ABCMdb

PMID: 10341008

Boucher D, Creveaux I, Grizard G, Jimenez C, Hermabessiere J, Dastugue B
Screening for cystic fibrosis transmembrane conductance regulator gene mutations in men included in an intracytoplasmic sperm injection programme.
Mol Hum Reprod. 1999 Jun;5(6):587-93., [PubMed]

Sentences

No. Mutations Sentence Comment

3 ABCC7 p.Arg117His ABCC7 p.Asn1303Lys In 10 out of 14 patients with CAVD, CFTR mutations were found; nine patients had one ∆∆F508 mutation and one patient had two CFTR mutations (N1303K/R117H). Login to comment 51 ABCC7 p.Gly551Asp ABCC7 p.Arg117His ABCC7 p.Trp1282* ABCC7 p.Arg553* ABCC7 p.Asn1303Lys ABCC7 p.Gly542* Each patient was tested for the nine most frequent cystic fibrosis-causing CFTR mutations: ∆F508, ∆I507, 1717-1G→A, G542X, G551D, R553X, W1282X, N1303K, 621ϩ1G→T and the three most frequent CFTR mutations involved in CBAVD (∆F508, R117H and the IVS8 polyT). Login to comment 53 ABCC7 p.Gly551Asp ABCC7 p.Arg117His ABCC7 p.Trp1282* ABCC7 p.Arg553* ABCC7 p.Asn1303Lys ABCC7 p.Gly542* The other mutations were detected using either heteroduplex analysis (∆I507), allele specific oligonucleotide (ASO) hybridization (G542X, 1717-1G→A, IVS8 polyT) (Kerem et al., 1990), restriction endonuclease analysis (G551D, R553X, W1282X) (Zielenski et al., 1991) or polymerase chain reaction (PCR)-mediated site-directed mutagenesis (621ϩ1G→T, R117H, N1303K) (Friedman et al., 1991). Login to comment 60 ABCC7 p.Gly551Asp ABCC7 p.Arg117His ABCC7 p.Trp1282* ABCC7 p.Arg553* ABCC7 p.Asn1303Lys Restriction digestion and electrophoresis (Figures 2 and 3) Detection of 621ϩ1G→T, R117H, G551D, R553X, W1282X and N1303K were performed using appropriate restriction enzymes (New England Biolabs, Ozyme, Saint Quentin Yvelines, France) as described in Table IV. Login to comment 64 ABCC7 p.Arg117His ABCC7 p.Asn1303Lys Clinical status, semen characteristics and concentrations of follicle stimulating hormone (FSH) in azoospermic patients No. Plasma FSH Semen volume Semen pH Semen fructose Semen Semen carnitine CFTR genotype (mIU/ml) (ml) (µmoles) α-glucosidase (mIU) (nmoles) Patients with congenital absence of the vas deferens (CAVD) 1 4.5 0.7 6.5 0 3 20 ∆F508/N 9T/5T 2 6.9 0.5 6.8 - - - ∆F508/N 9T/7T 3 1.9 0.2 6.5 Ͻ1 - - ∆F508/N 9T/5T 4 0.7 0.5 6.8 0 3 20 ∆F508/N 9T/7T 5 6.0 0.3 6.8 - - - ∆F508/N 9T/5T 6 3.6 0.5 6.5 - 3 20 ∆F508/N 9T/5T 7 4.0 1.5 6.5 - - - ∆F508/N 9T/5T 8 5.9 1.2 6.5 0 4 20 ∆F508/N 9T/7T 9 3.8 - - - - - ∆F508/N 9T/5T 10 5.0 - - 0 - 24 N1303K/ 9T/7T R117H *11 2.0 0.8 7.1 0 2 - N/N 9T/7T 12 4.3 0.3 7.4 0 - 3.6 N/N 7T/7T 13 1.0 0.4 6.5 0 - - N/N 9T/5T 14 2.0 1.3 6.8 0 5 - N/N 9T/5T Patients without CAVD 15 7.4 1.8 8.3 29 1 126 N/N 7T/7T 16 - 2.2 8.5 33 20 154 N/N 7T/7T 17 - 4.4 8.3 35 12 352 N/N 7T/7T 18 3.6 8.5 8.1 187 82 680 N/N 7T/7T 19 3.1 2.5 8.5 25 - 100 N/N 7T/7T 20 3.4 0.4 6.5 Ͻ1 7 28 N/N 7T/5T 21 1.6 1.4 8.3 4.2 5 126 N/N 7T/9T 22 3.1 3.0 8.7 78 2 270 N/N 7T/7T 23 0.9 3.0 9.0 - - - N/N 7T/7T 24 4.4 10.0 7.9 190 - 300 N/N 7T/7T 25 1.3 2.2 8.1 40 - 44 N/N 7T/7T 26 2.9 2.6 8.3 26 8 286 N/N 7T/7T 27 1.7 2.1 8.3 44 - 84 N/N 7T/7T 28 - 9.0 8.1 180 27 540 N/N 7T/7T 29 - 1.5 8.3 23 - 120 N/N 7T/7T 30 4.1 0.3 7.1 - - - N/N 7T/7T 31 4.9 1.7 8.3 20 20 - N/N 7T/7T 32 7.2 1.0 7.9 Ͻ1 3 30 N/N 7T/7T 33 9.0 1.5 6.5 Ͻ1 2 - N/N 7T/5T 34 11.7 5.0 7.9 90 - 400 N/N 7T/7T 35 7.2 4.6 8.1 92 132 1518 N/N 7T/9T 36 4.4 2.5 8.1 30 11 1510 N/N 7T/7T 37 14 3.6 8.3 50 27 900 N/N 7T/7T 38 4.9 6.0 7.9 72 - 300 N/N 7T/7T 39 6.0 1.8 8.3 31 17 252 N/N 7T/7T 40 14.3 1.5 8.3 22 1 90 N/N 7T/9T 41 6.6 2.2 - 27 - 290 N/N 7T/7T 42 4.8 0.5 6.8 0 3 42 N/N 7T/7T 43 13.9 2.0 