[Functional characterization of naturally occurring CFTR mutants: interest for cystic fibrosis]. / Caractérisation fonctionnelle de mutants CFTR naturels: intérêt pour la mucoviscidose.

Cystic fibrosis is caused by mutations in the CFTR gene (Cystic Fibrosis Transmembrane conductance Regulator) encodes a protein mainly functioning as a chloride channel that regulates chloride and sodium transport in secretory epithelial cells. The past several years with advances in DNA analysis have seen an increased knowledge of the mutational spectrum for cystic fibrosis. The functional characterization of some naturally occurring CFTR mutants led to classification of mutations according to the mechanisms by which they disrupt CFTR function. This article reports the strategies that are currently available to evaluate the deleterious consequences of naturally occurring CFTR mutants. A number of in silico tools and molecular approaches are presented. The accurate knowledge of CFTR mutations causing or non-causing disease present obvious interest in both clinical diagnosis and research fields. A better understanding of the molecular defects associated with various CFTR mutations will provide a basis for development of novel pharmacologic compounds intended to correct transcriptional abnormalities or improve protein processing/trafficking. This is illustrated by the functional characterization of the first sequence variation identified in the CFTR minimal promoter, which allowed to address in original way the important and poorly understood regulation of expression of the CFTR gene.

Heterotaxia syndrome and autosomal dominant inheritance.

Previous familial cases of recurrent heterotaxia have suggested an autosomal recessive or exceptionally X-linked or dominant inheritance. Here, we report six families including 18 affected members, consistent with autosomal dominant inheritance. Among these, four families have more than one case of heterotaxia. The other two families have one member with heterotaxia and at least one other affected member with an "isolated" heart malformation, which could be considered as a mild form of heterotaxia. In five families, the disorder is transmitted through two or three generations. In one family, the patients are of the same generation but are linked to each other by obligate carriers. We suggest a rule to classify these families with heart malformations, according to the etiologic factor involved (rule of precocity). This rule might be useful to other disruptions of morphogenetic processes.

DNA analysis of haemophilia A families from southern France. Experience of a hospital laboratory.

A sample of 101 individuals from 19 unrelated families from Southern France affected with haemophilia A was studied in our laboratory from 1990 to 1992. The aim of the analysis was to define the carrier status of women related to a haemophiliac, or to find an informative DNA marker for further prenatal diagnosis in obligate carriers. Three intragenic (BclT/intron 18, XbaI/intron 22, AlwNI/intron x7) and two extragenic polymorphisms (TaqI/St14, BglII/DX13) were used for this study. The tested population exhibited some original characteristics, including a lower rate of heterozygosity for the FVIII BclI polymorphism and a number of specificities for the St14 RFLP. We also compared the different methodologies available for each RFLP in a routine diagnostic service, and determined a strategy for linkage analysis in our population.

First functional polymorphism in CFTR promoter that results in decreased transcriptional activity and Sp1/USF binding.

Growing evidences show that functionally relevant polymorphisms in various promoters alter both transcriptional activity and affinities of existing protein-DNA interactions, and thus influence disease progression in humans. We previously reported the -94G>T CFTR promoter variant in a female CF patient in whom any known disease-causing mutation has been detected. To investigate whether the -94G>T could be a regulatory variant, we have proceeded to in silico analyses and functional studies including EMSA and reporter gene assays. Our data indicate that the promoter variant decreases basal CFTR transcriptional activity in different epithelial cells and alters binding affinities of both Sp1 and USF nuclear proteins to the CFTR promoter. The present report provides evidence for the first functional polymorphism that negatively affects the CFTR transcriptional activity and demonstrates a cooperative role of Sp1 and USF transcription factors in transactivation of the CFTR gene promoter.

Transcript analysis of CFTR frameshift mutations in lymphocytes using the reverse transcription-polymerase chain reaction technique and the protein truncation test.

mRNA transcripts of the cystic fibrosis transmembrane conductance regulator (CFTR) gene were analyzed from lymphocytes of two cystic fibrosis compound heterozygotes (394delTT/3195del6 and 1215delG/ 2423delG), of five related carriers heterozygous for one of these mutations, and of five normal individuals. After reverse transcription of total RNA and amplification by the polymerase chain reaction, fragments were investigated by sequencing and by the protein truncation test (PTT). The three frameshift mutants were correctly detected by PTT, as they introduced a premature termination codon resulting in shortened protein products. The PTT approach thus provides a simple and reliable alternative method for detecting frameshift, nonsense, or splice site mutations, and for ascertaining their putative effect on the reading frame of the mRNA. In addition, we have identified 6 alternatively spliced forms of CFTR mRNA, two of which (transcripts lacking 4 + 5 or 17B) have not been described previously.

