequencing (Kharrazi et al, 2015). If on sequencing CFTR (IVS8)-(TG)m-(T)n, poly T status revealed a 5T, TG tract length was reported (Strom et al, 2003;Kammesheidt et al, 2006;Keiles et al, 2012).…”

Section: Methodsmentioning

confidence: 99%

Phenotypes of California CF Newborn Screen-Positive Children with CFTR 5T Allele by TG Repeat Length

Salinas

Azen

Young

et al. 2016

Genetic Testing and Molecular Biomarkers

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Background: At the cystic fibrosis transmembrane conductance regulator (CFTR) gene (IVS8)-(TG)m(T)n locus, a lower number of thymidines (legacy names 9T vs. 7T vs. 5T) and a higher number of (TG) repeats (TG-11 vs. 12 vs. 13) are associated with decreasing translation of functional CFTR protein in vitro. Methods: Retrospective cohort study comparing phenotypes of California CF newborn screen-positive children (followed 2-8 years) who had two CF-causing mutations (diagnosed as CF) with those who had one mutation from a panel of 40 CF-causing mutations (CF40 mut ) and one (IVS8)-(TG)11, 12, or 13-5T mutation detected by sequencing (diagnosed as CFTR-related metabolic syndrome [cRMS]). Results: The study included 428 children, of which 234 had two CF-causing mutations, and were used to compare with the other 194 children with one CF-causing mutation and one isolated 5T allele [CF40 mut /(TG)13-5T = 21, CF40 mut /(TG)12-5T = 85, and CF40 mut /(TG)11-5T = 88]. Among children with CF40 mut /(TG)13-5T, 38% were diagnosed with CF by 8 years, based on sweat chloride results and clinical presentation. Six percent of those with CF40 mut /(TG)12-5T, and none with CF40 mut /(TG)11-5T, reached diagnostic criteria. Conclusions: CFTR (IVS8)-(TG)m-5T allele (TG) tract length determination provides valuable information in predicting the risk of developing a CF phenotype. Of the three types of 5T alleles evaluated, screen-positive children with genotype CF40 mut /(TG)13-5T progressed from CRMS to CF at a high rate, while there was little evidence of clinical disease in those with CF40 mut /(TG)11-5T. Additional data from longer follow-up intervals are needed to fully understand the natural history of individuals with a CF40 mut /(TG)m-5T genotype.

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

Cited by 56 publications

References 12 publications

Characterization of a Recurrent Novel Large Duplication in the Cystic Fibrosis Transmembrane Conductance Regulator Gene

Characterization of a Recurrent Novel Large Duplication in the Cystic Fibrosis Transmembrane Conductance Regulator Gene

Comprehensive and Rapid Genotyping of Mutations and Haplotypes in Congenital Bilateral Absence of the Vas Deferens and Other Cystic Fibrosis Transmembrane Conductance Regulator-Related Disorders

Phenotypes of California CF Newborn Screen-Positive Children with CFTR 5T Allele by TG Repeat Length

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