Novel length variant of the polypyrimidine tract within the splice acceptor site in intron 8 of the CFTR gene: consequences for genetic testing using standard assays

Viel, Marion; Leroy, C.; Georges, Marie des; Claustres, Mireille; Bienvenu, Thierry

doi:10.1038/sj.ejhg.5201261

Cited by 12 publications

(8 citation statements)

References 19 publications

(6 reference statements)

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“…As polyT detection is also included in many commercial kits such as ELUCIGENE CF-Poly-T, Tepnel diagnostics, UK; INNO-LiPA CFTR, Innogenetics, Belgium; Cystic fibrosis v3 5/7/9T OLA ASR, Abbott diagnostics, USA, it could also be investigated in segregation analysis in those laboratories using these commercial kits. However as in rare cases, other polyT alleles such as T2, T3 or T6 were reported [37][38][39] one should note that in rare cases, homozygosity for common alleles detected by these methods may be artifactual and due to the fact that all these polyT detection methods assume that only three common length variants exist in the general population. Preliminary molecular investigation of previously reported mutations with a frequency higher than 2% in Iranian population in these CF patients showed that the population is highly heterogeneous and except p.Phe508del, does not present the most frequent mutations reported worldwide or in other parts of Iran (data not shown).…”

Section: Discussionmentioning

confidence: 99%

Poly Thymidine Polymorphism and Cystic Fibrosis in a Non-Caucasian Population

et al. 2012

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Abstract.Background: Cystic fibrosis is a monogenic recessive disorder found predominantly in Caucasian population. This disease arises from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. In this study we consider poly T polymorphism c.1210-12T [5], c.1210-12T[7], c.1210-12T[9] (T5, T7, T9) in the intron 8 of CFTR gene in normal individuals and cystic fibrosis patients in the north of Iran. Material and methods: 40 CF patients and 40 normal individuals were screened for poly T polymorphism in intron 8 of CFTR gene using Reverse Dot Blot method which was also used to detect p.Phe508del among CF patients. Results: T7 allele is the most prevalent in both normal and CF patients. Its abundance is approximately 75%. T9 and T5 represent approximately 20% and 5% of alleles respectively. T7/ T7 genotype is the most present in both normal and CF patients with 72.5% and 60% prevalence respectively. p.Phe508del was present in 13 CFTR alleles belonging to 7 patients with either homozygote T9/ T9, T7/ T7 or compound heterozygote T7/ T9 genotypes. Conclusion: Contrary to the Caucasians, T7 allele is more frequent in Northern Iranian CF patients. The presence of p.Phe508del and T7 allele in the same framework is reported for the first time in this part of the world. Further investigations of other populations will help to understand whether p.Phe508del arose by selection pressure in this part of the world or was imported from European countries. The abundance of T5, T7, T9 alleles indicates that this polymorphism can be used as one of the informative markers for detection of normal and mutant alleles in prenatal diagnosis or carrier assessment in families with previous history of the disease in regions with high degree of CFTR mutation heterogeneity.

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Section: Discussionmentioning

confidence: 99%

Poly Thymidine Polymorphism and Cystic Fibrosis in a Non-Caucasian Population

et al. 2012

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“…However, these techniques are unable to determine the length variants localized at the polypyrimidine locus upstream to the splice acceptor site of intron 8 (polyTG followed by polyT repeats) that affect the splicing efficiency of exon 9 and act as genetic modifiers of CFTR function. Five variants, (TG)9 to (TG) 13, are known in the (TG)m tract, whereas up to seven different alleles have been reported in the Tn tract (common alleles with nine, seven, or five thymidines and rare alleles with three, 10 six, 11 10, 12 or 11 13 thymidines). In Caucasian populations, the frequency of IVS8-T5 allele in CBAVD patients (30%) is six times higher than in the general population (5%), and 34% of men with CBAVD have inherited a CFTR mutation on one gene and IVS8-T5 on the other one, making this combination the most common cause of CBAVD.…”

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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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“…In addition, as penetrance of T5 alleles seems to depend on the (TG)m repeat, results of these assays are only partly informative for genetic testing, and supplementary determination of the (TG)m repeat is indicated after the identification of a T5 allele. Sequencing-based methods include protocols in which the (TG)mTn region is first analysed using DGGE or SSCP/HD (single-strand conformation polymorphism/heteroduplex) analysis, followed by sequencing of differing banding patterns, 6,17,19,27 and protocols which take advantage of direct sequencing. 10,20,34 However, these sequencing-based methods have the drawback that electropherograms may be more difficult to interpret compared to the method presented here.…”

Section: Discussionmentioning

confidence: 99%

“…10 This finding is supported by studies proving a positive correlation between the length of the (TG)m repeat and the proportion of exon 9 skipping. 11,12 Several methods for the analysis of the polymorphic (TG)mTn locus have been proposed, including multistep sequencing with biotinylated primers and/or radioactive sequencing 13 -15 or the analysis of denaturating gradient gel electrophoresis (DGGE) patterns characterized by direct sequencing, 6,16,17,10 as well as a two-step procedure using nested PCR and XmnI digest 7 for the analysis of the Tn tract and subsequent determination of the (TG)m repeat by direct sequencing. 18,19 Alternatively, both the (TG)m repeat and Tn tract are assessed simultaneously by sequencing.…”

Section: Introductionmentioning

confidence: 99%

Rapid and reliable genotyping of polymorphic loci modifying correct splicing of CFTR pre-mRNA using mass spectrometry

et al. 2006

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We describe a fast and unambiguous method for haplotyping the (TG)mTn repeat in IVS8 and determining three other single nucleotide polymorphisms (SNPs) in exons 10, 14a and 24 in the cystic fibrosis transmembrane conductance regulator (CFTR) gene affecting correct splicing of the CFTR pre-mRNA using primer extension and mass spectrometry. The diagnostic products are generated by primer extension (PEX) reactions, which require a single detection primer complementary to a region downstream of a target strand's variable site. On addition of a polymerase and an appropriate mixture of dNTP's and 2 0 , 3 0 -dideoxynucleotide triphosphates (ddNTP's), the primer is extended through the mutation region until the first ddNTP is incorporated and the mass of the extension products determines the composition of the variable site. Analysis of patient DNA assigned the correct and unambiguous haplotype for the (TG)mTn repeat in intron 8 of the CFTR gene. Additional crucial SNPs influencing correct splicing in exon 10, 14 and 24 can easily be detected by biplexing the assay to genotype allelic variants important for correct splicing of the CFTR pre-mRNA. Different PEX reactions with subsequent mass spectrometry generate sufficient data, to enable unambiguous and easy haplotyping of the (TG)mTn repeat in the CFTR gene. The method can be easily extended to the inclusion of additional SNPs of interest by biplexing some of the PEX reactions. All experimental steps required for PEX are amenable to the high degree of automation desirable for a high-throughput diagnostic setting, facilitating the work of clinicians involved in the diagnosis of non-classic cystic fibrosis.

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Novel length variant of the polypyrimidine tract within the splice acceptor site in intron 8 of the CFTR gene: consequences for genetic testing using standard assays

Cited by 12 publications

References 19 publications

Poly Thymidine Polymorphism and Cystic Fibrosis in a Non-Caucasian Population

Poly Thymidine Polymorphism and Cystic Fibrosis in a Non-Caucasian Population

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

Rapid and reliable genotyping of polymorphic loci modifying correct splicing of CFTR pre-mRNA using mass spectrometry

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