Preimplantation genetic diagnosis for cystic fibrosis: the Montpellier center's 10‐year experience

This study provides an overview of 10 years of experience of preimplantation genetic diagnosis (PGD) for cystic fibrosis (CF) in our center. Owing to the high allelic heterogeneity of CF transmembrane conductance regulator (CFTR) mutations in south of France, we have set up a powerful universal test based on haplotyping eight short tandem repeats (STR) markers together with the major mutation p.Phe508del. Of 142 couples requesting PGD for CF, 76 have been so far enrolled in the genetic work‐up, and 53 had 114 PGD cycles performed. Twenty‐nine cycles were canceled upon in vitro fertilization (IVF) treatment because of hyper‐ or hypostimulation. Of the remaining 85 cycles, a total of 493 embryos were biopsied and a genetic diagnosis was obtained in 463 (93.9%), of which 262 (without or with a single CF‐causing mutation) were transferable. Twenty‐eight clinical pregnancies were established, yielding a pregnancy rate per transfer of 30.8% in the group of seven couples with one member affected with CF, and 38.3% in the group of couples whose both members are carriers of a CF‐causing mutation [including six couples with congenital bilateral absence of the vas deferens (CBAVD)]. So far, 25 children were born free of CF and no misdiagnosis was recorded. Our test is applicable to 98% of couples at risk of transmitting CF.     

Improving clinical preimplantation genetic diagnosis for cystic fibrosis by duplex PCR using two polymorphic markers or one polymorphic marker in combination with the detection of the DeltaF508 mutation

Cystic fibrosis (CF) is an autosomal recessive disease characterized by obstruction and chronic infection of the respiratory tract and pancreatic insufficiency. The first preimplantation genetic diagnosis (PGD) for CF was carried out in 1992. At our centre the first cycle was performed in 1993. However, the number of known CF mutations is >1000, so developing mutation-specific PCR protocols for PGD is unfeasible. This is why a number of marker-based duplex PCRs were developed at the single cell level. A duplex PCR of a mutation and one or two microsatellites is not only a diagnostic tool, but it can also be used as a control for allele drop-out and contamination. During PGD, embryos obtained in vitro are analysed for the presence or absence of a particular genetic disease, after which only embryos shown to be free of this disease are returned to the mother. In total, 22 PGD cycles with duplex PCR (IVS8CA/IVS17BTA, DeltaF508/IVS8CA, DeltaF508/IVS17BTA and D7S486/D7S490) were carried out in 16 couples, which resulted in four ongoing pregnancies and one miscarriage.     

Genetics: Preimplantation diagnosis for X and Y normality in embryos from three Klinefelter patients

In some 47,XXY Klinefelter patients without evidence of mosaicism,testicular spermatozoa can be successfully recovered and usedfor intracytoplasmlc sperm Injection (ICSI). To ensure the replacementof embryos with a normal X and Y chromosome pattern, preimplantationdiagnosis can be performed. This paper reports on three 47,XXYKlinefelter patients from whom it was possible to retrieve testicularspermatozoa in order to perform ICSI. From their healthy wivesa total of 27 oocyte-cumulus complexes were retrieved from which22 metaphase-II oocytes were obtained and injected; 19 of thesewere intact after injection. Two distinct pronuclel were observedin eight oocytes (42.1%) 18 h after injection. On day 3 of development,five embryos (62.5%) had reached at least the 6-cell stage andwere of sufficient quality to undergo biopsy and subsequentpreimplantation diagnosis for sex chromosome analysis by fluorescentin-situ hybridization. The four embryos diagnosed as normalwere transferred to three respective patients, resulting Inone biochemical pregnancy. The remaining cells of the fifthembryo were analysed afterwards, revealing a normal X and Ychromosome constitution. So far, in the five embryos diagnosed,a normal sex chromosome pattern has been observed.     

