共查询到20条相似文献,搜索用时 33 毫秒
1.
Vrettou C Tzetis M Traeger-Synodinos J Palmer G Kanavakis E 《Molecular human reproduction》2002,8(9):880-886
Cystic fibrosis (CF) is one of the most important genetic diseases requiring prevention programmes. Preimplantation genetic diagnosis (PGD) represents an alternative to prenatal diagnosis, and is especially appropriate for couples with an unsuccessful reproductive history. For clinical application, protocols must be optimized to minimize PCR failure, allelic drop-out (ADO) and contamination, while simultaneously detecting a wide spectrum of CF genotypes. We have developed a flexible multiplex PCR protocol allowing analysis of sequence variations in any combination amongst seven CFTR gene exons (4, 10, 11, 13 in two parts, 14b, 17b and 21) by nested PCR and denaturing gradient gel electrophoresis analysis, along with analysis of a fluorescently labelled intragenic microsatellite (IVS8CA). The experiments were carried out on 390 single lymphocytes from three CF patients, one heterozygote and one non-CF individual. PCR efficiency of the exons ranged from 90 to 100%, and ADO from 0 to 3.8%. IVS8CA was co-amplified with a PCR efficiency of 92.4 and 10.8% ADO. The present method overcomes the need for separate assays for each CFTR gene mutation. Additionally, it facilitates analysis of any informative linked polymorphic sequence variation (within the seven exons) along with analysis of a microsatellite, which is useful (when informative) for minimizing misdiagnosis and/or indirect diagnosis. This method proved robust and flexible for diagnosing diverse CF genotype combinations in single cells. 相似文献
2.
Multiplex PCR of polymorphic markers flanking the CFTR gene; a general approach for preimplantation genetic diagnosis of cystic fibrosis 总被引:10,自引:0,他引:10
Dreesen JC Jacobs LJ Bras M Herbergs J Dumoulin JC Geraedts JP Evers JL Smeets HJ 《Molecular human reproduction》2000,6(5):391-396
Cystic fibrosis (CF) is the first monogenic disorder for which single cell preimplantation genetic diagnosis (PGD) has been successfully applied. The spectrum of mutations in CF is extremely heterogeneous, and hence, the development of mutation-specific PGD protocols is impracticable. The current study reports the development and evaluation of a general multiplex marker polymerase chain reaction (PCR) protocol for PGD of CF. Four closely linked highly polymorphic (CA)(n) repeat markers D7S523, D7S486, D7S480 and D7S490, flanking the cystic fibrosis transmembrane regulator (CFTR) gene, were used. In 99% of the single cells tested (100 leukocytes and 50 blastomeres), multiplex PCR results were obtained and the overall allelic drop out (ADO) rate varied from 2 to 5%. After validation for the presence of ADO and additional alleles, 95% of the multiplex PCR results were accepted to construct the marker genotypes. Depending on the genotype of the couple, and taking into account the embryos lost for transfer due to validation criteria (5%), ADO (0-2%) and single recombination (1.1-3%), in general >90% of the embryos could be reliably genotyped by PGD using a single blastomere. The risk of misdiagnosis equals the chance of a double recombination between informative flanking markers and is <0.05%. Therefore, this polymorphic and multi-allelic marker system is a reliable and generally applicable alternative for mutation-directed PGD protocols. Furthermore, it provides a test for the origin of the detected genotype and also gives an indication of the chromosomal ploidy status of the blastomere tested. 相似文献
3.
Current concepts in preimplantation genetic diagnosis (PGD): a molecular biologist's view 总被引:5,自引:0,他引:5
Sermon K 《Human reproduction update》2002,8(1):11-20
The first clinically applied preimplantation genetic diagnosis (PGD) was reported more than a decade ago and since then PGD has known an exponential growth. This first report described the use of PCR to sex embryos from couples at risk for X-linked diseases. Not surprisingly, in the first years, the development of PCR-based tests led to PGD for well-known monogenic diseases such as cystic fibrosis and thalassaemia. When fluorescent in-situ hybridization (FISH) was introduced it quickly replaced PCR-based methods, which had led to misdiagnoses, for sexing of embryos. FISH was also quickly introduced for aneuploidy screening, which has as its main aim the improvement of IVF results in patients with poor reproductive outcome, and later for PGD in translocation carriers. In this review, PGD for patients with a pre-existing genetic risk will be discussed, i.e. the monogenic diseases and the translocations, as well as different biopsy methods and promising new developments. 相似文献
4.
