ABCMdb

PMID: 11597137

Manson A, Huxley C
Skipping of exon 9 of human CFTR in YAC-transgenic mice.
Genomics. 2001 Oct;77(3):127-34., [PubMed]

Sentences

No. Mutations Sentence Comment

30 ABCC7 p.Arg117His ABCC7 p.Arg117His Thus the mild R117H missense mutation occurs on both 7T and 5T haplotypes [22] and the phenotype of R117H mutations (as a compound heterozygote with a severe mutation such as èc;F508) is more severe when combined with the 5T allele (mostly pancreatic sufficient CF) than when combined with the 7T allele (mostly CBAVD) [22]. Login to comment 70 ABCC7 p.Arg117Thr We also introduced the R117T mutation in exon 4 to assess this mutation, but inadvertently also introduced another mutation at nt 413, C to T, also in exon 4, at the same time. Login to comment 71 ABCC7 p.Thr94Ile ABCC7 p.Thr94Ile This is a missense mutation replacing threonine 94 with isoleucine (T94I) and should have no effect on the splicing of exon 9. Login to comment 72 ABCC7 p.Arg117Thr ABCC7 p.Thr94Ile We generated one line of mice, A10, with the YAC carrying the TG12T5 splice site and the R117T and T94I mutations. Login to comment 73 ABCC7 p.Arg117His ABCC7 p.Thr94Ile Another, 7T44, was generated with a YAC carrying the original TG10T7 splice site with the R117H and T94I mutations. Login to comment 79 ABCC7 p.Arg117His ABCC7 p.Thr94Ile However, we cannot determine whether this is due to the T94I mutation, the R117H mutation, and/or low splicing efficiency. Login to comment 80 ABCC7 p.Arg117His ABCC7 p.Thr94Ile As further controls, two cell lines (F7T.2 and F7T.5) were made with the YAC carrying the R117H and T94I mutations and the 7T splice site. Login to comment 94 ABCC7 p.Arg117His ABCC7 p.Arg117His ABCC7 p.Arg117His ABCC7 p.Arg117His ABCC7 p.Thr94Ile ABCC7 p.Thr94Ile ABCC7 p.Thr94Ile ABCC7 p.Thr94Ile mRNA (n)a of transgeneb TgN(yCFTR)T30Clh T30 TG10T7 2 0.108 (2) 11% TgN(yCFTR)T57Clh T57 TG10T7 1 0.109 (2) 22% TgN(yCFTR)A10Clh A10 TG12T5, R117H, T94I 6 0.205 (2) 7% TgN(yCFTR)7T44Clh 7T44 TG10T7, R117H, T94I nd nd nd Cell line name F7T.2 TG10T7, R117H, T94I 2 0.178 (2) 18% F7T.5 TG10T7, R117H, T94I 8 0.825 (3) 21% a Number of independent cDNA and RT-PCR reactions. Login to comment 125 ABCC7 p.Arg117His ABCC7 p.Thr94Ile The level of skipping of exon 9 in the lung, trachea, gall bladder, duodenum, colon, uterus, and epididymis of A10 mice (5Ts, R117H, and T94I) was quite uniform at about 91%. Login to comment 127 ABCC7 p.Arg117His ABCC7 p.Thr94Ile A10 differs from T30 not only at the splice site of exon 9, but also in having the R117H and T94I mutations in exon 4. Login to comment 128 ABCC7 p.Arg117His ABCC7 p.Thr94Ile To determine whether the R117H and T94I mutations were affecting splicing of exon 9, we also determined the level of skipping of exon 9 in the duodenum and testis of a 7T44 mouse. Login to comment 130 ABCC7 p.Arg117His ABCC7 p.Thr94Ile Although slightly higher than the values obtained for T30 mice, the R117H and T94I mutations do not account for the much higher levels of skipping in the A10 line than the T30 line. Login to comment 139 ABCC7 p.Arg117His ABCC7 p.Arg117His ABCC7 p.Thr94Ile Our original intention was to also study the R117H missense mutation that was introduced into exon 4, but another missense mutation, T94I, was inadvertently introduced into exon 4 at the same time so the effect of the R117H mutation could not be studied. Login to comment 141 ABCC7 p.Arg117His ABCC7 p.Thr94Ile In this case, the presence of the R117H and T94I mutations in exon 4 has little effect on the level of skipping of exon 9 as indicated by the similar splicing in the lines T30 and 7T44, which differ only by the two mutations in exon 4 (Fig. 5). Login to comment 176 ABCC7 p.Arg117His This may be true for otherwise normal alleles and mild mutations such as R117H in which the CFTR-related phenotype has been shown to be more severe when there is less full-length mRNA. Login to comment 198 ABCC7 p.Arg117His The YAC y37AB12 was then modified by homologous recombination in the yeast host to change the splice acceptor to the 5T allele and to introduce the R117H mutation. Login to comment 200 ABCC7 p.Arg117His This YAC was then modified to carry the R117H mutation in exon 4 and the 5T splicing variant in intron 8 using the "pop-in pop-out" procedure as described [44]. Login to comment 201 ABCC7 p.Arg117His Patient DNAs with the R117H mutation and the 5T splice acceptor allele were kindly provided by Liz Kay (North West Thames Regional Genetic Service). Login to comment 202 ABCC7 p.Arg117His The exon 4 region carrying the R117H mutation was amplified using the primers 5b18;-TCACATATGGTATGACCCTC-3b18; and 5b18;-TTGTACCAGCTCACTACCTA-3b18; [45]. Login to comment 210 ABCC7 p.Arg117His PCR and sequencing was used to confirm the clones which had been modified with the R117H mutation. Login to comment 212 ABCC7 p.Arg117His On later analysis it transpired that a point mutation (nt 413, C to T), in addition to the desired R117H mutation, had been cloned and introduced. Login to comment 213 ABCC7 p.Thr94Ile This point mutation causes a missense mutation, T94I, in the coded protein. Login to comment