Rapid detection of single nucleotide deletions: application to the beta 6 (-A) mutation of the beta-globin gene and to cystic fibrosis.

The formation of heteroduplexes from the amplified products of homologous alleles has been shown to be useful in the identification of heterozygotes carrying deletion or insertion mutations. Here, we describe an improved procedure that allows the detection of single base pair (bp) deletions on nondenaturing polyacrylamide gels. Carriers for a common Mediterranean beta-thalassemic mutation, beta6 (-A), could be easily detected by use of this method, as could carriers of a 1-bp deletion in the cystic fibrosis gene.

A naturally occurring sequence variation that creates a YY1 element is associated with increased cystic fibrosis transmembrane conductance regulator gene expression.

We have identified previously a novel complex mutant allele in the cystic fibrosis transmembrane conductance regulator (CFTR) gene in a patient affected with cystic fibrosis (CF). This allele contained a mutation in CFTR exon 11 known to cause CF (S549R(T>G)), associated with the first alteration described so far in the minimal CFTR promoter region (-102T>A). Studies on genotype-phenotype correlations revealed striking differences between patients carrying mutation (S549R(T>G)) alone, who had a severe disease, and patients carrying the complex allele (-102(T>A)+S549R(T>G)), who exhibited milder forms of CF. We thus postulated that the sequence change (-102T>A) may attenuate the effects of the severe (S549R(T>G)) mutation through regulation of CFTR expression. Analysis of transiently transfected cell lines with wild-type and -102A variant human CFTR-directed luciferase reporter genes demonstrates that constructs containing the -102A variant (which creates a Yin Yang 1 (YY1) core element) increases CFTR expression significantly. Electrophoretic mobility shift assays indicate that the -102 site is located in a region of multiple DNA-protein interactions and that the -102A allele recruits specifically an additional nuclear protein related to YY1. The finding that the YY1-binding allele causes a significant increase in CFTR expression in vitro may allow a better understanding of the milder phenotype observed in patients who carry a severe CF mutation within the same gene.

Factor IX gene mutations causing haemophilia B: comparison of SSC screening versus systematic DNA sequencing and diagnostic applications.

The search for mutations of the factor IX gene responsible for haemophilia B should nowadays be used routinely for the molecular diagnosis of this inherited disorder, i.e. carrier detection and prenatal diagnosis. A number of methodologies have been proposed, most of them being delicate or expensive. We have used a simple strategy based on a preliminary screening of eight factor IX gene fragments by single-strand conformation analysis (SSCA), followed by direct sequencing of fragments displaying an abnormal migration pattern. Carrier testing is then performed by use of an enzyme restriction site altered by the mutation or by the SSCA itself. By using this strategy we were able readily to identify the factor IX molecular defect of nine unrelated haemophilia B patients from southern France. We validated the efficiency and reliability of the SSC-based detection of mutations by sequencing all the polymerase chain reaction (PCR) fragments studied in the haemophilic patients. No other sequence alteration could be found except the one detected by SSC analysis. We conclude that this method can be advantageously used for diagnosis purposes in a routine laboratory involved in haemophilia B diagnosis and report nine previously undescribed haemophilia B families with their factor IX mutation.

Direct carrier testing of haemophilia B by SSCP.

Haemophilia B is due to multiple molecular defects in the factor IX gene. Most of them are single base substitutions, and can now be identified by direct sequencing of the coding sequence of the factor IX gene, preceded or not by a screening strategy. In some instances the mutation alters an enzyme recognition site and this allows rapid and accurate carrier testing and prenatal diagnosis in the affected pedigree. This was not the case for the previously described nt 31119 (G-->A) mutation that we found in an extended haemophilia B pedigree, during the search for mutations in the factor IX gene in patients from Southern France. We first detected this mutation by single stranded conformation polymorphism (SSCP) and then identified it by DNA sequencing. Carriership could be easily determined in the females of the pedigree by analysis of the SSCP patterns. Our results indicate that the SSCP analysis of amplified genomic DNA fragments can be successfully used as a diagnosis approach for direct carrier testing and prenatal diagnosis.

Mutations in the cystic fibrosis gene in patients with congenital absence of the vas deferens.

