Allele-Specific Oligonucleotide (ASO) test for Cystic Fibrosis

    Cystic Fibrosis (CF) is an autosomal recessive disease, characterized by accumulation of mucous in the lungs. The most common allele in Europeans that leads to CF has a 3bp DNA deletion, and is called 508. In the pedigree, the birth of an affected daughter (II-1) to two unaffected parents indicates that both are heterozygotes. The clinical question is whether the two unaffected siblings are carriers for CF.

    An oligonucleotide probe is constructed with a sequence complementary to the exact DNA sequence of the allele with the 508 deletion: an allele-specific oligonucleotide (ASO). DNA is obtained from all family members and spotted onto a Southern hybridization filter. The filter is probed with a radioactive ASO. Hybridization of the ASO to the filter shows as a spot on the autoradiogram, which indicates the presence of the 508 allele. The molecular phenotypes of the heterozygous parents and the affected daughter are as predicted: each carries (at least) one 508 allele, and therefore tests positive. The unaffected son (II-2) tests negative for the 508 allele and is therefore "homozygous standard", whereas his unaffected sister (II-3) tests positive for 508, and is therefore a "carrier" for CF.

    Because the ASO method avoids electrophoresis and size determinations, the tests are rapid and suitable for large-scale population screens. The test as described has two weaknesses as a diagnostic tool. First, about 30% of CF cases in persons of European ancestry are due to alternative alleles other than 508, and will not be detected with this specific test. Second, the hybridization test is subject to "false negatives": perhaps II-2 is a carrier, but the ASO hybridization failed for technical reasons. The test can then be improved by adding a parallel ASO "control" test for the "standard" allele: