ABCMdb

PMID: 11810271

Tzetis M, Efthymiadou A, Doudounakis S, Kanavakis E
Qualitative and quantitative analysis of mRNA associated with four putative splicing mutations (621+3A-->G, 2751+2T-->A, 296+1G-->C, 1717-9T-->C-D565G) and one nonsense mutation (E822X) in the CFTR gene.
Hum Genet. 2001 Dec;109(6):592-601. Epub 2001 Nov 6., [PubMed]

Sentences

No. Mutations Sentence Comment

1 ABCC7 p.Asp565Gly ABCC7 p.Glu822* We studied four subjects with 621+3A→G, two with 2751+2T→A, one with 296+1G→C, two with 1717-9T→C-D565G and seven with E822X and compared the results with CFTR mRNA from normal subjects. Login to comment 2 ABCC7 p.Asp565Gly Our results showed that mutations 621+3A→G, 2751+2T→A, and 296+1G→C, which disrupt the 5` splice donor sites of introns 4, 14a, and 2, respectively, and 1717-9T→C-D565G, which possibly disrupts the exonic splicing enhancer sequences of exon 12 (owing to the missense mutation in cis), lead to the production of aberrantly spliced mRNA in nasal epithelial cells. Login to comment 5 ABCC7 p.Asp565Gly The complex allele 1717-9T→C-D565G results in aberrant splicing of CFTR mRNA with production of transcripts lacking exon 12 (major product), with minor amounts of transcripts revealing joint exon 11 and 12 skipping. Login to comment 6 ABCC7 p.Glu822* Nonsense mutation E822X results in a severe reduction in mRNA levels to about 6% of wild type. Login to comment 19 ABCC7 p.Asp565Gly ABCC7 p.Asp565Gly ABCC7 p.Glu822* ABCC7 p.Glu822* Ultimately, however, all the disease-causing mutations result in defective cAMP-regulated Cl-secretion by epithelial cells, though for various reasons, namely defective pro- Maria Tzetis · Alexandra Efthymiadou · Stavros Doudounakis · Emmanuel Kanavakis Qualitative and quantitative analysis of mRNA associated with four putative splicing mutations (621+3A→G, 2751+2T→A, 296+1G→C, 1717-9T→C-D565G) and one nonsense mutation (E822X) in the CFTR gene Hum Genet (2001) 109:592-601 DOI 10.1007/s00439-001-0631-0 Received: 18 June 2001 / Accepted: 13 September 2001 / Published online: 6 November 2001 ORIGINAL INVESTIGATION M. Tzetis · A. Efthymiadou · E. Kanavakis (✉) Department of Medical Genetics, Athens University, "Aghia Sophia" Children`s Hospital, Thivon & Livadias, Athens, 11527, Greece e-mail: ekanavak@cc.uoa.gr, Tel.: +30-1-7467460, Fax: +30-1-7795553 S. Doudounakis Cystic Fibrosis Unit, "Aghia Sophia" Children`s Hospital, Athens, Greece (c) Springer-Verlag 2001 tein production (class I), defective protein processing (class II), defective regulation (class III), defective conduction (class IV), or defective synthesis (class V) (Welsh and Smith 1993; Zielenski and Tsui 1995). Login to comment 30 ABCC7 p.Asp565Gly ABCC7 p.Asp565Gly We have investigated three putative splicing mutations, 621+3A→G, 2751+2T→A, 296+1G→C, which disrupt the 5` splice donor sites of introns 4, 14a, and 2, respectively, and the complex allele 1717-9T→C-D565G, which should disrupt the 3` acceptor splice site of intron 10 and possibly cause skipping of exon 12 owing to missense mutation D565G. Login to comment 31 ABCC7 p.Glu822* Additionally we studied one nonsense mutation, E822X. Login to comment 36 ABCC7 p.Asp565Gly ABCC7 p.Glu822* Patients and controls Nasal epithelial cells were collected from four subjects with 621+3A→G (all compound heterozygotes), two with 2751+2T→A (one compound heterozygote and one carrier), one with 296+1G→C, two with 1717-9T→C-D565G (both only carriers), and seven with E822X (three compound heterozygotes, two homozygotes, and two carriers). Login to comment 37 ABCC7 p.Asp565Gly A patient with the 621+1G→T mutation and one with the 1898+1G→T were also studied for the appropriate cDNA fragment and the results were compared with those of mutations 621+3A→G and D565G, respectively. Login to comment 54 ABCC7 p.Glu822* ABCC7 p.Glu822* Samples for the E822X mutation were reverse-transcribed into cDNA with an initial set of primers spanning exons 13 to 14a (Table 2), one of which contains a 3` mismatch which, in combination with the normal sequence at E822 (G@2596; E822X: G→T@2596), creates a restriction site for Hph1. Login to comment 58 ABCC7 p.Glu822* The resulting PCR products derived from cDNA templates for each of the above mutations, including the digested product for the E822X mutation, were electrophoresed on a 6% acrylamide denaturing gel using an automatic DNA sequencer (Vistra, model 725-Molecular Dynamics). Login to comment 59 ABCC7 p.Trp1282* ABCC7 p.Asp565Gly 593 594 Table1Genotype/phenotypeoftheCFTRpatientsa DevelopmentPulmonaryfunctionCFTRgenotypeAge (yrs) SexAgeat diagnosis Sweattest (mEq/l) Meconium ileusHeight (%-ile) Weight (%-ile) Pancreatic status FEV1 (%) FVC (%) Other Bacterial pathogens Other clinical features F508del/621+3AÆÆÆÆG6FBirth108.5Yes<50%>50%PI10398-Sa,Klebsiella- F508del/621+3AÆÆÆÆG7M2mos93.6No>10%~25%PI131132-Sa,Hi,Sa- 1898+1GÆÆÆÆT/621+3AÆÆÆÆG18F3mos82.1No>97%<90%PI7370Bronchi- ectasis Sa,PaDiabetes W1282X/621+3AÆÆÆÆG2F8mos100.9No<75%>75%PI---Hi,Psputida- F508del/2751+2TÆÆÆÆA5MBirth85.7Yes75%75%PI---Sa,Pa- 3120+1GÆA/296+1GÆÆÆÆC33M27yrs93.1No>75%~50%PI133128-Pa- 1717-9TÆÆÆÆC-D565G/Nb 7F5yrs41.4No??PS? Login to comment 60 ABCC7 p.Glu822* ABCC7 p.Glu822* ABCC7 p.Glu822* ABCC7 p.Glu822* ABCC7 p.Glu822* ?--Recurrent episodes ofpneu- monia E822X/D110E21M11mos127No<50%50%-75%PS100%110%-Pa,Sa E822X/F508del8M5yrs113No<75%90%-97%PI141%133%Nasal polyposis Sa,Klebsiella pneumoniae Gallblad- derstones, rectal proptosis E822X/N1303K11F1mo102.5/116.1/ 104.2 No<25%<50%PI24%27%-Pa,SaDIOS, hepatic disease E822X/E822X4MBirth83/82.9Yes50%25%-50%PI---Sa,Hi,Pa E822X/E822X15F1mo129.4/136.5/ 130.5 No>10%>10%PI50%54%Pa, xanthomonas maltiphilia,Sa PI,Pancreaticinsufficiency;PS,pancreaticsufficiency;Sa,Staphylococcus;Hi,Haemophilus;Pa,Pseudomonasaureus;Ps,Pseudomonas;DIOS,distalintestinalobstruction syndrome aHeterozygotesforthemutationsstudiedarenotincludedinthetable bNotaCFpatient,heterozygotewithrecurrentepisodesofpneumonia Each analysis for each sample was repeated three times to ensure accurate quantitation and all measurements were combined to reach the final result for each of the fragments analyzed. Login to comment 91 ABCC7 p.Asp565Gly ABCC7 p.Asp565Gly C Sequencing results of alternatively spliced transcript (253 bp), lacking all of exon 4 1717-9T→C-D565G cDNA from nasal epithelial cells of two heterozygotes with 1717-9T→C-D565G, two controls, and a patient with the 1898+1G→T mutation was amplified with primers F2/B21 spanning exons 10 to 13. Login to comment 98 ABCC7 p.Glu822* ABCC7 p.Glu822* E822X One-step extension of the fluorescent primer E822Xrev (cDNA products of seven subjects for mutation E822X as well as two controls), followed by Hph1 digestion of the product, allowed differentiation between cDNA derived from the normal (79 bp plus 31 bp) and mutant (110 bp) alleles (Fig.4). Login to comment 99 ABCC7 p.Glu822* Quantitation of cDNA produced from the E822X allele indicated that the mutation is associated with severely reduced mRNA levels (6.3%±1.9%) compared with normal, and this should result in the production of minimal amounts of truncated protein. Login to comment 100 ABCC7 p.Asp565Gly ABCC7 p.Glu822* Discussion Analysis of