Three Novel CFTR Polymorphic Repeats Improve Segregation Analysis for Cystic Fibrosis

Abstract

Background: Molecular diagnosis for cystic fibrosis (CF) is based on the direct identification of mutations in the CFTR gene [cystic fibrosis transmembrane conductance regulator (ATP-binding cassette sub-family C, member 7)] (detection rate about 90% with scanning procedures) and on segregation analysis of intragenic polymorphisms for carrier and prenatal diagnosis in about 20% of CF families in which 1 or both causal mutations are unknown. Methods: We identified 3 novel intragenic polymorphic repeats (IVS3polyA, IVS4polyA, and IVS10CA repeats) in the CFTR gene and developed and validated a procedure based on the PCR followed by capillary electrophoresis for large-scale analysis of these polymorphisms and the 4 previously identified microsatellites (IVS1CA, IVS8CA, IVS17bTA, and IVS17bCA repeats) in a single run. We validated the procedure for both single- and 2-cell samples (for a possible use in preimplantation diagnosis), and on a large number of CF patients bearing different genotypes and non-CF controls. Results: The allelic distribution and heterozygosity results suggest that the 3 novel polymorphisms strongly contribute to carrier and prenatal diagnosis of CF in families in which 1 or both causal mutations have not been identified. At least 1 of the 4 previously identified microsatellites was informative in 78 of 100 unrelated CF families; at least 1 of all 7 polymorphisms was informative in 98 of the families. Finally, the analysis of haplotypes for the 7 polymorphisms revealed that most CF mutations are associated with different haplotypes, suggesting multiple slippage events but a single origin for most CFTR mutations. Conclusions: The analysis of the 7 polymorphisms is a rapid and efficient tool for routine carrier, prenatal, and preimplantation diagnosis of CF.

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

“…Also, even if both mutations are detected, it is still recommended to perform fetus genotyping during prenatal diagnosis both directly by mutation analysis and indirectly by studying multiple inter or intrageneic microsatellites or RFLPs to reduce errors due to methods of mutation detection, maternal contamination or human manipulations [21]. As most microsatellites analysis necessitate either the use of capillary electrophoresis or sequencing [22,23] which may not be available in many laboratories, using polymorphic markers easily detectable with routine laboratory methods such as RFLP or Reverse Dot Blot could be advantageous for those laboratories.…”

Biochemical and Molecular Genetic Testing Used in the Diagnosis and Assessment of Cystic Fibrosis

“…There was the identification of a total of 28 different mutations in Slovak CF patients, and 17 different polymorphisms (Kolesár et al, 2008). Elce et al (2009) reported three novel CFTR polymorphic repeats (IVS3polyA, IVS4polyA, and IVS10CA repeats) which improve segregation analysis for CF. They also developed and validated a procedure based on PCR followed by capillary electrophoresis (CE) for largescale analysis of these polymorphisms.…”

“…They also developed and validated a procedure based on PCR followed by capillary electrophoresis (CE) for largescale analysis of these polymorphisms. The allelic distribution and heterozygosity results suggest that the 3 novel intragenic polymorphic repeats strongly contribute to carrier and prenatal diagnosis of CF in families in which 1 or both causal mutations have not been identified (Elce et al, 2009). A universal array-based multiplexed test for CF carrier screening using the Tag-It multiplex mutation platform and the Cystic Fibrosis Mutation Detection Kit have been introduced.…”

Do Common Variants Separate between Obese Melanocortin-4 Receptor Gene Mutation Carriers and Non-Carriers? The Impact of Cryptic Relatedness

“…The classification of the mutations into functional categories and the subsequent analyses according to these categories was driven by both statistical and biological considerations: (i) from a statistical point of view, we aimed to avoid problems of misclassification by comparing obese carriers of MC4R mutations that were functionally characterized as ‘like wild type’ in the used in vitro system to obese carriers without MC4R mutations which could contribute to a diluted overall effect if present; (ii) from a biological point of view to discriminate between mutations having a relevant effect on receptor function in comparison to wild type (WT) and MC4R mutations without a relevant functional effect in comparison to WT. For some disease-causing genes a haplotype was reported to be associated with several mutations [16,17,18]. In this context, cystic fibrosis can serve as an example, as it has been shown that certain risk microsatellite alleles are indicative of disease-causing mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR) suggesting a single origin for most CFTR mutations [16,17].…”

“…For some disease-causing genes a haplotype was reported to be associated with several mutations [16,17,18]. In this context, cystic fibrosis can serve as an example, as it has been shown that certain risk microsatellite alleles are indicative of disease-causing mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR) suggesting a single origin for most CFTR mutations [16,17]. …”