ABCMdb

PMID: 21097845

Sheridan MB, Hefferon TW, Wang N, Merlo C, Milla C, Borowitz D, Green ED, Mogayzel PJ Jr, Cutting GR
CFTR transcription defects in pancreatic sufficient cystic fibrosis patients with only one mutation in the coding region of CFTR.
J Med Genet. 2011 Apr;48(4):235-41. Epub 2010 Nov 20., [PubMed]

Sentences

No. Mutations Sentence Comment

15 ABCC7 p.Arg117His ABCC7 p.Arg117His ABCC7 p.Ala455Glu Most PS-CF patients have two mutations in the coding regions of the CFTR gene.5 6 At least one mutation permits residual CFTR function, allowing the patient to escape the classic CF phenotype.7 8 While the 23 mutation ACMG panel includes several mutations frequently associated with PS-CF (eg, R117H [p.Arg117His], A455E [p.Arg455Glu], 2789+5G/A [c.2657+5G/A], and 3849+10kbC/T [c.3717+12191C/T]), it is not uncommon for a PS-CF patient to carry a rare CFTR mutation that is not present in the panel.9 Detection of less common mutations can be accomplished by use of extended mutation panels10 or by comprehensive analysis of the coding regions of the CFTR gene using scanning11e13 or DNA sequencing methods.14 Scanning techniques detect 85e99% of CFTR mutations15e17 while DNA sequencing has a higher sensitivity because it allows analysis of individual nucleotides.18 19 However, these techniques do not identify gene rearrangements and mutations in non-coding regions that affect splicing or expression of RNA transcripts. Login to comment 39 ABCC7 p.Arg764* RNA from a healthy R764X (p.Arg764X) carrier was used as a control. Login to comment 40 ABCC7 p.Arg764* RNA from a healthy R764X (p.Arg764X) carrier was used as a control. Login to comment 47 ABCC7 p.Arg553* ABCC7 p.Arg553* The patient`s other mutation, (R553X [p.Arg553X]) is associated with a low amount of CFTR transcript due to nonsense mediated RNA decay (NMRD) (figure 2B).37 38 The RNA transcripts lacking exon 5 (exon5À), were 2.7 times more abundant than the transcripts containing exon 5 (exon5+/R553X) (figure 2B). Login to comment 48 ABCC7 p.Arg553* ABCC7 p.Arg553* The patient`s other mutation, (R553X [p.Arg553X]) is associated with a low amount of CFTR transcript due to nonsense mediated RNA decay (NMRD) (figure 2B).37 38 The RNA transcripts lacking exon 5 (exon5À), were 2.7 times more abundant than the transcripts containing exon 5 (exon5+/R553X) (figure 2B). Login to comment 61 ABCC7 p.Arg553* ABCC7 p.Arg553* ABCC7 p.Arg553* ABCC7 p.Arg553* ABCC7 p.Pro205Ser ABCC7 p.Pro205Ser ABCC7 p.Arg764* ABCC7 p.Ser492Phe ABCC7 p.Ser492Phe To detect these transcripts, we amplified Table 1 CFTR genotypes and clinical characteristics of patients with one CFTR mutation after CFTR screening or scanning ID Sweat [ClL ]* FEV1 y Pseudomonas infection NPD CBAVD CFTR genotype upon entry to study (method) CFTR genotype after sequencing 1 52 57 Yes ND ND DF508/unknown, IVS8 5T/9T (c.1210-12T[5]/c.1210-12T[9]) (comprehensive scan) DF508/IVS8-TG12-5T 2 98 79 No CF Yes DF508/unknown (comprehensive scan) DF508/S492F [p.Ser492Phe] 3 89 62 No ND NA DF508/unknown (screened e 86 mutations) DF508/P205S [p.Pro205Ser] 4z 65 58 Yes ND NA R553X/unknown (screened e 86 mutations) R553X/711+3 A/G 5z 66 82 Yes ND Yes R553X/unknown (screened e 70 mutations) R553X/711+3 A/G 6 72 71 Yes CF No DF508/unknown{ DF508/unknownyy 7 59 106 Yes Abnormalx NA DF508/unknown{ DF508/unknownyy 8 37 85 No CF NA DF508/unknown{ DF508/unknownyy 9 40 112 No ND ND DF508/unknown (comprehensive scan) DF508/unknown 10 66 ND Yes ND ND 621+1G/T/unknown (comprehensive scan) 621+1G/T/ unknownyy 11 58 ND No ND ND NA** R764X/unknownyy *Sweat [ClÀ ] concentration is expressed as mmol/l. Login to comment 63 ABCC7 p.Arg553* ABCC7 p.Arg553* ABCC7 p.Arg553* ABCC7 p.Arg553* ABCC7 p.Pro205Ser ABCC7 p.Pro205Ser ABCC7 p.Arg764* ABCC7 p.Ser492Phe ABCC7 p.Ser492Phe To detect these transcripts, we amplified Table 1 CFTR genotypes and clinical characteristics of patients with one CFTR mutation after CFTR screening or scanning ID Sweat [ClL ]* FEV1 y Pseudomonas infection NPD CBAVD CFTR genotype upon entry to study (method) CFTR genotype after sequencing 1 52 57 Yes ND ND DF508/unknown, IVS8 5T/9T (c.1210-12T[5]/c.1210-12T[9]) (comprehensive scan) DF508/IVS8-TG12-5T 2 98 79 No CF Yes DF508/unknown (comprehensive scan) DF508/S492F [p.Ser492Phe] 3 89 62 No ND NA DF508/unknown (screened e 86 mutations) DF508/P205S [p.Pro205Ser] 4z 65 58 Yes ND NA R553X/unknown (screened e 86 mutations) R553X/711+3 A/G 5z 66 82 Yes ND Yes R553X/unknown (screened e 70 mutations) R553X/711+3 A/G 6 72 71 Yes CF No DF508/unknown{ DF508/unknownyy 7 59 106 Yes Abnormalx NA DF508/unknown{ DF508/unknownyy 8 37 85 No CF NA DF508/unknown{ DF508/unknownyy 9 40 112 No ND ND DF508/unknown (comprehensive scan) DF508/unknown 10 66 ND Yes ND ND 621+1G/T/unknown (comprehensive scan) 621+1G/T/ unknownyy 11 58 ND No ND ND NA** R764X/unknownyy *Sweat [ClÀ ] concentration is expressed as mmol/l. Login to comment 75 ABCC7 p.Arg764* The remaining patient (patient 11) carries R764X (table 1). Login to comment 77 ABCC7 p.Arg764* ABCC7 p.Arg764* ABCC7 p.Arg764* Amplification of the region containing R764X (exons 13e14a) from a healthy carrier of R764X revealed minimal amounts of transcript bearing the R764X mutation, indicating that this mutation induces NMRD (red peak, third sequence trace, figure 4). Login to comment 78 ABCC7 p.Arg764* ABCC7 p.Arg764* However, transcript bearing R764X in patient 11 is present at a higher level than the RNA transcript from the other CFTR gene (red peak versus blue peak in bottom trace, figure 4). Login to comment 80 ABCC7 p.Arg764* ABCC7 p.Arg764* ABCC7 p.Arg764* Amplification of the region containing R764X (exons 13e14a) from a healthy carrier of R764X revealed minimal amounts of transcript bearing the R764X mutation, indicating that this mutation induces NMRD (red peak, third sequence trace, figure 4). Login to comment 81 ABCC7 p.Arg764* However, transcript bearing R764X in patient 11 is present at a higher level than the RNA transcript from the other CFTR gene (red peak versus blue peak in bottom trace, figure 4). Login to comment 82 ABCC7 p.Arg553* These studies support the observation that patients with clinical features of CF and one Figure 2 Reverse transcriptase (RT)-PCR analysis of patient 4 carrying R553X and 711+3 A to G. Login to comment 85 ABCC7 p.Arg553* These studies support the observation that patients with clinical features of CF and one Figure 2 Reverse transcriptase (RT)-PCR analysis of patient 4 carrying R553X and 711+3 A to G. Login to comment 87 ABCC7 p.Arg553* ABCC7 p.Arg553* ABCC7 p.Arg553* ABCC7 p.Arg553* ABCC7 p.Arg553* ABCC7 p.Arg553* ABCC7 p.Pro205Ser ABCC7 p.Ser492Phe A Splicing of RNA from CFTR gene with R553X: Genomic DNA Transcript including exon 5 and R553X GTA R553X R553X (R553X) Splicing of RNA from CFTR gene with 711+3 A>G: Transcript missing exon 5 (90 bp)Transcript including exon 5 GTG (Exon 5+) (Exon 5-) 4 5 6b6a 11 6a 6b54 11 Genomic DNA4 5 6b6a 11 6a 6b54 11 6a 6b4 11 B PeakHeight Exon 