Non-invasive prenatal diagnosis (NIPD) of cystic fibrosis: an optimized protocol using MEMO fluorescent PCR to detect the p.Phe508del mutation.

BACKGROUND: Analysis of cell-free foetal DNA (cff-DNA) in maternal plasma is very promising for early diagnosis of monogenic diseases; in particular, cystic fibrosis (CF). However, NIPD of single-gene disorders has been limited by the availability of suitable technical platforms and the need to set up patient or disease-specific custom-made approaches. METHODS: To make research applications more readily accessible to the clinic, we offer a simple assay combining two independent methods to determine the presence or absence of paternally inherited foetal allele p.Phe508del (the most frequent mutation in CF patients worldwide). The first method detects the presence or absence of a p.Phe508del allele by Mutant Enrichment with 3'-Modified Oligonucleotide PCR coupled to Fragment Length Analysis (MEMO-PCR-FLA). The second method detects the p.Phe508del allele with classical Multiplex Fluorescent PCR including five intragenic and extragenic STR markers of the CFTR locus and a specific SRY sequence. RESULTS: We collected 24 plasma samples from 23 women carrying foetuses at risk for CF and tested each sample using both methods. Our new procedures were successfully applied to 10 couples where fathers carried the p.Phe508del mutation and mothers were carrying a different mutation in the CFTR gene. These simple tests provided clear positive or negative results from the maternal plasma of the pregnant women. We confirmed the presence of cff-DNA in the studied samples by the identification of a tri-allelic DNA profile using a miniSTR kit. All results were correlated with chorionic villus sampling or amniocentesis analyses. CONCLUSIONS: This NIPD approach, easily set up in any clinical laboratory where prenatal diagnosis is routinely performed, offers many advantages over current methods: it is simple, rapid, and cost-effective. It opens up the possibility for testing a large number of couples with offspring at risk for CF.

[Maternal serum screening for Down's syndrome: a user's club experience]. / Dépistage du risque de trisomie 21 foetale par les marqueurs sériques maternels: expérience d'un cercle d'utilisateurs.

Screening of Down syndrome using maternal serum markers requires specific quality management. We report here a user's club experience (Down's Club Abbott) in surveying performances of AxSYM total beta hCG and AFP reagents combined to Maciel software. Regular analysis was carried out during three years (1998 to 2000). Values from five laboratories were collected to achieve calculation of medians for each parameter, multiple of the medians and initial positive rate (cut-off of 1/250 established in France). The large size of the observed population (3,1020 women during the studied period) increased the performances of statistical evaluations. In July 1999, five months after a change in reagents leading to new medians, these latter were recalculated in agreement with the manufacturer. Our experience exhibits that only the concomitant analysis of all parameters (medians, multiple of medians and initial positive rate) can allow the monitoring of a potential drift (bound or not to reagents). Moreover, such user's clubs enhance the individual quality management of the laboratories and allow a good follow-up of the performances of the testing in time.

[Value of a genetic evaluation before an intracytoplasmic sperm injection procedure]. / Intérêt d'une évaluation génétique lors d'une procédure d'ICSI.

Intracytoplasmic sperm injection (ICSI) still needs to be validated as far as safety is concerned. Results concerning foetal karyotypes currently seem to show an increase in sexual chromosomal abnormalities. In view of the heterogeneity of publications concerning genetic tests, it seems necessary to define a protocol with a limited time span, which will allow the extensive collection of data. The analysis of the results will secondarily allow a common procedure to be established. We propose a protocol limited in time, aiming to evaluate the chromosomal status at birth by a systematic karyotype of umbilical cord blood.

Localization of rDNA transcription sites in nucleoli of human Sertoli cells: an EM quantitative autoradiographic study using 3H-uridine.

The sites of rDNA transcription within human Sertoli cell nucleoli have been localized using EM autoradiography after a 45-min pulse of 3H-uridine and an exposure time of 6 months. Two successive quantitative image analyses, one derived from the 50% probability circle method and the other from the cross-fire method, allowed us to estimate the radioactivity incorporated within each nucleolus compartment. This study demonstrated that rDNA transcription occurred mainly at the border between fibrillar centers and dense fibrillar components and to a lesser extent within the dense fibrillar component. The other Sertoli cell nucleoli compartments did not incorporate 3H-uridine and therefore were not involved in rDNA transcription.