CFTR mRNA from nasal epithelial cells, which are the cells biologically relevant to the disease process in CF, shows that mutations 621+3A→G, 2751+2T→A, 296+1G→C, and double allele 1717-9T→C-D565G result in the production of aberrantly spliced mRNA transcripts and that nonsense mutation E822X results in severe reduction of CFTR mRNA. Login to comment 111 ABCC7 p.Asp565Gly The reduc- 598 Fig.3A-D mRNA results for mutations 296+1G→C and 1717-9T→C-D565G. Login to comment 113 ABCC7 p.Asp565Gly A mRNA RT-PCR products analysed by 2% agarose gel electrophoresis. M, Marker lane, phiX174/HaeIII DNA; lane 1: normal control; lane 2: sample with mutation 296+1G→T. B mRNA RT-PCR products analysed by 2% agarose gel electrophoresis. M, Marker lane, phiX174/HaeIII DNA; lanes 1 and 2: samples with double allele 1717-9T→C-D565G; lane 3: sample with mutation 1898+1G→T; lane 4: normal control. Login to comment 129 ABCC7 p.Asp565Gly Two aberrantly spliced transcripts were produced by double allele 1717-9T→C-D565G (mRNA transcripts lacking exon 12 as a major product and a minor aberrant transcript lacking both exons 11 and 12), while mutation 1818+1G→T produced aberrant transcripts lacking exon 12 only. Login to comment 133 ABCC7 p.Asp565Gly However, the high percentage of aberrant transcripts produced lacking exon 12, comparable to those produced from the 1818+1G→T, which is a true splicing mutation, could be the result of D565G (A→G@1826). Login to comment 135 ABCC7 p.Glu822* Splicing 599 Fig.4A,B mRNA results for mutation E822X. Login to comment 137 ABCC7 p.Glu822* ABCC7 p.Glu822* A mRNA RT-PCR products analysed by 2% agarose gel electrophoresis. M, Marker lane, phiX174/ HaeIII DNA; lanes 1, 2 and 6: samples homozygous for mutation E822X; lanes 3, 4, 7, 8 and 9: samples heterozygous for E822X; lane 5: normal control; 110 bp: mutant transcript; 79 bp: normal transcript. Login to comment 138 ABCC7 p.Glu822* B Schematic representation of method of analysis used (showing location of primers) for the RT-PCR for the study of mutation E822X. Login to comment 140 ABCC7 p.Glu822* Underlined and bold nucleotides represent primer mismatch (G→A) and E822X mutation (G→T) enhancers are usually located within 100 nucleotides of the 3` splice site and their potential target is thought to be U2AF, which binds to the 3` splice site (Graveley et al. 1998). Login to comment 141 ABCC7 p.Asp565Gly Further studies are needed in order to confirm the effect of D565G mutation on such sequences in exon 12. Login to comment 142 ABCC7 p.Glu822* Transcripts produced from the E822X allele indicated that the mutation is associated with severely reduced mRNA levels (6.3%), resulting in the production of minimal amounts of truncated protein. Login to comment 145 ABCC7 p.Glu822* Nonsense mutation E822X is common in the Greek population, accounting for 1.7% of CF chromosomes. Login to comment 146 ABCC7 p.Glu822* The patients with E822X were diagnosed between birth and 5 years with high sweat test values and were all pancreatic insufficient except for the patient carrying a mild mutation (D110 E) in trans (Table 1). Login to comment 147 ABCC7 p.Asp565Gly In conclusion, the splicing mutations (621+3A→G, 2751+2T→A, 296+1G→C, and 1717-9T→C-D565G) all lead to the production of alternatively spliced CFTR transcripts. Login to comment 149 ABCC7 p.Glu822* The nonsense mutation (E822X) results in undetectable CFTR mRNA in nasal epithelial cells, possibly owing to the nonsense-mediated mRNA decay pathway; therefore minimal amounts of truncated protein are expected to be produced from the mutant allele. Login to comment