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 beta subunits of the epithelial sodium channel.
BACKGROUND
The epithelial sodium channel (ENaC) is composed of three homologous subunits: alpha, beta, and gamma. Mutations in the Scnn1b and Scnn1g genes, which encode the beta 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 beta 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 beta-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 beta-ENaC, only one family showed a different CDCE profile. Two members of this family (n = 5) were heterozygous at 1781C-->T (T594M).


CONCLUSIONS
CDCE rapidly detects point mutations in these candidate disease genes.
Xue
Ming-Zhan
MZ
Institute of Pharmacology and Toxicology, Rue du Bugnon 27, 1005 Lausanne, Switzerland.
Bonny
Olivier
O
Morgenthaler
Stephan
S
Bochud
Murielle
M
Mooser
Vincent
V
Thilly
William G
WG
Schild
Laurent
L
Leong-Morgenthaler
Phaik-Mooi
PM
D019075:Electrophoresis, Capillary; D053503:Epithelial Sodium Channels; D005091:Exons; D005260:Female; D006801:Humans; D008297:Male; D010375:Pedigree; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide; D021122:Protein Subunits; D015222:Sodium Channels / Q000097:blood* / Q000235:genetics*
D053503:Epithelial Sodium Channels; D021122:Protein Subunits; C507045:SCNN1A protein, human; C507048:SCNN1B protein, human; C507052:SCNN1G protein, human; D015222:Sodium Channels