ABCMdb

PMID: 16635477

Lucarelli M, Narzi L, Piergentili R, Ferraguti G, Grandoni F, Quattrucci S, Strom R
A 96-well formatted method for exon and exon/intron boundary full sequencing of the CFTR gene.
Anal Biochem. 2006 Jun 15;353(2):226-35. Epub 2006 Apr 5., [PubMed]

Sentences

No. Mutations Sentence Comment

5 ABCC7 p.Gly1244Arg A novel G1244R disease causing mutation, leading to a CF phenotype with pancreatic sufficiency but early onset of pulmonary involvement, was detected in the subject with an uncertain diagnosis. Some discrepancies between our results and previously published CFTR sequence were found. Login to comment 26 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.Gln493* ABCC7 p.Val520Phe ABCC7 p.Gly85Glu ABCC7 p.Ser549Asn ABCC7 p.Tyr122* ABCC7 p.Arg560Thr None of these subjects showed any clinical manifestations of CF, nor were any positive for CFTR mutations when analyzed by means of the PCR/OLA/SCS method (Celera Diagnostics) [21], which searches for the most common worldwide 31 CFTR mutations (G85E, R117H, Y122X, 621+1G->T, 711+1G->T, 1078delT, R347P, R347H, R334W, A455E, DF508, DI507, Q493X, V520F, 1717-1G->A, G542X, G551D, R553X, R560T, S549R(T->G), S549N, 1898+1G->A, 2183AA->G, 2789+5G->A, R1162X, 3659delC, 3849+10kbC->T, 3849+4A->G, W1282X, 3905insT, N1303K), including the 12 most common in Italy [1,22]. Login to comment 31 ABCC7 p.Gly1244Arg To verify the absence of the novel G1244R mutation in the general population, the exon 20 of a further 90 subjects from the general population was analyzed. Login to comment 99 ABCC7 p.Gly1244Arg DHPLC assay design With the aim of optimizing a rapid method for the search of the novel G1244R mutation on a large number of samples, a DHPLC assay was designed and performed by a WAVE DNA Fragment Analysis System on a DNASep column (Transgenomic). Login to comment 122 ABCC7 p.Gly1244Arg ABCC7 p.Gly1244Arg Novel G1244R mutation and biochemical and clinical characterization of a subject with uncertain diagnosis The application of this protocol to the subject with an uncertain diagnosis allowed us to detect the novel CFTR mutation G1244R (3862 G->A, glycine to arginine) in exon 20 (Fig. 2A) [1] in the critical NBF2 domain. Login to comment 123 ABCC7 p.Gly1244Arg The analysis of allelic segregation in the parents assigned the 3849+10kbC->T (carried by the mother) and G1244R (carried by the father) mutations to different alleles. Login to comment 125 ABCC7 p.Gly1244Arg The novel G1244R mutation was not found in the other 73 unknown CF alleles analyzed, nor was it found in the 232 alleles from the general population. Login to comment 127 ABCC7 p.Gly1244Arg The biochemical and clinical characteristics of the 3849+10KbC->T/G1244R genotype are as follows. Login to comment 139 ABCC7 p.Gly1244Glu ABCC7 p.Thr338Ile ABCC7 p.Leu1065Pro ABCC7 p.Arg1066Cys ABCC7 p.Arg334Leu ABCC7 p.Arg117Cys ABCC7 p.Leu997Phe ABCC7 p.Leu1077Pro ABCC7 p.Ala349Val ABCC7 p.Asp110His ABCC7 p.His139Arg ABCC7 p.Tyr849* In this work, we found a limited subset of 13 mutations (not included in the PCR/OLA/SCS assay) in 7 CFTR exons, significantly improving the sensitivity of standard assays: D110H, R117C, and H139R (exon 4); R334L, T338I, and A349V (exon 7); S549R(A->C) (exon 11); Y849X (exon 14a); L997F (exon 17a); L1065P, R1066C, and L1077P (exon 17b); and G1244E (exon 20). Login to comment 153 ABCC7 p.Gly1244Arg Several findings suggest that the novel G1244R should be classified as a disease-causing mutation. Login to comment 155 ABCC7 p.Gly1244Glu ABCC7 p.Gly1244Val The importance of the 1244 codon, which can be classified as a mutational hot spot, is also highlighted by the fact that this same codon can be affected by two other mutations, G1244V [32] and G1244E [33], both of which already have been shown to cause disease. Login to comment 156 ABCC7 p.Gly1244Arg The G1244R mutation was absent from the 232 alleles in the general population and in the other affected subjects studied, and it was found as the only mutation on one allele of 1 other subject studied, with no other mutation on this allele being detectable by extended sequencing. Login to comment 159 ABCC7 p.Gly1244Arg The G1244R mutation by sequencing (A) and by DHPLC (B). Login to comment 162 ABCC7 p.Gly1244Arg Nevertheless, additional functional studies should be performed to assess the exact clinical severity of the G1244R mutation. Login to comment 163 ABCC7 p.Gly1244Arg The fact that this subject escaped neonatal screening demonstrates that both the G1244R and 3849+10kbC->T mutations yield a negative neonatal IRT dosage; with regard to the latter mutation, this finding is in contrast to some previous reports [39]. Login to comment 165 ABCC7 p.Gly1244Glu ABCC7 p.Gly1244Arg The frequency of the G1244R mutation must be calculated on a larger number of CF subjects to ascertain whether it is a rare mutation; however, the frequency of the G1244E mutation, one of the other mutations in the same codon, was found to be approximately 0.3%, which is relatively high. Login to comment