5-Exon 5+ /R553X including exon 5 390bp missing exon 5 300bp Table 2 Functional consequences of CFTR mutations identified after CFTR sequencing ID Second CFTR mutation identified after sequencing Amino acid conservation* Functional consequence References 1 TG12-5T NA Missplicingeloss of exon 9 from CFTR transcript Cuppens et al 199835 Groman et al 200436 2 S492F Conserved in mammalian orthologues except platypus None described Ferec et al31 Wine et al 200139 3 P205S Conserved in mammalian orthologues Defective biosynthesis, decreased amount of fully glycosylated CFTR Sheppard et al 199634 4, 5 711+3 A/G NA Missplicingeloss of exon 5 from CFTR transcript This study (figure 2) *Sequences were obtained from GenBank. Login to comment 90 ABCC7 p.Arg553* ABCC7 p.Arg553* ABCC7 p.Arg553* ABCC7 p.Arg553* ABCC7 p.Arg553* ABCC7 p.Arg553* ABCC7 p.Pro205Ser ABCC7 p.Ser492Phe A Splicing of RNA from CFTR gene with R553X: Genomic DNA Transcript including exon 5 and R553X GTA R553X R553X (R553X) Splicing of RNA from CFTR gene with 711+3 A>G: Transcript missing exon 5 (90 bp)Transcript including exon 5 GTG (Exon 5+) (Exon 5-) 4 5 6b6a 11 6a 6b54 11 Genomic DNA4 5 6b6a 11 6a 6b54 11 6a 6b4 11 B PeakHeight Exon 5-Exon 5+ /R553X including exon 5 390bp missing exon 5 300bp Table 2 Functional consequences of CFTR mutations identified after CFTR sequencing ID Second CFTR mutation identified after sequencing Amino acid conservation* Functional consequence References 1 TG12-5T NA Missplicingeloss of exon 9 from CFTR transcript Cuppens et al 199835 Groman et al 200436 2 S492F Conserved in mammalian orthologues except platypus None described Ferec et al31 Wine et al 200139 3 P205S Conserved in mammalian orthologues Defective biosynthesis, decreased amount of fully glycosylated CFTR Sheppard et al 199634 4, 5 711+3 A/G NA Missplicingeloss of exon 5 from CFTR transcript This study (figure 2) *Sequences were obtained from GenBank. Login to comment 116 ABCC7 p.Arg764* ABCC7 p.Arg764* Control Genomic DNA Patient 11 Genomic DNA C:T ratio= 1.21 Healthy R764X Carrier cDNA C:T ratio= 2.6 Patient 11 cDNA C:T ratio= 0.31 R764X C>T Figure 4 Quantification of CFTR RNA transcripts in cells from patient 11. Login to comment 118 ABCC7 p.Arg764* The R764X mutation is caused by the replacement of a C nucleotide at 2422 by a T nucleotide (compare upper and second tracings). Login to comment 119 ABCC7 p.Arg764* cDNA was generated from nasal epithelial cell RNA obtained from a healthy carrier of the R764X mutation and from patient 11. Login to comment 120 ABCC7 p.Arg764* ABCC7 p.Arg764* ABCC7 p.Arg764* The C: T ratio indicates the amount of DNA or cDNA derived from the wild type CFTR gene in the healthy carrier or the CFTR gene without a coding region mutation in the patient (C) to the amount of DNA or cDNA derived from the CFTR gene with the R764X mutation (T). Login to comment 122 ABCC7 p.Arg764* The R764X mutation is caused by the replacement of a C nucleotide at 2422 by a T nucleotide (compare upper and second tracings). Login to comment 123 ABCC7 p.Arg764* cDNA was generated from nasal epithelial cell RNA obtained from a healthy carrier of the R764X mutation and from patient 11. Login to comment 124 ABCC7 p.Arg764* The C: T ratio indicates the amount of DNA or cDNA derived from the wild type CFTR gene in the healthy carrier or the CFTR gene without a coding region mutation in the patient (C) to the amount of DNA or cDNA derived from the CFTR gene with the R764X mutation (T). Login to comment