Clinical Chemistry
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Clinical Chemistry 55: 1372-1379, 2009. First published May 14, 2009; 10.1373/clinchem.2008.119545
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(Clinical Chemistry. 2009;55:1372-1379.)
© 2009 American Association for Clinical Chemistry, Inc.

Molecular Diagnostics and Genetics

Three Novel CFTR Polymorphic Repeats Improve Segregation Analysis for Cystic Fibrosis

Ausilia Elce1,2, Angelo Boccia1,2, Giuseppe Cardillo1,2, Sonia Giordano1,2,a, Rossella Tomaiuolo1,2,3,a, Giovanni Paolella1,2,3 and Giuseppe Castaldo1,2,3,4

1 Dipartimento di Biochimica e Biotecnologie Mediche, Università di Napoli Federico II, Naples, Italy; 2 CEINGE-Biotecnologie Avanzate scarl, Naples, Italy; 3 Facoltà di Scienze Biotecnologiche, Università di Napoli Federico II, Naples, Italy; 4 SEMM, Naples, Italy.

aAddress correspondence to: G.C. at CEINGE-Biotecnologie Avanzate scarl, Via Comunale Margherita n. 482, 80145, Naples, Italy. Fax +39-0813737808; e-mail castaldo{at}dbbm.unina.it. G.P. at CEINGE-Biotecnologie Avanzate scarl, Via Comunale Margherita n. 482, 80145, Naples, Italy. Fax +39-0813737808; e-mail paolella{at}dbbm.unina.it.

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.






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