Strategies and clinical outcome of 250 cycles of Preimplantation Genetic Diagnosis for single gene disorders 总被引:4,自引:0,他引:4
Fiorentino F Biricik A Nuccitelli A De Palma R Kahraman S Iacobelli M Trengia V Caserta D Bonu MA Borini A Baldi M 《Human reproduction (Oxford, England)》2006,21(3):670-684
BACKGROUND: We report on our experience with preimplantation genetic diagnosis (PGD) for single gene disorders (SGDs), from 1999 to 2004, describing strategies and overall clinical outcome of 250 cycles in 174 couples for 23 different genetic conditions. METHODS: PGD cycles included 15 for autosomal dominant, 148 for autosomal recessive and 19 for X-linked SGDs. In addition, 68 cycles of PGD for SGDs were performed in combination with HLA matching. The strategy in each case used an initial multiplex PCR, followed by minisequencing to identify the mutation(s) combined with multiplex PCR for closely linked informative markers to increase accuracy. Linkage analysis, using intragenic and/or extragenic polymorphic microsatellite markers, was performed in cases where the disease-causing mutation(s) was unknown or undetectable. RESULTS: In 250 PGD cycles, a total of 1961 cleavage stage embryos were biopsied. PCR was successful in 3409 out of 3149 (92.4%) biopsied blastomeres and a diagnosis was possible in 1849 (94.3%) embryos. Four hundred and twenty-seven embryos were transferred in 211 cycles, resulting in 71 pregnancies (33.6% per embryo transfer), including 15 biochemical pregnancies, six spontaneous miscarriages, two ectopic pregnancies, which were terminated, and nine pregnancies which are still ongoing. The remaining pregnancies were confirmed to be unaffected and went to term without complications, resulting in the birth of 35 healthy babies. CONCLUSIONS: Minisequencing for mutation detection combined with multiplex fluorescence PCR for linkage analysis is an efficient, accurate and widely applicable strategy for PGD of SGDs. Our experience provides a further demonstration that PGD is an effective clinical tool and a useful option for many couples with a high risk of transmitting a genetic disease. 相似文献
5.
Simultaneous DNA'fingerprinting', diagnosis of sex and single-gene defect status from single cells 总被引:1,自引:0,他引:1
Findlay Ian; Urquhart Andy; Quirke Phil; Sullivan Kevin; Rutherford Anthony J.; Litford Richard J. 《Molecular human reproduction》1995,1(2):85-93
Sex and cystic fibrosis status have been previously diagnosedseparately at the single cell level. We have developed a sensitive,reliable, accurate and rapid (within 56 h) system forthe simultaneous diagnosis of sex, cystic fibrosis and a DNAfingerprint within a single reaction from a varietyof single cells. As contamination cannot be totally excluded,particularly at the single cell level, DNA fingerprintingcan be used to assess the risk of contamination. High sensitivitywith single cells is combined with very high specificity (estimatedmatching probability of 107108), allowingthe source of the amplified cell to be identified with a veryhigh degree of probability. Fluorescent primers were multiplexedfor six tetranucleotide microsatellite sequences to determinethe DNA fingerprint; the amelogenin gene was used to diagnosesex, and primers for the CFTR region were used to determinecystic fibrosis (CF) status. Analysis of the fluorescent productwas undertaken using an automated DNA sequencer with Genescansoftware. This technique has many applications such as prenataland preimplantation diagnosis, forensic identification of smallor degraded samples, and detection of contamination sources.DNA fingerprints of single haploid spermatozoa and other cellscan be assessed, so ensuring the detection of both diploid andhaploid contamination during preimplantation diagnosis. cystic fibrosis/DNA fingerprinting/fluorescent PCR/preimplantation diagnosis/sexing 相似文献
6.
