ABCMdb

PMID: 12544470

Strom CM, Huang D, Chen C, Buller A, Peng M, Quan F, Redman J, Sun W
Extensive sequencing of the cystic fibrosis transmembrane regulator gene: assay validation and unexpected benefits of developing a comprehensive test.
Genet Med. 2003 Jan-Feb;5(1):9-14., [PubMed]

Sentences

No. Mutations Sentence Comment

54 ABCC7 p.Gly551Asp ABCC7 p.Arg117His ABCC7 p.Arg117His ABCC7 p.Arg117His ABCC7 p.Asn1303Lys ABCC7 p.Asn1303Lys ABCC7 p.Gly542* ABCC7 p.Ile148Thr ABCC7 p.Ile148Thr ABCC7 p.Arg1066Cys Table 2 Mutant samples used for validation of sequencing assay Mutation expected wt/wt (3 patients) delta F508/wt (2 patients) R117H/wt (3 patients) 2789 ϩ 5 G 3 A/2789 ϩ 5 G 3 A (both parents confirmed carriers) R117H/delta F508 (2 patients) delta F508/I148T delta F508/R1066C delta F508/3848 ϩ 10 kb C 3 T delta F508/G542X R117H/I148T (2 patients) 2307 delA/N1303K deltaF 508/711 ϩ 1 G 3 T deltaF 508/1898 ϩ 1 G 3 A G551D/N1303K 2789 ϩ 5G3A. Login to comment 65 ABCC7 p.Asp806Gly ABCC7 p.Phe191Leu For the 10 unknown chromosomes, 7 described mutations were found and 2 novel missense mutations, D806G and F191L, were discovered. Login to comment 72 ABCC7 p.Ala455Glu Seven of the nine involved the A455E mutation, and the remaining two involved the 1898 ϩ 1 G3A mutation. Login to comment 73 ABCC7 p.Ala455Glu Weak positive lines for the A455E mutation were observed in five instances. Login to comment 75 ABCC7 p.Ala455Glu ABCC7 p.Gly463Asp ABCC7 p.Ser459Phe Sequencing analysis revealed novel missense mutations or sequence variations in all five cases: two patients had 1508 C3T (S459F) occurring four codons upstream from A455E, two had 1496 C3T (A455 V), and one had 1520G3A (G463D). Login to comment 76 ABCC7 p.Ala455Glu Whether these base changes represent novel CF mutations or polymorphism cannot be determined at this time, but the sequence analysis prevented the misdiagnosis of the patients as heterozygotes for A455E. Login to comment 77 ABCC7 p.Ala455Glu In two cases there was no A455E signal in either the mutant or wild-type line. Login to comment 79 ABCC7 p.Val456Ala In the other patient, sequencing revealed homozygosity for a novel missense mutation V456A. Login to comment 81 ABCC7 p.Ala455Glu ABCC7 p.Ala455Glu Because of these unusual results at the A455E site, we sequenced that exon for every patient previously found to be heterozygous for A455E. Login to comment 82 ABCC7 p.Ala455Glu Ten patients were sequenced and the genotype A455E/wt was confirmed, indicating that the assay performance for this allele is acceptable when the expected intensity of the mutant band is observed. Login to comment 83 ABCC7 p.Ala455Glu These data suggest that caution must be taken when weak A455E mutant bands are observed. Login to comment 90 ABCC7 p.Ala455Glu ABCC7 p.Ala455Glu ABCC7 p.Ala455Glu ABCC7 p.Ala455Glu ABCC7 p.Ala455Glu ABCC7 p.Ala455Glu ABCC7 p.Ala455Glu ABCC7 p.Gly1244Glu ABCC7 p.Pro205Ser ABCC7 p.Arg1066Cys ABCC7 p.Leu1077Pro ABCC7 p.Val456Ala ABCC7 p.Asp806Gly ABCC7 p.Phe191Leu ABCC7 p.Gly463Asp ABCC7 p.Ser459Phe ABCC7 p.Ser459Phe ABCC7 p.Ala455Val ABCC7 p.Ala455Val Table 4 Results of sequencing of patient samples Description Prior genotype Sequencing CommentAllele 1 Allele 2 Confirmed CF wt/delta F508 delta F508 P205S Known mutation Confirmed CF wt/3849 ϩ 10 kb 3849 ϩ 10 kb L1077P Known mutation C 3 T C 3 T Confirmed CF wt/delta F508 delta F508 R1066C Known mutation Confirmed CF wt/delta F508 delta F508 D806G Novel missense Confirmed CF wt/wt 3154delG 3154delG Both parents confirmed carriers Confirmed CF delta F508/wt delta F508 G1244E Known mutation Confirmed CF wt/wt wt F191L Novel missense Borderline sweat test wt/wt wt wt Table 5 Resolution of ambiguities on linear array assay using sequencing Linear array result Resolution Weak mutant A455E line 1508 C 3 T (S459F) polymorphism or novel mutation Weak mutant A455E line 1508 C 3 T (S459F) polymorphism or novel mutation Weak mutant A455E line wt/1496 C 3 T (A455V) polymorphism or novel mutation Weak mutant A455E line wt/1496 C 3 T (A455V) polymorphism or novel mutation Weak mutant A455E line wt/1520 G 3 A (G463D) polymorphism or novel mutation No A455E mutant or wt line Homozygous 1499 T 3 C (V456A) polymorphism or novel mutation No A455E mutant or wt line Homozygous 1497 C 3 A polymorphism (no amino acid change) Weak wt 1898 ϩ 1 G 3 A line wt/E587A novel missense mutation or polymorphism Weak 1898 ϩ 1 G 3 A line wt/1898 ϩ 1 G 3 C-different mutation; G 3 C NOT G 3 A DISCUSSION The ACMG recommended panel of CF mutations has rapidly become the standard of care for US carrier screening. Login to comment 97 ABCC7 p.Asp1270Asn ABCC7 p.Ile148Thr However, previous studies have shown that two such mutations, I148T and D1270N, are probably polymorphisms or low penetrance alleles.7 For those reasons, caution must be used when interpreting the presence of a rare or novel mutation found in the sequencing assay. Login to comment 109 ABCC7 p.Ala455Glu ABCC7 p.Ala455Glu ABCC7 p.Ala455Glu In fact, in our current assay an almost equal number of patients with positive mutant lines for the A455E mutation had sequence changes near the A455E locus as had true A455E mutations. Login to comment 127 ABCC7 p.Asp1270Asn ABCC7 p.Ile148Thr The discovery that the I148T and D1270N mutations are likely to be polymorphisms and not causative CF mutations points out the danger in assuming that all mutations listed in the CF database are causative mutations. Login to comment 132 ABCC7 p.Ile148Thr ABCC7 p.Ile148Thr A new use for single exon sequencing is the recent observation that I148T is only a CF chromosome in a haplotype that includes the 9T IVS8 polymorphism and 3199del6.11 In patients with I148T and 9T, single exon sequencing can determine whether 3199del6 is present. Login to comment