ABCMdb

PMID: 15269305

Pont-Kingdon G, Jama M, Miller C, Millson A, Lyon E
Long-range (17.7 kb) allele-specific polymerase chain reaction method for direct haplotyping of R117H and IVS-8 mutations of the cystic fibrosis transmembrane regulator gene.
J Mol Diagn. 2004 Aug;6(3):264-70., [PubMed]

Sentences

No. Mutations Sentence Comment

15 ABCC7 p.Arg117His Two of these mutations, R117H in exon 4 and the 5T polymorphism of the polythymidine tract in intron 8 (IVS-8 5T polymorphism) have a phenotypic synergic effect. Login to comment 16 ABCC7 p.Arg117His The mutation R117H which accounts for approximately 0.8% of mutant alleles, changes an arginine to an histidine in a transmembrane domain of the protein, altering the conductance of the ion channel.16 The IVS-8 polymorphism affects the splicing efficiency of intron 8.17,18 A tract of 7T or 9T at the 3Ј end of intron 8 insures proper splicing of the intron while a 5T results in a majority of mRNA lacking exon 9.19 -22 Each mutation is independently considered mild because in both cases, residual activity of the ion channel remains. Login to comment 17 ABCC7 p.Arg117His Therefore, an individual carrying the R117H mutation and the IVS-8 5T polymorphism on two different chromosomes (in trans) is not affected by, nor considered a carrier of classic CF23 although this individual might present with atypical CF. Login to comment 18 ABCC7 p.Arg117His ABCC7 p.Arg117His ABCC7 p.Arg117His In contrast, a gene with both R117H and IVS-8 5T (in cis) is severely affected.24 Both R117H and the IVS-8 5T variants have been found at higher frequencies in individuals with atypical CF than in the normal population.22,25-27 If an individual is heterozygous for both R117H and the IVS-8 5T variant, it is necessary to establish if both mutations are in cis or in trans to correctly analyze the Supported by the Institute for Clinical and Experimental Pathology, LLC, ARUP Laboratories. Login to comment 26 ABCC7 p.Arg117His Since the R117H and IVS-8 loci are separated by 17.7 kilobases (Cystic Fibrosis Mutation database at http://genet.sickkids.on.ca/cftr), the assay described here is based on long-range allele-specific PCR. Login to comment 29 ABCC7 p.Arg117His Materials and Methods Patient Samples Genomic samples previously genotyped as R117H heterozygous were selected for the development of this assay and de-identified following Institutional Review Board protocol. Login to comment 40 ABCC7 p.Arg117His Determination of R117H and IVS-8 Haplotype Using Oligonucleotide Ligation Assay At the end of the PCR, products are analyzed using the Cystic Fibrosis Assay version 3.0 (Celera Diagnostics, Alameda, CA). Login to comment 49 ABCC7 p.Arg117His The R117H and the IVS-8 polymorphism are analyzed concurrently using as template 1.5 ␮l of the 17.7-kb PCR product previously diluted with 3.5 ␮l of "diluent for purified DNA" buffer. Login to comment 52 ABCC7 p.Arg117His The R117H and the IVS-8 loci are analyzed in the green channel. Login to comment 65 ABCC7 p.Arg117His ABCC7 p.Arg117His Results Long-Range Allele-Specific PCR Three independent long-range PCRs are performed per sample: one reaction amplifies both R117H alleles of the heterozygous R117H sample, while the other two are allele-specific (Figure 1). Login to comment 68 ABCC7 p.Arg117His The forward primer of the "RH" PCR (R117H-F) anneals in exon 4, 5Ј of the mutation site. Login to comment 77 ABCC7 p.Arg117His The sample shown in Figure 2 is heterozygous for the R117H mutation and carries the 5T/7T IVS-8 polymorphisms. Login to comment 85 ABCC7 p.Arg117His "RH" is the PCR that amplifies both the wild-type and the R117H mutant allele. Login to comment 89 ABCC7 p.Arg117His Primers Sequences PCR Primer Sequence Predicted Tm (°C) R allele H allele "RH" R117H-F 5Ј-TGTAGGAAGTCACCAAAGCAGTAC 58.2 58.2 "Halsp" Halsp-F 5Ј-CCCGGATAACAAGGAGGAACA 55.8 (1.9) 57.7 "Ralsp" Ralsp-F 5Ј-CCCGGATAACAAGGAGGAACG 58.1 55.8 (2.3) IVS8-R 5Ј-CAACCGCCAACAACTGTCC 57.7 57.7 Bold characters indicate the allele-specific nucleotide. Login to comment 101 ABCC7 p.Arg117His Haplotyping of R117H Samples Sixteen samples with a variety of IVS-8 genotypes were haplotyped (Table 3). Login to comment 105 ABCC7 p.Arg117His In addition to the R117H and IVS-8 loci, the OLA analysis reveals results for other CF mutation loci included in the CF version 3.0 kit from Celera. Login to comment 106 ABCC7 p.Arg117His In the same channel as R117H and IVS-8, the two extra peaks correspond to the wild-type alleles of the 621 ϩ 1G Ͼ T and 711 ϩ 1G Ͼ T mutations. Login to comment 110 ABCC7 p.Arg334Trp ABCC7 p.Arg347Pro