Diagnosis and preventing inherited disease: Allelic drop-out and preferential amplification in single cells and human blastomeres: implications for preimplantation diagnosis of sex and cystic fibrosis

Previously the diagnosis of sex and cystic fibrosis status hasbeen studied on single cells using the polymerase chain reaction(PCR). It has been suggested that allelic drop-out (PCR failureof one allele) and/or preferential amplification (hypo-amplificationof one allele) may contribute to poor reliability and misdiagnosis,although this remains controversial as some reports suggestthat allelic drop-out does not occur. We investigated an improvedmethod of diagnosing sex and cystic fibrosis in single cellsusing a new technology (fluorescent PCR) to determine the baselevel of PCR artefacts (allelic drop-out and preferential amplification)which, in combination with improved sensitivity, should improvePCR reliability and accuracy. Fluorescent PCR gives high reliability(97%) and accuracy rates (97%) in somatic cells for both sexand cystic fibrosis diagnosis and its lower detection thresholdallows allelic drop-out and preferential amplification to beeasily distinguished. We also achieved high reliability andaccuracy in diagnosing cystic fibrosis in human blastomeres.This study confirms earlier reports of both allelic drop-outand preferential amplification in single cell analysis. We demonstratethat both allelic drop-out and preferential amplification occurin somatic cells and suggest these are separate phenomena. Preferentialamplification appeared common in single cell PCR while allelicdropout apparently occurred at random in each allele. Preferentialamplification was mainly amplification of the larger allele.We suggest that some inaccuracy/misdiagnosis may be due to bothpreferential amplification as well as allelic drop-out. Otherfindings were variability in drop-out between PCR and that amplificationof signals from human blastomeres may be linked to embryo quality.We suggest that allelic drop-out is dependent on the numberof cells within the sample.     

Biosensor technology for real‐time detection of the cystic fibrosis W1282X mutation in CFTR

In the present paper, biospecific interaction analysis (BIA) was performed using surface plasmon resonance (SPR) and biosensor technologies to detect the Trp1282Ter mutation (W1282X) of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene. We first immobilized on a SA5 sensor chip a single‐stranded biotinylated oligonucleotide containing the sequence involved in this mutation, and the efficiency of hybridization of oligonucleotide probes differing in length was determined. Second, we immobilized on different SA5 sensor chips biotinylated polymerase‐chain reaction (PCR) products from a normal subject as well as from heterozygous and homozygous W1282X samples. The results obtained show that both allele‐specific 10‐ and 12‐mer oligonucleotides are suitable probes to detect W1282X mutations of the cystic fibrosis gene under standard BIA experimental conditions. During the association phase performed at 25°C, discrimination between mismatched and full matched hybrids was readily and reproducibly observed by using the 10‐mer W1282X probes. By contrast, when the 12‐mer DNA probes were employed, discrimination between mismatched and full matched hybrids was observed during the dissociation phase. Taken together, the results presented suggest that BIA is an easy, speedy, and automatable approach to detect point mutations leading to cystic fibrosis. By this procedure, it is possible to perform real‐time monitoring of hybridization between target single stranded PCR products obtained by using as substrates DNA isolated from normal or heterozygous subjects, and homozygous W1282X CF samples and oligonucleotide probes, therefore enabling a one‐step, non‐radioactive protocol to perform diagnosis. Hum Mutat 18:70–81, 2001. © 2001 Wiley‐Liss, Inc.     

The experience of 3 years of external quality assessment of preimplantation genetic diagnosis for cystic fibrosis

Preimplantation genetic diagnosis (PGD) was first performed over 20 years ago and has become an accepted part of genetic testing and assisted reproduction worldwide. The techniques and protocols necessary to carry out genetic testing at the single-cell level can be difficult to master and have been developed independently by the laboratories worldwide offering preimplantation testing. These factors indicated the need for an external quality assessment (EQA) scheme for monogenic disease PGD. Toward this end, the European Society for Human Reproduction and Embryology came together with United Kingdom National External Quality Assessment Services for Molecular Genetics, to create a pilot EQA scheme followed by practical EQA schemes for all interested parties. Here, we detail the development of the pilot scheme as well as development and findings from the practical (clinical) schemes that have followed. Results were generally acceptable and there was marked improvement in results and laboratory scores for those labs that participated in multiple schemes. Data from the first three schemes indicate that the EQA scheme is working as planned and has helped laboratories improve their techniques and result reporting. The EQA scheme for monogenic PGD will continue to be developed to offer assessment for other monogenic disorders.     

