Bernard Mercier

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.

Detection of a cystic fibrosis modifier locus for meconium ileus on human chromosome 19q13

Detection of a cystic fibrosis modifier locus for meconium ileus on human chromosome 19q13

Mutations in the cationic trypsinogen gene and evidence for genetic heterogeneity in hereditary pancreatitis

Hereditary pancreatitis (HP) is a rare inherited disorder, characterised by recurrent episodes of pancreatitis often beginning in early childhood. The mode of inheritance suggests an autosomal dominant trait with incomplete penetrance. The gene, or at least one of the genes, responsible for hereditary pancreatitis has been mapped to the long arm of chromosome 7 and a missense mutation, an arginine to histidine substitution at residue 117 in the trypsinogen cationic gene (try4) has been shown to segregate with the HP phenotype. The aim of this work was to investigate the molecular basis of hereditary pancreatitis. This study was performed on 14 HP families. The five exons of the trypsinogen cationic gene were studied using a specific gene amplification assay combined with denaturing gradient gel electrophoresis (DGGE). The present paper describes three novel mutations, namely K23R and N29I and a deletion –28delTCC in the promoter region. We also found a polymorphism in exon 4, D162D. In eight of these families we found a mutation which segregates with the disease. A segregation analysis using microsatellite markers carried out on the other families suggests genetic heterogeneity in at least one of them. Our findings confirm the implication of the cationic trypsinogen gene in HP and highlight allelic diversity associated with this phenotype. We also show that the pattern of inheritance of HP is probably complex and that other genes may be involved in this genetic disease.

Mutational analysis of the human pancreatic secretory trypsin inhibitor (PSTI) gene in hereditary and sporadic chronic pancreatitis

Editor—Hereditary pancreatitis (HP) is an autosomal dominant disease with about 80% penetrance that mainly afflicts white families.1 Although pancreatitis was hypothesised to result from inappropriate activation of pancreatic zymogens by Chiara2 in 1896, and the genetic nature of HP was identified by Comfort et al 3 in 1952, the precise mechanism underlying the pathogenesis of HP has remained a mystery until recently. By familial linkage analysis, a genetic defect was mapped to chromosome 7q35 by Le Bodic et al ,4 and independently confirmed by two other groups in 1996.5 6 Soon after, a single G to A mutation resulting in an arginine (R) to histidine (H) substitution (R117H) in the third exon of the cationic trypsinogen gene was identified as being associated with HP by Whitcomb et al .7 Trypsinogen is synthesised in the acinar cells of the pancreas and is activated into trypsin upon cleavage of the activation peptide by enterokinase. Trypsin plays a central role in pancreatic exocrine physiology by acting as the trigger enzyme which leads to the activation of all the pancreatic digestive proenzymes as well as trypsinogen itself. When the R117H mutation was identified, Whitcomb et al 7 concluded that this mutation did not affect the tertiary structure of trypsin, nor alter its catalytic activity or interfere with trypsin inhibitor binding, since the three dimensional position of R117 was located on the opposite surface of the trypsin molecule to the catalytic and trypsin inhibitor binding sites. They hypothesised instead that the R117H mutation eliminated a “fail safe” mechanism for the inactivation of trypsin by abolishing an important autolytic site. Thus, the stabilised mutant enzyme would disrupt the trypsin activation/inhibition balance and trigger the pancreatic autodigestion process which results in pancreatitis under certain conditions. This model coincided with Chiaras …

Neonatal screening for cystic fibrosis in Brittany, France: assessment of 10 years' experience and impact on prenatal diagnosis

BACKGROUND Neonatal screening for cystic fibrosis has been a subject of debate over the past few years. This study assesses 10 years of neonatal screening in Brittany, France, and examines its impact on prenatal screening of subsequent pregnancies in couples with an affected child. METHODS The study included all the neonates screened for cystic fibrosis in Brittany from Jan 1, 1989, to Dec 31, 1998. The screening consisted of an immunoreactive trypsinogen assay from dried blood spots, plus, from 1993, mutation analysis. Data were collected on incidence of cystic fibrosis, and genotypic and biochemical characteristics. The use of prenatal screening of subsequent pregnancies in affected families was also investigated. FINDINGS Of the 343,756 neonates screened, 118 children with cystic fibrosis were identified, giving an incidence of one in 2913. All mutated alleles were characterised: 34 different mutations resulting in 36 genotypes were detected. The introduction of DNA analysis into the protocol greatly reduced the recall rate and increased the sensitivity of the test. The mean cost of the screening programme was US

Family history as a risk factor for venous thromboembolism

2.32 per screened child. 39 (34%) of the families identified by neonatal screening opted for subsequent prenatal diagnosis at least once. 12 couples would have benefited from this procedure while their first child was still symptom-free. 42 healthy children were born, and 18 pregnancies were terminated (therapeutic abortion rate of 100%). INTERPRETATION We have shown the feasibility of neonatal screening for cystic fibrosis in Brittany. Through the detection of a large range of mutations, neonatal screening provides the opportunity for more reliable prenatal diagnosis and cascade screening. The neonatal screening programme described here could provide a good model for other countries intending to initiate such a scheme.

