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Clinical Chemistry 48: 718-728, 2002;
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(Clinical Chemistry. 2002;48:718-728.)
© 2002 American Association for Clinical Chemistry, Inc.

Use of Constant Denaturant Capillary Electrophoresis of Pooled Blood Samples to Identify Single-Nucleotide Polymorphisms in the Genes (Scnn1a and Scnn1b) Encoding the {alpha} and ß Subunits of the Epithelial Sodium Channel

Ming-Zhan Xue1, Olivier Bonny2, Stephan Morgenthaler3, Murielle Bochud2, Vincent Mooser2, William G. Thilly4, Laurent Schild1 and Phaik-Mooi Leong-Morgenthaler1a

1 Institute of Pharmacology and Toxicology, Rue du Bugnon 27, 1005 Lausanne, Switzerland.

2 Department of Internal Medicine, Centre Hospitalier Universitaire Vaudois (CHUV), 1005 Lausanne, Switzerland.

3 Department of Mathematics, Swiss Federal Institute of Technology-Lausanne, 1015 Lausanne, Switzerland.

4 Program in Epidemiology and Human Genetics, Massachusetts Institute of Technology, Cambridge, MA 02139.

aAuthor for correspondence. Fax 41-21-692-5355; e-mail PhaikMooi.Morgenthaler-Leong{at}ipharm.unil.ch.

Background: The epithelial sodium channel (ENaC) is composed of three homologous subunits: {alpha}, ß, and {gamma}. Mutations in the Scnn1b and Scnn1g genes, which encode the ß and the {gamma} subunits of ENaC, cause a severe form of hypertension (Liddle syndrome). The contribution of genetic variants within the Scnn1a gene, which codes for the {alpha} subunit, has not been investigated.

Methods: We screened for mutations in the COOH termini of the {alpha} and ß subunits of ENaC. Blood from 184 individuals from 31 families participating in a study on the genetics of hypertension were analyzed. Exons 13 of Scnn1a and Scnn1b, which encode the second transmembrane segment and the COOH termini of {alpha}- and ß-ENaC, respectively, were amplified from pooled DNA samples of members of each family by PCR. Constant denaturant capillary electrophoresis (CDCE) was used to detect mutations in PCR products of the pooled DNA samples.

Results: The detection limit of CDCE for ENaC variants was 1%, indicating that all members of any family or up to 100 individuals can be analyzed in one CDCE run. CDCE profiles of the COOH terminus of {alpha}-ENaC in pooled family members showed that the 31 families belonged to four groups and identified families with genetic variants. Using this approach, we analyzed 31 rather than 184 samples. Individual CDCE analysis of members from families with different pooled CDCE profiles revealed five genotypes containing 1853G->T and 1987A->G polymorphisms. The presence of the mutations was confirmed by DNA sequencing. For the COOH terminus of ß-ENaC, only one family showed a different CDCE profile. Two members of this family (n = 5) were heterozygous at 1781C->T (T594M).

Conclusion: CDCE rapidly detects point mutations in these candidate disease genes.




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