Detection of the cystic fibrosis delta-F508 mutation at autopsy by site-directed mutagenesis

In Causasian populations cystic fibrosis (CF) is the most common autosomal recessive disorder. CF was previously considered rare in Mexico; however, the reported frequency is about 1% in autopsies. This discrepancy appears to be due to the inability to diagnose the illness during life. It is now known that in developing countries a great number of affected children die without the benefit of CF diagnosis and appropriate treatment. In this study we have used the PCR-mediated sitedirected mutagenesis technique for the detection of the delta-F508 mutation in a 6-month old Mexican boy who died without definitive diagnosis. The tissue available from the child was a formaldehyde fixed paraffin-embedded liver. We identified the delta-F508 mutation in homozygous form in the propositus and in a heterozygous form in his parents. This represents the first report of CF molecular diagnosis in Mexico. © 1993 Wiley-Liss, Inc.     

Endocrine and exocrine pancreatic function and the ΔF508 mutation in cystic fibrosis

The relationship between the cystic fibrosis (CF) genotype and endocrine and exocrine pancreatic function was studied in 215 CF patients. In the 211 patients with the delta F508 mutation, endocrine pancreatic function (oral glucose tolerance; WHO criteria) was normal in 72.5%, impaired in 12.3%, and diabetic in 15.2% of the patients, with no difference between CF patients homozygous (N = 163, median age 15 years, range 2-40) or heterozygous (N = 48, 18 years, 3-40; age difference not significant) for the delta F508 mutation. Exocrine pancreatic sufficiency (no need for pancreatic enzyme substitution) was found in 0.6% of the patients homozygous for the delta F508 mutation and in 10.4% of the heterozygotes (p less than 0.01). Homozygous patients with pancreatic insufficiency took more pancreatic enzyme capsules (median 42 per day, range 0-192) than the heterozygotes (29 per day, 0-300; p less than 0.001). The four patients (1.9%) without the delta F508 mutation had normal glucose tolerance but exocrine pancreatic insufficiency. In conclusion, the major mutation genotype in CF (delta F508) affects the severity of the exocrine pancreatic insufficiency, whereas endocrine pancreatic function is unrelated to this genotype.     

Frequency of the ΔF508 and exon 11 mutations in Norwegian cystic fibrosis patients

Eiklid K, Tranebjærg L, Eiken HG, Pedersen JC, Michalsen H, Fluge G, Schwartz M, Nilsen BR, Bolle R, Skyberg D, Boman H, Berg K. Frequency of the ΔF508 and exon 11 mutations in Norwegian cystic fibrosis patients.
Clin Genet 1993: 44: 12–14. © Munksgaard, 1993
We have searched for the ΔF508 mutation in 77 Norwegian cystic fibrosis patients. Of the 154 chromosomes tested, 93 (60%) carried the ΔF508 mutation. Haplotypes at the D7S23 locus (KM19 and XV2C markers) were determined. Of 81 chromosomes with the F508 mutation, the B haplotype was found on 77. We found three patients with the G551D and one patient with the R553X mutation in exon 11 of the CFTR locus.     

Quantitative immunoassays for diagnosis and carrier detection in cystic fibrosis

Quantitative immunoprecipitation and immunoradiometric assays have been developed for a protein present in the serum of cystic fibrosis homozygotes, and to a lesser extent in the serum of heterozygotes. When tested on a panel of sera from 14 cystic fibrosis patients, 29 heterozygotes and 23 controls, the immunoprecipitation assay allowed correct assignments to be made on 94% of occasions with one batch of antiserum and 95% with another. With the same panel of sera, the immunoradiometric assay allowed 94% correct assignments. It is suggested that such accuracy is the maximum that can be expected in the present state of knowledge of cystic fibrosis.     

