ABCMdb

PMID: 17949287

Lebo RV, Bixler M, Galehouse D
One multiplex control for 29 cystic fibrosis mutations.
Genet Test. 2007 Fall;11(3):256-68., [PubMed]

Sentences

No. Mutations Sentence Comment

103 ABCC7 p.Gly551Asp ABCC7 p.Arg553* ABCC7 p.Gly542* ABCC7 p.Arg560Thr For example, the Intron 10/Exon 11 fragment spans 5 common mutation sites: 1717-1G Ǟ A, G542X, G551D, R553X, and R560T, while the ⌬I507 and ⌬F508 mutations in Exon 10 overlap by one basepair and each delete three basepairs. Login to comment 105 ABCC7 p.Gly551Asp ABCC7 p.Arg553* ABCC7 p.Gly542* ABCC7 p.Arg560Thr Because the G551D and R553X mutations are within four basepairs, these mutations were also synthesized on independently cloned Intron 10/Exon 11 fragments, both of which carried three other mutations: 1717-1G Ǟ A, G542X, and R560T (Fig. 2, fragments 1 and 3). Login to comment 118 ABCC7 p.Ala455Glu ABCC7 p.Ala455Glu ABCC7 p.Asn1303Lys ABCC7 p.Asn1303Lys Replacing modified sequences near the A455E and N1303K mutations in clone #9 The initial clone 9 insert consisted of three amplicon fragments: (Fig. 2, #9, left) the 5Ј CFTR genomic segment with A455E from exon 9 and 5T from intron 8; (Fig. 2, #9, center) a middle segment with the 3849ϩ10kbC Ǟ T mutation in intron 19; and (Fig. 2, #9, right) the 3Ј insert with the N1303K mutation from exon 21 that all tested appropriately on the Innogenetics test strips (Fig. 3, f 9 strip pair). Login to comment 120 ABCC7 p.Ala455Glu However, 10 nucleotide substitutions were found 34 to 257 basepairs downstream of the A455E site. Login to comment 122 ABCC7 p.Asn1303Lys In addition, 11 nucleotide substitutions were found in the exon 21/intron 21 fragment beginning 110 bp downstream from the N1303K mutant site. Login to comment 133 ABCC7 p.Ala455Glu ABCC7 p.Arg334Trp ABCC7 p.Arg347Pro ABCC7 p.Asn1303Lys ABCC7 p.Arg1162* Instead, our laboratory has prepared multiplex controls from aliquots of lin- MULTIPLEX CYSTIC FIBROSIS CONTROL 261 1 2 3 4 5 6 7 8 9 EXON/INTRON 4F4 R4 F11 R11INTRON 10/EXON 11 EXON 7R7 F7 R5 F9INTRON 5 R347P R334W 1078delT EXON 10R10 F10 R11 F11EXON 11/INTRON 10 INTRON 16R16 F16 R14b F14bINTRON 14b EXON 19F19 R19 F20 R20EXON 20 R1162X 3659delC INTRON 8/EXON 9F9 R9 F119 R119INTRON 19 5T A455E F21 R21EXON 21 N1303K EXON 10F10 R10 F3 R3EXON 3 1507 INTRON 12F12 R12 EXON 13R13 F13 2184delA FIG. 2. Login to comment 165 ABCC7 p.Gly551Asp ABCC7 p.Arg553* ABCC7 p.Gly542* ABCC7 p.Arg560Thr As part of this validation, two different Intron10/Exon11 fragments were sequenced and tested: both contain the 1717-1G Ǟ A, G542X, and R560T mutations, and the first also contains the G551D mutation (Fig. 2, clone 1; Fig. 3, f1), while the second also contains the R553X mutation (Fig. 2, clone 3; Fig. 3, f3). Login to comment 166 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly542* ABCC7 p.Arg560Thr When tested individually, clone 1 hybridized uniquely to the G551D mutant allelic site as well as to the other three mutations (1717-1G Ǟ A, G542X, and R560T), but not to the wild-type (normal) R553 allelic site because the G551D mutation sequence interferes with the binding to the wild type R553 probe on the strip (Fig. 3, f1, left strip). Login to comment 167 ABCC7 p.Ile148Thr The I148T mutation included in clone 1 is also detected on the same strip (Fig. 3, f1, left strip). Login to comment 168 ABCC7 p.Arg553* In contrast, clone 3 with the R553X mutation hybridized to the wild-type G551 allele as well as the mutant LEBO ET AL.264 FIG. 3. Login to comment 169 ABCC7 p.Arg553* (Continued) R553X allele (Fig. 3, f3, right strip). Login to comment 170 ABCC7 p.Arg553* Thus, the R553X sequence does not prevent hybridization to the G551 probe at the hybridization stringency used in the assay. Login to comment 173 ABCC7 p.Gly551Asp All of the mutations in the clone mix appear homozygous (mutant band present, wild-type band not present) with the exception at the G551D mutation site, which appears heterozygous (mutant and wild-type bands present). Login to comment 174 ABCC7 p.Gly551Asp This is expected, since, as discussed above, clone 1 contains the G551D mutation sequence and clone 3 contains the G551 wild-type sequence. Login to comment 181 ABCC7 p.Gln552* The four additional mutant locations that do not label, CFTR⌬2,3(21kb), 3272-26A Ǟ G, 3199⌬6, and Q552X, are located in regions of the CF gene not spanned by any of the clones and no bands were seen at either of these wild-type or mutant allelic sites. Login to comment 203 ABCC7 p.Gly551Asp ABCC7 p.Arg553* The G551/R553 gene region is represented by four bead populations, one for each normal allele, G551 and R553, and one for each mutant allele, G551D and R553X. Login to comment 204 ABCC7 p.Arg553* ABCC7 p.Arg553* A known artifact of the TmBiosciences methodology is that the R553X sequence will interfere with the binding of the normal G551 extension primer so that the normal G551 allele will not be detected on the template containing the R553X allele. Login to comment 205 ABCC7 p.Arg553* This occurs in patient genomic DNA positive for the R553X allele as well as in our control mix (Fig. 4, panel 2, unpublished results). Login to comment 228 ABCC7 p.Gly551Asp ABCC7 p.Arg553* See results for a further description of the G551D and R553X heterozygous sites on clones 1 and 3. Login to comment 229 ABCC7 p.Val520Phe ABCC7 p.Ser549Arg ABCC7 p.Ser549Asn ABCC7 p.Tyr122* ABCC7 p.Ser1255* The nine normal sequences detected at other tested mutation sites (3876delA; S1255X; 2307insA; 1898ϩ5G Ǟ T; S549R; S549N; V520F; Y122X; and 394delTT) reflected the correct sequence in this mixture of nine cloned sequences. Login to comment