7.8 - - - N/N 7T/7T 44 3.9 3.9 8.3 47 11 - N/N 7T/7T 45 1.5 1.8 8.6 11 - - N/N 7T/7T 46 4.2 3.0 8.5 25 28 210 N/N 7T/7T 47 4.0 5.8 7.9 122 42 696 N/N 7T/9T 48 16.3 5.0 8.7 145 68 900 N/N 7T/7T 49 7.2 4.0 7.5 - - - N/N 7T/5T 50 8.0 4.3 8.1 34 200 1284 N/N 7T/7T 51 23.9 0.8 9.0 5.6 17 184 N/N 7T/7T 52 - 2.2 8.5 35 - 308 N/N 7T/9T 53 17.8 3.1 8.1 31 50 527 N/N 7T/7T Fructose, α glucosidase and carnitine are expressed per ejaculate. Login to comment 67 ABCC7 p.Gly542* ASO hybridization PCR-amplified DNA (6 µl) of exon 9 or exon 11 was denatured and vacuumblotted onto nylon membrane(Hybond Nϩ, Amersham Pharmacia Biotech, Orsay, France) and hybridized with oligonucleotides which recognize the presence of 5T, 7T or 9T for detection of the IVS8 polyT-stretch, normal or mutated allele for 1717-1G→A and G542X. Login to comment 74 ABCC7 p.Arg117His ABCC7 p.Asn1303Lys One patient was a compound heterozygote, R117H/N1303K. Login to comment 94 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Arg117His ABCC7 p.Arg117His ABCC7 p.Trp1282* ABCC7 p.Trp1282* ABCC7 p.Arg553* ABCC7 p.Arg553* ABCC7 p.Asn1303Lys ABCC7 p.Asn1303Lys ABCC7 p.Gly542* ABCC7 p.Gly542* Methods for detecting mutations Mutation Method R117H PCR-mediated-site-directed mutagenesis, HaeII digestion N: 113 ϩ 24 bp R117H: 137 bp 621ϩ1G→T MseI digestion N: 269 ϩ 33 bp 621ϩ1G→T: 215 ϩ 54 ϩ 33 bp IVS8polyT ASO hybridization: hybridization at 50°C 5T: TGT GTG TGT TTT TAA CAG washing at 55°C 7T: TGT GTG TTT TTT TAA CAG washing at 51°C 9T: GTG TGT TTT TTT TTA ACA G washing at 55°C ∆I507 Heteroduplex DNA formation ∆F508 Heteroduplex DNA formation (see Figure 1) 17171G→A ASO hybridization, hybridization at 42°C, washing at 54°C N: TTT GGT AAT AGG ACA TCT CC 17171G→A: TTT GGT AAT AAG ACA TCT CC G542X ASO hybridization, hybridization at 42°C, washing at 49°C N: ACC TTC TCC AAG AAC T G542X: ACC TTC TCA AAG AAC T G551D DpnII digestion: N: 425 bp G551D: 243 ϩ 182 bp R553X HincII digestion N: 239 ϩ 186 bp R553X: 425 bp W1282X MnlI digestion N: 178 ϩ 172 ϩ 123 bp W1282X: 301 ϩ 172 bp N1303K PCR-mediated-site-directed mutagenesis, BstNI digestion N: 266 ϩ 23 bp N1303K: 289 bp The underlining indicates the location of nucleotide substitution in normal and mutated allele. Login to comment 99 ABCC7 p.Arg117His ABCC7 p.Asn1303Lys Our results are in accordance with these data, since nine of the 14 patients (64.2%) with CAVD were heterozygous for one CFTR mutation (∆F508) and one patient with CBAVD (7.1%) was a compound heterozygote (R117H/N1303K) and was 7T/9T. Login to comment 100 ABCC7 p.Asn1303Lys N1303K is classified as a severe mutation with respect to pancreatic status in CF patients. Login to comment 101 ABCC7 p.Arg117His R117H associated with a 5T variant is known to lead to a mild phenotype in CF patients. Login to comment 102 ABCC7 p.Arg117His In CAVD without CF, R117H is probably associated with the 7T allele allowing correct splicing of exon 9 and translation into a CFTR protein which is partially functional (Kiesewetter et al., 1993). Login to comment 105 ABCC7 p.Arg117His Detection of the R117H mutation by polymerase chain reaction (PCR). Login to comment 109 ABCC7 p.Arg117His Lanes 2 and 7: heterozygote carrier for the R117H mutation. Login to comment 127 ABCC7 p.Asn1303Lys Detection of the mutation N1303K by polymerase chain reaction (PCR). Login to comment 130 ABCC7 p.Asn1303Lys Lane 1: 1 kb DNA ladder (Life Technologies, Cergy-Pontoise, France); Lanes 2 and 6: heterozygote for the N1303K mutation; Lanes 3, 4, 5, 7: normal homozygote; Lane 8: BstNI cut control DNA; Lane 9: uncut control DNA. Login to comment