A rapid,efficient, and sensitive assay for simultaneous detection of multiple cystic fibrosis mutations

We describe the use of DGGE multiplex systems for rapid analysis of 15 exons of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, in which about half of the known CF molecular defects are clustered. We have previously determined the spectrum of mutation affecting the CFTR gene in the French population using a strategy based on denaturing gradient gel electrophoresis (DGGE) of amplified gene segments. Analysis of CF patients' DNA with five DGGE multiplex systems permitted us to characterize nearly 35% of non-ΔF508 CF alleles and increased the CF allele detection rate to almost 82% in this population. This simple and rapid multiplex analysis strategy, which allows a significant proportion of the most frequent CF mutations in Caucasians to be detected, will be helpful in the implementation of genetic screening programs. © 1993 Wiley-Liss, Inc.     

Detection of the ΔF508 (F508del) mutation of the cystic fibrosis gene by surface plasmon resonance and biosensor technology

In the present paper, we applied surface plasmon resonance (SPR) and biosensor technologies for biospecific interaction analysis (BIA) to detect ΔF508 mutation (F508del) of the cystic fibrosis transmembrane regulator (CFTR) gene in both homozygous as well as heterozygous human subjects. The proposed method is divided into three major steps. The first step is the immobilization on a SA5 sensor chip of two biotinylated oligonucleotide probes (one normal, N‐508, and the other mutant, ΔF508) that are able to hybridize to the CFTR gene region involved in F508del mutation. The second step consists of the molecular hybridization between the oligonucleotide probes immobilized on the sensor chips and (1) wild‐type or mutant oligonucleotides, as well as (2) single‐stranded DNA obtained by asymmetric polymerase chain reaction (PCR), performed using genomic DNA from normal individuals and from F508del heterozygous and F508del homozygous patients. The third, and most important, step consists of the evaluation of differential stabilities of DNA/DNA molecular complexes generated after hybridization of normal and ΔF508 probes immobilized on the sensor chips. The results obtained strongly suggest that the proposed procedure employing SPR technology enables a one‐step, nonradioactive protocol for the molecular diagnosis of F508del mutation of the CFTR gene. This approach could be of interest in clinical genetics, as the hybridization step is oftenly required to detect microdeletions present within PCR products. Hum Mutat 13:390–400, 1999. © 1999 Wiley‐Liss, Inc.     

Improved detection of cystic fibrosis mutations in infertility patients with DNA sequence analysis

BACKGROUND: Accurate determination of mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene is critical for genetic counselling and treatment of obstructive azoospermia. Of concern is that detection rates with routine CFTR mutation panels vary widely depending on patient ancestry; and such panels have limited value for azoospermic patients, who are more likely to carry rare mutations. An alternative approach offers comprehensive, CFTR mutation analysis by a DNA sequence method. We investigated whether this method could improve CFTR detection rates in men with obstructive azoospermia in a prospective study of men with obstructive azoospermia and their partners who were referred for genetic counselling and testing at one of two institutions. METHODS: Sixteen patients with congenital absence of the vas deferens (CAVD, n = 14) or idiopathic obstructive azoospermia (n = 2) were studied. DNA from all patients was analysed for mutations by the DNA sequence method. In addition to this method, six men underwent CFTR analysis by a common 25 or 31 mutation panel coupled with poly T analysis. In 10 subjects, common mutation panel findings were inferred from DNA sequence method results. RESULTS: Overall, 12/16 (75%) azoospermic patients had one or more CFTR mutations and/or 5T alleles, including 12 mutations in 10 patients (two compound heterozygotes) and seven 5T alleles in six patients (one homozygote). The sequence method detected all mutations and three variants of unknown significance. By comparison, the common mutation panels detected only 3/12 mutations (25%) and 0/3 variants. CONCLUSION: The DNA sequence method detects more CFTR mutations than common mutation panels. Given the serious, clinical consequences of transmitting such mutations, this study underscores the importance of accurate, CFTR mutation detection in men with obstructive azoospermia and their partners.     

