Marie Desgeorges

Analysis of the whole CFTR coding regions and splice junctions in azoospermic men with congenital bilateral aplasia of epididymis or vas deferens

Several recent studies have demonstrated the presence of mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene in healthy males with infertility caused by congenital absence of the vas deferens (CBAVD), previously recognized as an idiopathic genetic condition distinct from CF. In order to document further the genetic commonality of these two disorders, we undertook a double screening of the entire coding and flanking sequences of the CFTR gene, by using single-strand conformational polymorphism analysis and denaturing gradient gel electrophoresis in 12 unrelated infertile men with abnormalities of the vas deferens and/or epididymis. This strategy allowed us to identify 11 DNA sequence alterations considered as CF-causing mutations and several variations. Despite this double analysis, only two patients out of eight with CBAVD could be demonstrated as compound heterozygotes for CF mutations.

Linkage disequilibrium between the M470V variant and the IVS8 polyT alleles of the CFTR gene in CBAVD.

Congenital bilateral absence of the vas deferens (CBAVD) is a cause of male sterility mostly resulting from mutations in the cystic fibrosis transmembrane regulator (CFTR) gene. The most common defect is the 5T variant at the branch/acceptor site of intron 8, which induces high levels of exon 9 skipping leading to non-functional protein. However, this 5T variant has incomplete penetrance and variable expressivity, suggesting that some other regulatory factors may modulate the splicing of exon 9. To identify such factors, we report here the genetic analysis of a polymorphic locus, M470V, located in exon 10 of the CFTR gene in 60 patients with CBAVD, compared to a normal control population. The statistical analysis showed strong linkage disequilibrium between the 5T allele and the V allele of the M470V polymorphism in the CBAVD population, but not in the normal population. The V allele in a gene carrying 5T could, however, contribute to lowering the level of normal transcripts, as already suggested by in vitro transcriptional studies. These genetic findings, together with previous studies, suggest involvement of the M470V variant in the modulation of the splicing of exon 9 of the CFTR gene.

Comprehensive CFTR gene analysis of the French cystic fibrosis screened newborn cohort: implications for diagnosis, genetic counseling, and mutation-specific therapy

Purpose:Newborn screening (NBS) for cystic fibrosis (CF) was implemented throughout France in 2002. It involves a four-tiered procedure: immunoreactive trypsin (IRT)/DNA/IRT/sweat test was implemented throughout France in 2002. The aim of this study was to assess the performance of molecular CFTR gene analysis from the French NBS cohort, to evaluate CF incidence, mutation detection rate, and allelic heterogeneity.Methods:During the 8-year period, 5,947,148 newborns were screened for cystic fibrosis. The data were collected by the Association Française pour le Dépistage et la Prévention des Handicaps de l’Enfant. The mutations identified were classified into four groups based on their potential for causing disease, and a diagnostic algorithm was proposed.Results:Combining the genetic and sweat test results, 1,160 neonates were diagnosed as having cystic fibrosis. The corresponding incidence, including both the meconium ileus (MI) and false-negative cases, was calculated at 1 in 4,726 live births. The CF30 kit, completed with a comprehensive CFTR gene analysis, provides an excellent detection rate of 99.77% for the mutated alleles, enabling the identification of a complete genotype in 99.55% of affected neonates. With more than 200 different mutations characterized, we confirmed the French allelic heterogeneity.Conclusion:The very good sensitivity, specificity, and positive predictive value obtained suggest that the four-tiered IRT/DNA/IRT/sweat test procedure may provide an effective strategy for newborn screening for cystic fibrosis.Genet Med 17 2, 108–116.

Germinal mosaicism from grand-paternal origin in a family with Duchenne muscular dystrophy.

SummaryWe have identified a Duchenne muscular dystrophy (DMD) pedigree with an unexpected pattern of inheritance. Using marker restriction fragment length polymorphisms detected by probes that lie within and outside the DMD gene, we could demonstrate that the maternal grandfather has transmitted two distinct types of X chromosomes to his offspring. This original observation may be explained by postulating that the DMD mutation must have occurred during mitosis in early germline proliferation, leading to a germline mosaicism within this male ancestor.

Analysis of 30 known cystic fibrosis mutations: 10 mutations account for 27% of non-ΔF508 chromosomes in Southern France

We have analyzed 131 unrelated families from Southern France for 29 known cystic fibrosis (CF) mutations identified in 8 exons of the cystic fibrosis transmembrane regulator gene. All these mutations were detected by amplification of DNA by the polymerase chain reaction (PCR) followed by restriction enzyme digestion or hybridization with allele specific oligoprobes. The most frequent mutations after the ΔF508 deletion (frequency: 63%) were G542X (5.3%), AI507 (1.1%), and N1303K (0.76%). Seven other mutations (621 + 1G → T, Y122X, R347P, R334W, S549N, G551D, R1162X) were each identified in only one CF chromosome. Apart from G542X, most of the other mutations identified in this study were found to be associated with 7-(GATT)-repeats allele of IVS6A. In Southern France, only 73% of CF chromosomes could be identified by the analysis of 30 mutations.

Non-invasive prenatal diagnosis of monogenic disorders: an optimized protocol using MEMO qPCR with miniSTR as internal control

Abstract Background: Analysis of circulating cell-free fetal DNA (cffDNA) in maternal plasma is very promising for early diagnosis of monogenic diseases. However, this approach is not yet available for routine use and remains technically challenging because of the low concentration of cffDNA, which is swamped by the overwhelming maternal DNA. Methods: To make clinical applications more readily accessible, we propose a new approach based on mutant enrichment with 3′-modified oligonucleotides (MEMO) PCR along with real-time PCR to selectively amplify from the maternal blood the paternally inherited fetal allele that is not present in the maternal genome. Results: The first proof of concept of this strategy was displayed for cystic fibrosis by the accuracy of our detection of the p.Gly542* mutation used as the initial developmental model. Subsequently, a retrospective study of plasmas originating from two pregnant women carrying a fetus with private mutation confirmed the effectiveness of our method. We confirmed the presence of cffDNA in the studied samples by the identification of a tri-allelic DNA profile using a miniSTR kit. Conclusions: This new non-invasive prenatal diagnosis test offers numerous advantages over current methods: it is simple, cost effective, time efficient and does not require complex equipment or bioinformatics settings. Moreover, our assays for different private mutations demonstrate the viability of this approach in clinical settings for monogenic disorders.

Cystic fibrosis typing with DNA probes and screening for ΔF508 deletion in families from Southern France

SummaryA sample of 235 individuals from 49 French cystic fibrosis (CF) families with at least one living affected child was typed with probes for restriction fragment length polymorphisms (RFLPs) known to be linked to the CF gene, and was screened for the ΔF508 mutation. Using a combination of six probes, 44 out of the 49 families were sufficiently informative to enable prenatal diagnosis or carrier determination. As in many other populations, linkage disequilibrium was found between the CF locus and the haplotype B (XV2c: allele 1; KM19: allele 2), which accounts for about 78% of CF chromosomes in our families. The ΔF508 deletion was present in 64.3% of CF chromosomes.