ABCC7 p.Ile148Thr Mutations at these positions (621 ϩ 1G Ͼ T, 711 ϩ 1G Ͼ T, R347P, I148T, R334W, and 1078delT) could be detected and associated with one of the haplotypes. Login to comment 111 ABCC7 p.Arg117His In this analysis, the samples shown in A and C (samples 1 and 4) carries the 5T allele on the wild-type chromosome (R117) therefore R117H and 5T are in trans. Login to comment 117 ABCC7 p.Arg117His Genotypes of R117/R117H locus and the 5T/7T/9T locus were confirmed by sequencing the extremities of each PCR obtained from sample 1 and 2 (data not shown). Login to comment 118 ABCC7 p.Arg117His ABCC7 p.Arg117His ABCC7 p.Arg117His Accuracy and Reproducibility Several methods are available for genotyping independently both R117H and IVS-827,31 but determination of the phase relies on family studies or unequivocal results such as homozygosity of the IVS-8 genotype or compound heterozygous with delF508-9T.24,27 Although R117H and the 5T IVS-8 polymorphism are found at relatively high frequency in populations affected with CF or with atypical CF,22,25-27 they are relatively rare in the normal population and we found only five R117H/5T-positive samples in more than 10000 CF tests. Login to comment 121 ABCC7 p.Arg117His The parent carrying the R117H mutation is IVS-8-7T homozygous and the other parent has the 5T allele (data not shown). Login to comment 122 ABCC7 p.Arg117His Thus, this linkage study confirms the molecular diagnosis that R117H and 5T are in trans in this sample. Login to comment 123 ABCC7 p.Arg117His Additionally, sample 5 and 16 (Table 3) and the Coriell cell line control GM13591 (Bernacki SH, Beck JC, Muralidharan K, Schaefer F, Shrimpton AE, Pont-Kingdon G, Stenzel TT, submitted) are compound heterozygous R117H and DelF508. Login to comment 125 ABCC7 p.Arg117His ABCC7 p.Arg117His Molecular haplotyping determined the haplotypes as R117H-7T/DelF508-9T (sample 16) and R117H-5T/DelF508-9T (sample 5 and GM13591). Login to comment 131 ABCC7 p.Arg117His ABCC7 p.Arg117His ABCC7 p.Arg117His ABCC7 p.Arg117His ABCC7 p.Arg117His ABCC7 p.Arg117His ABCC7 p.Arg117His ABCC7 p.Arg117His ABCC7 p.Arg117His ABCC7 p.Arg117His ABCC7 p.Arg117His ABCC7 p.Arg117His ABCC7 p.Arg117His ABCC7 p.Arg117His ABCC7 p.Arg117His ABCC7 p.Arg117His ABCC7 p.Arg117His ABCC7 p.Arg117His ABCC7 p.Gly542* Genotypes and Haplotypes of R117H Samples Sample Genotype Haplotype Interpretation R chromosome (wild type) H chromosome (mutant) 1 R117H-5T/7T R-5T H-7T Trans 2 R117H-5T/7T R-7T H-5T Cis 3 R117H-5T/9T R-9T H-5T Cis 4 R117H-5T/7T R-5T H-7T Trans 5 R117H/Del F508-7T/9T R-9T H-5T Cis 6 R117H-7T/7T failed H-7T na 7 R117H-7T/7T R-7T H-7T na 8 R117H-7T/9T R-9T H-7T na 9 R117H-7T/7T R-7T H-7T na 10 R117H-7T/7T failed H-7T na 11 R117H-7T/7T R-7T H-7T na 12 R117H-7T/7T R-7T H-7T na 13 R117H-7T/7T R-7T H-7T na 14 R117H-7T/9T R-9T H-7T na 15 R117H-7T/9T R-9T H-7T na 16 R117H/DelF508-7T/9T R-9T H-7T na 17 R117H/G542X-7T/9T R-9T H-7T na na, not applicable. Login to comment 133 ABCC7 p.Arg117His Discussion This test allows the direct haplotyping of the R117H mutation and the 5T polymorphism of intron 8 of the CFTR gene. Login to comment 134 ABCC7 p.Arg117His Both the R117H mutation and the IVS-8 variant have been found at a higher frequency in individuals affected with classic or atypical cystic fibrosis. Login to comment 136 ABCC7 p.Arg117His If an individual is found to have the R117H mutation and the IVS-8 5T polymorphism, the molecular test can establish the cis/trans status of both mutations without the need to obtain DNA from parents. Login to comment 137 ABCC7 p.Arg117His Haplotyping technologies have rarely been applied to clinical testing for several reasons: methods are labor intensive and highly sophisticated,12,32 rely on extreme dilution of DNA not practical in a clinical setting,5,11 are limited to short fragments of DNA,6,8 -10,13 or are not accurate enough to determine haplotypes from specific individuals.33 A recent, elegant molecular approach that uses post-PCR ligation has been described and would also allow the amplification and haplotyping of the R117H mutation and the IVS-8 5T polymorphism.15 This approach has the disadvantage of requiring additional post-PCR steps before analysis. Login to comment 154 ABCC7 p.Arg117His The analysis of the R117H loci from each PCR provides an internal control. Login to comment 155 ABCC7 p.Arg117His An additional probe set, distinguishing the R117H mutation from the wild type, could also be added to the FRET probe assay and multiplexed with the IVS-8 probe. 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