BACKGROUND: Congenital bilateral absence of the vas deferens (CBAVD) is a form of male infertility in which mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene have been identified. The molecular basis of CBAVD is not completely understood. Although patients with cystic fibrosis have mutations in both copies of the CFTR gene, most patients with CBAVD have mutations in only one copy of the gene. METHODS: To investigate CBAVD at the molecular level, we have characterized the mutations in the CFTR gene in 102 patients with this condition. None had clinical manifestations of cystic fibrosis. We also analyzed a DNA variant (the 5T allele) in a noncoding region of CFTR that causes reduced levels of the normal CFTR protein. Parents of patients with cystic fibrosis, patients with types of infertility other than CBAVD, and normal subjects were studied as controls. RESULTS: Nineteen of the 102 patients with CBAVD had mutations in both copies of the CFTR gene, and none of them had the 5T allele. Fifty-four patients had a mutation in one copy of CFTR, and 34 of them (63 percent) had the 5T allele in the other CFTR gene. In 29 patients no CFTR mutations were found, but 7 of them (24 percent) had the 5T allele. In contrast, the frequency of this allele in the general population was about 5 percent. CONCLUSIONS: Most patients with CBAVD have mutations in the CFTR gene. The combination of the 5T allele in one copy of the CFTR gene with a cystic fibrosis mutation in the other copy is the most common cause of CBAVD: The 5T allele mutation has a wide range of clinical presentations, occurring in patients with CBAVD or moderate forms of cystic fibrosis and in fertile men.

Complex allele [-102T>A+S549R(T>G)] is associated with milder forms of cystic fibrosis than allele S549R(T>G) alone.

We recently reported a novel complex allele in the cystic fibrosis transmembrane regulator (CFTR) gene, combining a sequence change in the minimal CFTR promoter (-102T>A) and a missense mutation in exon 11 [S549R(T>G)]. Here we compare the main clinical features of six patients with cystic fibrosis (CF) carrying the complex allele [-102T>A+S549R(T>G)] with those of 16 CF patients homozygous for mutation S549R(T>G) alone. Age at diagnosis was higher, and current age was significantly higher (P=0.0032) in the group with the complex allele, compared with the S549R/S549R group. Although the proportion of patients with lung colonization was similar in both groups, the age at onset was significantly higher in the group with the complex allele (P=0.0022). Patients with the complex allele also had significantly lower sweat test chloride values (P=0.0028) and better overall clinical scores (P=0.004). None of the 22 patients reported in this study had meconium ileus. All 16 patients homozygous for S549R(T>G), however, were pancreatic insufficient, as compared with 50% of patients carrying the complex allele (P=0.013). Moreover, the unique patient homozygous for [-102T>A+S549R(T>G)] presented with a mild disease at 34 years of age. These observations strongly suggest that the sequence change (-102T>A) in the CFTR minimal promoter could attenuate the severe clinical phenotype associated with mutation S549R(T>G).

A new approach for identifying non-pathogenic mutations. An analysis of the cystic fibrosis transmembrane regulator gene in normal individuals.

Given q as the global frequency of the alleles causing a disease, any allele with a frequency higher than q minus the cumulative frequency of the previously known disease-causing mutations (threshold) cannot be the cause of that disease. This principle was applied to the analysis of cystic fibrosis transmembrane conductance regulator (CFTR) mutations in order to decide whether they are the cause of cystic fibrosis. A total of 191 DNA samples from random individuals from Italy, France, and Spain were investigated by DGGE (denaturing gradient gel electrophoresis) analysis of all the coding and proximal non-coding regions of the gene. The mutations detected by DGGE were identified by sequencing. The sample size was sufficient to select essentially all mutations with a frequency of at least 0.01. A total of 46 mutations was detected, 20 of which were missense mutations. Four new mutations were identified: 1341+28 C/T, 2082 C/T, L1096R, and I11131V. Thirteen mutations (125 G/C, 875+40 A/G, TTGAn, IVS8-6 5T, IVS8-6 9T, 1525-61 A/G, M470V, 2694 T/G, 3061-65 C/A, 4002 A/G, 4521 G/A, IVS8 TG10, IVS8 TG12) were classified as non-CF-causing alleles on the basis of their frequency. The remaining mutations have a cumulative frequency far exceeding q; therefore, most of them cannot be CF-causing mutations. This is the first random survey capable of detecting all the polymorphisms of the coding sequence of a gene.