ABCMdb

PMID: 8829658

Cronin MT, Fucini RV, Kim SM, Masino RS, Wespi RM, Miyada CG
Cystic fibrosis mutation detection by hybridization to light-generated DNA probe arrays.
Hum Mutat. 1996;7(3):244-55., [PubMed]

Sentences

No. Mutations Sentence Comment

80 ABCC7 p.Arg553* ABCC7 p.Arg553* Following cleavage from the synthesis support and deprotection, the labeled oligonucleotides were purified by reverse-phase high-performance liquid chromatograph (HPLC).. Primary Sample Characterization A homozygous R553X mutant hybridization target was generated by constructing exon 11 as two long oligonucleotides, one with the R553X mutant sequence, with an overlap at their 3` ends. Login to comment 112 ABCC7 p.Arg553* Hybridization targets were prepared from normal human genomic DNA and from a synthetic, homozygous R553X exon 11 mutant generated by PCR. Login to comment 113 ABCC7 p.Arg553* The C+T base substitution characteristic of the R553X mutation is clearly evident in comparing the wild-type and mutant target hybridization images shown in Figure 2A,B. Login to comment 123 ABCC7 p.Arg553* Figure 3E3-D show typical results from hybridizing oligonucleotide targets complementary to the wild-type or mutant sequence or an equal mixture of both to the specialized probe array specific for the exon ll R553X (C+T) point mutation. Login to comment 145 ABCC7 p.Arg553* The arrow in the third row indicates the R553X C+T mutation position. Login to comment 147 ABCC7 p.Arg553* B:Image of the CFTR exon 11tiled array hybridized with a hornozygous R553X target. Login to comment 155 ABCC7 p.Gly551Asp ABCC7 p.Gly480Cys One sample was compound heterozygous for G480C (G+T) in exon 10and G551D (G-A) in AL. n-2 n-1 n n+l n+2 A GGTCWCGAGCAAG GGTCAATGAGCAAG AGGTCAACGAGCAAG GGTCAATGAGCAAG GGTCAACGAGCAAG GGTCWTGAGCAAG - GGTCUTGAGCAAG GGTCAAZGAGCAAG GGTCAACGAGCAAG GGTCAACGAGCAAG GGTCAAZGAGCAAG B C D FIGURE 3. Login to comment 156 ABCC7 p.Arg553* A: Diagram of the R553X mutation specific DNA probe set. Login to comment 175 ABCC7 p.Gly551Asp ABCC7 p.Gly480Cys In Figure 4A, probe sets specific for the G480C and G551D mutations are indicated along with diagrams showing the expected heterozygote hybridization patterns. Login to comment 178 ABCC7 p.Gly551Asp ABCC7 p.Gly480Cys All of these probes sets, except for G480C and G551D, give complete homozygous wild type hybridization patterns. Login to comment 180 ABCC7 p.Gly480Cys In particular, there is hybridization with the C probes in the "n" position of the G480C array. Login to comment 187 ABCC7 p.Phe508Cys Another important aspect of this homozygous deletion mutant hybridization is the absence of hybridization patterns in the A1507 and F508C probe sets. Login to comment 189 ABCC7 p.Phe508Cys Full-length exon lO/exon 11 targets were used in this experiment, however, the AF508 deletion, the A1507 deletion, and the F508C polymorphism all occur within the space of a six nucleotide sequence. Login to comment 191 ABCC7 p.Phe508Cys As a result, the A1507 and F508C sets do not contain any probes that are fully complementary with a AF508 target. Login to comment 192 ABCC7 p.Phe508Cys A homozygous AF508 target will not hybridize significantly with any probes in the A1507 and F508C sets. Login to comment 203 ABCC7 p.Gly551Asp ABCC7 p.Gly542* Figure 5 shows a typical image from this experiment made from CHO sample nine which had two exon 11 mutations, G542X and G551D. Login to comment 212 ABCC7 p.Gly551Asp ABCC7 p.Gly480Cys A Image of the specialized mutation specific array hybridizedwith exon 10and exon 11targets multiplexedfrom acompoundheterozygous genomic DNAsample with G551D and G480C mutations. Login to comment 213 ABCC7 p.Gly551Asp ABCC7 p.Gly480Cys Diagrams of the G551D and G480C mutation subarrays indicating probes fully complementaryto the wild-type and mutant sequences are at the sides of the