Prenatal diagnosis of cystic fibrosis by using linked DNA markers in 138 pregnancies at l-in-4 risk

Prenatal diagnosis was carried out in 138 pregnancies at 1-in-4 risk for cystic fibrosis (CF) by using closely linked DNA markers, including XV-2c and KM-19. In fully informative families, 25 of 123 (20%) fetuses were predicted to be affected; 16 of these 25 pregnancies were terminated and 9 were continued. Postnatal sweat tests are completed in 42 cases; the diagnoses were confirmed in 4 of 4 infants predicted to be affected and in 37 of 38 infants predicted to be unaffected. One infant predicted to be a carrier had an abnormal sweat test after birth, but the mother also had an abnormal sweat test, and there was no evidence of an error in linkage analysis. The data indicate that prenatal diagnosis using linkage analysis is fully informative in most families and is highly reliable with either chorionic villus sampling or amniocentesis. Although Outcome data are available on only 42 pregnancies, based on our experience, on general principles of linkage of the known DNA markers with CF we recommend that DNA analysis replace microvillar intestinal enzyme analysis for 1-in-4 risk pregnancies when DNA is available from the propositus.     

Complete mutational screening of the cystic fibrosis transmembrane conductance regulator gene: cystic fibrosis mutations are not involved in healthy men with reduced sperm quality

Based on the analysis of the most frequent mutations responsible for cystic fibrosis (CF), a higher than expected frequency of CF mutations was recently reported in men with infertility due to reduced sperm quality. To further document whether this condition is associated with severe or mild abnormalities of cystic fibrosis transmembrane conductance regulator (CFTR) functions, we carried out a complete scanning of CFTR sequences using a strategy that detects almost all 850 mutations and 150 polymorphisms reported to date in the CFTR gene. We have investigated a cohort of 56 patients with severe oligoasthenoteratozoospermia (OAT) and 50 controls from southern France for CFTR gene mutations and variations. The frequencies of CF-causing mutations and CFTR variations identified in this OAT sample did not differ significantly from the frequencies found in the normal population. However, we observed a 1.7-fold increase in the proportion of homozygotes for a specific CFTR haplotype (TG11-T7-G1540) in the OAT group (P = 0.025). Our results do not confirm a link between CF mutations and reduced sperm quality. Further studies are needed to substantiate the hypothesis that a combination of variants affecting expression and function of the CFTR protein is associated with male infertility.     

Prenatal diagnosis of cystic fibrosis by trehalase enzyme assay in amniotic fluid

Amniocentesis and amniotic fluid trehalase enzyme assay were offered to 14 pregnant women at a 1 in 4 risk for a child with cystic fibrosis. Twelve of these pregnancies were screened at the 18th week of gestation; ten proceeded to term, seven following the finding of a normal trehalase activity and three despite the low enzyme level in amniotic fluid. In all ten cases prenatal diagnosis proved to be correct. In two cases with low enzyme activity parents opted for termination at the 19th week, and with PAS-Alcian Blue staining some slight histochemical lesions characteristic of cystic fibrosis were seen in the exocrine glands, including the pancreas and intestinal mucosa, of both fetuses. The total protein content in the meconium of these fetuses was significantly higher than in the controls. Results suggest that trehalase assay in the amniotic fluid is a potential prenatal test for cystic fibrosis and it appears that in fetuses with cystic fibrosis some histochemical and biochemical abnormalities can be observed as early as the 19th week of gestation. The role of ultrasound examination as an additional procedure for the prenatal diagnosis of cystic fibrosis is also discussed.     

Preimplantation genetic diagnosis: the ethics of intermediate cases

According to the current guiding principle regarding preimplantation genetic diagnosis (PGD), the technique should focus on the diagnosis of genetic defects which (may) affect the health of this particular potential child--the so-called 'medical model'. I argue in favour of a more permissive view, also allowing PGD of characteristics which may be relevant for the health of 'third parties'. Two cases are analysed: PGD/HLA typing in order to save a sib, and PGD/sex selection in order to prevent the birth of healthy female carriers of X-linked recessive disorders, who are at high risk of conceiving affected sons. While these cases are at odds with the medical model stricto sensu, they do have a link with health problems. In the first case, the health benefit hoped for is intrafamilial, in the second case the health benefit is transgenerational. These cases illustrate that the traditional dichotomy between the medical model on the one hand and the 'designer' or autonomy model on the other hand is simplistic--they represent an intermediate category.     