Sensitivity of the denaturing gradient gel electrophoresis technique in detection of known mutations and novel Asian mutations in the CFTR gene

INTRODUCTION There are very few data assessing a family history of venous thromboembolism (VTE) as a risk factor for VTE. This question is nonetheless of interest as inherited risk factors are involved but at least partly unknown. METHODS The E.D.I.TH. study is a prospective hospital-based case-control study. The family history was assessed by using a standard questionnaire, considering the total number of the first-degree relatives and the number of these relatives who had suffered from VTE. We analysed 698 first VTE cases and their matched controls, 507 pairs without and 191 pairs with a major acquired risk factor (active malignancy, surgery or plaster cast in the past three months, pregnancy or delivery in the past three months). RESULTS A family history of VTE was associated with VTE occurrence, irrespective of carrying or not factor V Leiden mutation or G20210A prothrombin gene mutation and irrespective of the presence or absence of major acquired risk factors; adjusted conditional odds ratio: 2.7 (95%CI, 1.8-3.8). CONCLUSION A family history might well be considered when estimating type and duration of prophylaxis for VTE specifically in patients with active cancer or who experienced surgery. Family history of VTE could be added to a prior VTE history to define a concept of clinical thrombophilia which is not necessarily related to carrying a known inherited risk factor.

Simultaneous screening for HBV DNA and HCV RNA genomes in blood donations using a novel TaqMan PCR assay.

More than 500 mutations have been identified in the CFTR gene, making it an excellent system for testing mutation scanning techniques. To assess the sensitivity of denaturing gradient gel electrophoresis (DGGE), we collected a representative group of 202 CFTR mutations. All mutations analyzed were detected by scanning methods other than the DGGE approach evaluated in this study. DGGE analysis was performed on 24 of the 27 exons and their flanking splice site sequences. After optimization, 201 of the 202 control samples produced an altered migration pattern in the region in which an alteration occurred. The remaining sample was sequenced and found not to have the reported mutation. The ability of DGGE to identify novel mutations was evaluated in three Asian CF patients with four unknown CF alleles. Three novel Asian mutations were detected—K166E, L568X, and 3121‐2 A→G (in homozygosity)—accounting for all CF alleles. These results indicate that an optimized DGGE scanning strategy is highly sensitive and specific and can detect 100% of mutations. Hum Mutat 9:136–147, l997.

Identification of 12 novel mutations in the CFTR gene

The risk of contracting hepatitis from blood transfusions is estimated to be 1 in 63000 in the case of hepatitis B virus (HBV) and 1 in 103000 in the case of hepatitis C virus (HCV). In some countries (Germany, USA and England, for example), molecular protocols are evaluated to detect viral genomes in blood donations in order to reduce the seroconversion period. However, no such method is available currently to screen large series samples for HBV and HCV. While strategies involving the pooling of plasma samples have been proposed and tested in Germany, there is the question of sensitivity. We developed a novel approach to screen for HBV and HCV based on the TaqMan technology that allows for the quantification of an amplified fragment during PCR analysis (Lee et al., 1993). This approach is more sensitive than other quantification methods. As a first step primers and probes were designed to detect the different sub-types of HBV and HCV genomes. We then optimized the reaction conditions in order to screen for the two viruses at the same time. The observed sensitivity is less than 50 molecules per ml for HBV and less than 50 molecules per ml for HCV. This assay is, to our knowledge, the first that allows the simultaneous detection of DNA and RNA viral genomes. In conclusion, this TaqMan approach could be used as a single test to screen for HBV and HCV genomes in a series of 96 samples in less than 5 h. Such an approach is a first step for development of automation allowing a systematic screening of blood donations.

Seroprevalence of human herpes virus 8 antibody in populations at high or low risk of transfusion, graft, or sexual transmission of viruses

Over 200 mutations, besides the deletion delta F508, have been identified in the CFTR gene and are known to cause CF. In order to characterize the molecular defects of non delta F508 CF chromosomes of various French origin, we have combined the techniques of denaturing gradient gel electrophoresis (DGGE) and direct sequencing to screen for mutations in the whole coding sequence of the CFTR gene corresponding to the 27 exons and their exon-intron boundaries. This approach enabled us to identify 12 novel mutations which are described here. We have systematically tested a large number of other nucleotide changes distributed in the 27 exons, each of them was clearly detected. These data support the notion that the DGGE conditions we have defined for screening coding sequence of the CFTR gene allows the identification of most of, if not all, the CFTR gene mutations.