image. Login to comment 215 ABCC7 p.Gly551Asp G551D 5`GTGG- AGATCAACGA3'. Login to comment 217 ABCC7 p.Gly480Cys G480C 5'TCAGAGTGTAAAAT3`. Login to comment 219 ABCC7 p.Gly551Asp ABCC7 p.Gly480Cys A total of 15of the 37 mutation-specific probe sets hybridize with exon 10and exon 11targets (Table 1).All except G551D and G480C display wild-type hybridization patterns. Other hybridization signals that do not form complete patterns (as compared with examples in Fig. 3)result from cross-hybridization with probes that are partially complementary to the target. Login to comment 228 ABCC7 p.Phe508Cys F508C 5`AAATATCATCTGTGGTGTT3`.Underlined bases are interrogated in the three mutation arrays and deletions are in lowercase letters. Login to comment 231 ABCC7 p.Phe508Cys The AF508 probe set shows a homozygous mutant pattern: however, the two neighboring probe sets, A1507 and F508C, have no probes fully complementary to the mutant target and do not show any significanthybridization (seetext fordetailed explanation). Login to comment 238 ABCC7 p.Gly551Asp ABCC7 p.Arg117His ABCC7 p.Ala455Glu ABCC7 p.Trp1282* ABCC7 p.Arg553* ABCC7 p.Arg334Trp ABCC7 p.Arg347Pro ABCC7 p.Arg347His ABCC7 p.Asn1303Lys ABCC7 p.Gly542* ABCC7 p.Arg1162* ABCC7 p.Ile148Thr ABCC7 p.Gln493* ABCC7 p.Val520Phe ABCC7 p.Gly85Glu ABCC7 p.Arg560Thr ABCC7 p.Arg1066Cys ABCC7 p.Tyr1092* ABCC7 p.Gln552* ABCC7 p.Phe508Cys ABCC7 p.Leu1077Pro ABCC7 p.Gly480Cys ABCC7 p.Ala559Thr Cystic Fibrosis Mutation-Specific DNA Probe Array" Mutation Exon and column Tested Subarrayhow G85E R117H I148T 621 -+ l(G+T) 711 + 1(G+T) R334W R347H R347P 1078 delT A455E G480C Q493X A1507 F508C AF508 V520F G542X S549R(T-+ G) G551D Q552X R553X A559T R560T 1898 + l(G-,A) 2184 del A 2789 + 5(G+ A) R1066C L1077P Y1092X R1162X 3659 del C 1717-1(& A) 3272 - 26(A+ G) 3 4 4 in 4 in 5 7 7 7 7 9 10 10 10 10 10 10 in 10 11 11 11 11 11 11 11 in 12 13 in 14b in 17a 17b 17b 17b 19 19 * * * * * * * * * * * * * * * * * * * * * * * * * * * * 3849 + lOkb C-, T in 19 9,3 W1282X 20 994 3905insT 20 10.1 * N1303K 21 10,2 * * * "Row and column locations for each of the mutation specific,40 probe sets included in the specialized probe array design. Login to comment 247 ABCC7 p.Phe508Cys Their CFTR exon 10 DNA was cloned and sequenced, resulting in their correct assignment as AF508/F508C compound het- TABLE 2. Login to comment 248 ABCC7 p.Gly551Asp ABCC7 p.Arg553* ABCC7 p.Arg553* ABCC7 p.Gly542* ABCC7 p.Gly542* Results From Unknown Patient Sample CFTR Genotyping" Sample CHO 1 CHO 2 CHO 3 CHO 4 CHO 5 CHO 6 CHO 7 CHO 8 CHO 9 CHO 10 Exon 10 genotype Wild-type AF508 Wild-type Wild-type AF508 Wild-type Wild-type AF508 Wild-type Wild-type Exon 11genotype Wild-type G542X Wild-Type Wild-Type G551D R553X G542X R553X G542WG551D Wild-type "Results summary from blind analysis of ten patient samples for CFTR exon 10and 11 mutations. Login to comment 252 ABCC7 p.Phe508Cys erozygotes (Kobayashi et al., 1990).Hybridization tests without probes specific for both sequence variants cannot effectively discriminate between AF508 homozygotes and AF508/F508C heterozygous samples. Login to comment 265 ABCC7 p.Gly551Asp ABCC7 p.Gly542* Image of a specialized mutation array hybridized with exon lO/exon 11targets prepared from a compound heterozygote for exon 11 mutations G542X and G551D (Children`s Hospital of Oakland, Sample 9). Login to comment 268 ABCC7 p.Gly542* G542X: 5'TAGTTClTTGAGAAGGT3`.Wild type: 5'GAGTGGAGGTCAACGAG3`. Login to comment 269 ABCC7 p.Gly551Asp G551D: 5`GAGTG- GAGATCAACGAGJ`. Login to comment 280 ABCC7 p.Gly551Asp ABCC7 p.Gly542* All except G542X and G551D display homozygous wild-type hybridization patterns. Other hybridization signals that appear as incomplete hybridization patterns compared with examples in Figure 3result from cross-hybridization with probes partially complementary to the target. Login to comment