Preimplantation diagnosis: Primer extension preamplification for detection of multiple genetic loci from single human blastomeres

A new technology called primer extension preamplification (PEP),which has been applied to single spermatozoa, increases theamount of polymerase chain reaction (PCR) templates by amplifyingDNA of the whole genome. The current investigation was aimedat applying PEP to single human blastomeres. Two blastomereswith nuclei from arrested embryos were selected for this study.Using three different PEP protocols (experiments I, II and III),DNA from single blastomeres was amplified using 15-base oligonucleotiderandom primers. The efficiency of the procedure was determinedby further amplifications of aliquots of the PEP products withtwo specific sequences. Three aliquots from each PEP productwere used as PCR templates for the human X chromosome (X) orthe exon 10 of the cystic fibrosis gene (CF). PCR amplifiedproducts were analysed by gel electrophoresis. In experimentI, when X primers were used, positive signals were detectedin all 10 embryos (100%), 90.0% (18/20) of the blastomeres,and in 80.0% (96/120) of the replicates. When CF primers wereamplified, all embryos (100%, 10/10), 90.9% (18/20) of the blastomeresand 78.3% (47/60) of the replicates were positive. In experimentII, efficiency was significantly reduced when total time forthe procedure was minimized from 8 h to 5 h and 45 min. Althoughthe time was further reduced to 4 h and 40 min in experimentIII, the efficiency remained the same as in experiment I whenthe volume of PEP was reduced from 60 µl (experimentsI and II) to 40 µl. One out of 132 control replicates(0.8%) was contaminated. The present study indicates that PEPcan be successfully applied to single blastomeres allowing forsimultaneous detection of approximately 20 DNA sequences.     

Nine mutations in the cystic fibrosis (CF) gene account for 80% of the CF chromosomes in French patients

Thirteen mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene have been screened in a French sample of 185 cystic fibrosis (CF) patients, together with their respective associated RFLP haplotypes at the linked D7S23 locus (XV2C and KM19 markers). The respective frequencies of the mutations showed that 9 of them account for 80% of the CF chromosomes. Implications for prenatal diagnosis and heterozygote detection are defined and discussed. The well-known great excess of RFLP B marker within CF chromosomes is partially explained by two already characterized mutations highly associated with haplotype B: delta F508 and G542X. Similarly, the excess of haplotype D within CF chromosomes is partially explained by the association between delta I507 and this haplotype. These results may suggest the existence of two still untested or uncharacterized mutations, whose frequencies could be near 1%, one which would be associated with haplotype B and a second which would be associated with haplotype D. The possible cause of the specific association between most of the main different CF mutations and the RFLP haplotype B is discussed.     

Attitudes of parents of cystic fibrosis children towards neonatal screening and antenatal diagnosis

Information on parents' attitudes towards neonatal screening for cystic fibrosis (CF) and antenatal diagnosis by chorion villus biopsy (CVS) has been derived from a detailed questionnaire administered to parents of CF babies diagnosed early following newborn screening (18 babies), and later on account of clinical criteria (11 babies). Screening was by measurement of immunoreactive trypsin (IRT) on Guthrie card blood spots, which was the basis of the Wales/West Midlands IRT Screening Survey, 1985-1989. Families questioned were from Wales. Most parents supported screening: parents of 15/18 (83%) screened babies and 10/11 (91%) unscreened babies. Following antenatal diagnosis, 15/29 (52%) of families would abort a CF foetus. Neither standard of education nor social class correlated with attitudes to screening or antenatal diagnosis, although these factors were related to the parents' knowledge of CF in general. Several families emphasised the importance of minimal delay between the initial mention of the possibility of CF on IRT testing and confirmation (or otherwise) of the diagnosis. Four mothers acknowledge temporary rejection of their babies during the period of uncertainty or following the procedures of diagnosis, emphasising that neonatal screening for CF can have a psychological impact on the parent-child bonding. Although most families supported neonatal screening for CF, this study underlines some of the difficulties which may be encountered during the procedure of screening for CF by IRT.