Mutation analysis for heterozygote detection and the prenatal diagnosis of cystic fibrosis

The cystic fibrosis gene was recently cloned, and a three-base deletion removing phenylalanine 508 from the coding region was identified as the mutation on the majority of cystic fibrosis chromosomes. We used the polymerase chain reaction and hybridization with allele-specific oligonucleotides to analyze the presence or absence of this mutation on 439 cystic fibrosis chromosomes and 433 normal chromosomes from non-Ashkenazic white families. This mutation was present on 75.8 percent of the cystic fibrosis chromosomes. Using the DNA markers XV-2c and KM-19, we found that 96 percent of cystic fibrosis chromosomes with the mutation had a single DNA haplotype that occurs frequently with cystic fibrosis chromosomes. This haplotype was also found on 54 percent of the cystic fibrosis chromosomes without the three-base deletion. The three-base deletion was found on only 30.3 percent of cystic fibrosis chromosomes from Ashkenazic families, although the common cystic fibrosis haplotype was present on 97 percent of cystic fibrosis chromosomes from Ashkenazic families. The ability to detect the common mutation causing cystic fibrosis represents a major improvement in prenatal diagnosis and heterozygote detection, particularly in families in which no DNA sample is available from the affected child, and provides an improved method of testing for spouses of carriers of cystic fibrosis. Mutation analysis introduces the possibility of population-based screening programs for carriers, which on the basis of the sample in this study, would currently identify about 57 percent of the non-Ashkenazic white couples at risk.published_or_final_versio

Molecular cloning and sequence analysis of the murine cDNA for the cystic fibrosis transmembrane conductance regulator

We have cloned the mouse homolog of the human cystic fibrosis transmembrane conductance regulator (CFTR) using clones isolated from a mouse lung cDNA library and using amplification of cDNA to isolate specific regions. The cDNA was 6304 bp in length and encoded a polypeptide of 1476 amino acids. Comparison of the deduced amino acid sequence showed that the mouse protein has high homology to the human protein; overall identity was 78.3%. The amino acid identity was high for both transmembrane domains (first transmembrane domain, 86.7%; second transmembrane domain, 81.1%) and for both ATP-binding folds (first ATP-binding fold, 80.5%; second ATP-binding fold, 83.9%), suggesting the functional importance of these regions. On the other hand, the R domain was less well conserved (68.9% identity). All of the published missense mutation sites and the site of the common ΔF508 mutation were conserved between human and mouse.link_to_subscribed_fulltex

A cluster of cystic fibrosis mutations in the first nucleotide-binding fold of the cystic fibrosis conductance regulator protein

The gene responsible for cystic fibrosis (CF) has recently been identified and is predicted to encode a protein of 1,480 amino acids called the CF transmembrane conductance regulator (CFTR). Several functional regions are thought to exist in the CFTR protein, including two areas for ATP-binding, termed nucleotide-binding folds (NBFs), a regulatory (R) region that has many possible sites for phosphorylation by protein kinases A and C, and two hydrophobic regions that probably interact with cell membranes. The most common CF gene mutation leads to omission of phenylalanine residue 508 in the putative first NBF, indicating that this region is functionally important. To determine whether other mutations occur in the NBFs of CFTR, we determined the nucleotide sequences of exons 9, 10, 11 and 12 (encoding the first NBF) and exons 20, 21 and 22 (encoding most of the second NBF) from 20 Caucasian and 18 American-black CF patients. One cluster of four mutations was discovered in a 30-base-pair region of exon 11. Three of these mutations cause amino-acid substitutions at residues that are highly conserved among the CFTR protein, the multiple-drug-resistance proteins and ATP-binding membrane-associated transport proteins. The fourth mutation creates a premature termination signal. These mutations reveal a functionally important region in the CFTR protein and provide further evidence that CFTR is a member of the family of ATP-dependent transport proteins.link_to_subscribed_fulltex