First preimplantation genetic diagnosis of hereditary retinoblastoma using informative microsatellite markers 总被引:2,自引:0,他引:2
Girardet A Hamamah S Anahory T Déchaud H Sarda P Hédon B Demaille J Claustres M 《Molecular human reproduction》2003,9(2):111-116
Retinoblastoma is a malignant intra-ocular tumour of developing retina initiated by inactivation of both alleles of the retinoblastoma susceptibility (RB1) gene. This paper reports the first clinical experience of preimplantation genetic diagnosis (PGD) for hereditary retinoblastoma using two highly polymorphic microsatellite markers RB1.20 and D13S284, located within and close to the RB1 gene respectively. Duplex PCRs were tested on more than 300 single lymphocytes from heterozygous individuals at both loci, in order to test the accuracy and reliability of the single-cell protocol. This procedure requires a nested PCR and the analysis of fluorescently labelled PCR products on an automatic DNA sequencer. Amplification efficiency and allele drop-out rates ranged from 96.7 to 98.4%, and 3.7 to 5.4% respectively. This test was found to be accurate and reliable enough to be applied to the study of human blastomeres. Subsequently, this approach was used in a PGD treatment cycle for a couple who already had a child affected with hereditary retinoblastoma and found to be informative for both microsatellite markers. 相似文献
7.
Biopsies of human embryonic cell preparations previously analysed by
cytogenetic and/or fluorescent in-situ hybridization (FISH) chromosome
probes provide a unique reference DNA resource for the archival
preimplantation genetic diagnosis (PGD) of the transferred embryo. DNA
polymerase chain reaction (PCR) may be utilized on these fixed cell
preparations to verify equivocal FISH/PGD results. Retrospective PCR
screens of the genotype of biopsied embryonic cell(s) may be of benefit in
the case of a suspected genetic mutation. Currently, carrier detection or
linkage analysis is often not possible because of early death of the fetus,
or of patients with a lethal disease. Alternatively, fixed/stained 'failed
fertilized' oocytes provide a resource to extend genetic analysis to
infertile patients. A successful research is described which minimizes loss
of individual analysed fixed/stained oocytes, metaphase chromosomes, and
embryonic cell samples. Initial DNA amplification takes place in situ using
a modified PCR protocol. Comparative cellular studies using primer sets
previously used for PGD analyses show that 65% of the preparations
amplified unequivocally using the modified protocol and primers for a CA
repeat motif gene sequence, in comparison with 81% using the original PCR
protocol. With further refinement and optimization, the methods outlined
have the potential to retrospectively screen archival fixed chromosomes,
gametes, and embryonic cells for clinical application, and enable the
further study of the fixed human preimplantation embryo at the
morphological, cell and molecular level.
相似文献
8.
One major limitation of pre-implantation genetic diagnosis (PGD) practice comes from the need to develop single cell PCR protocols. For a disease such as cystic fibrosis (CF), for which almost 1000 mutations have been identified, the development of a mutation based PGD protocol is impracticable. An elegant way to overcome this problem is to set up an indirect diagnosis using polymorphic markers allowing the identification of the pathogenic haplotype instead of the mutation. We present here a new PGD protocol for CF. Our strategy is based on a multiplex fluorescent PCR co-amplifying the DeltaF508 mutation and two CFTR intragenic polymorphic microsatellites (IVS8CA and IVS17bCA). Such an approach is justified since in 91% of the cases at least one partner of the couple carries the DeltaF508 mutation. The use of intragenic markers reduces the risk of misdiagnosis due to meiotic recombination. In 97% of the single lymphoblasts (151/155) tested a PCR signal was obtained. A complete haplotyping was achieved in 137/151 (91%) lymphoblasts and a 6% rate of allele drop out (ADO) was observed. Three cases were performed. Case one was at risk of transmitting mutations DeltaF508 and R1162X, case 2 DeltaF508 and R1066C and case 3 DeltaF508 and 1341+1A. Considering these three cases and the re-analysis of the affected embryos, we have analysed 62 blastomeres from which we had PCR signal for 58 (94%) and a complete haplotype for 49 (84%). With the degree of polymorphism of the markers used in this work (48 and 39%) and the fact that we co-amplified the F508 locus our test should be suitable for nearly 80% of the couples requesting PGD for CF. This fluorescent multiplex PCR indirect diagnosis provides also a safer test since it allows the confirmation of the diagnosis, the detection of contamination and could give an indication on the ploidy of the embryos tested. 相似文献
9.
10.
Birth of healthy female twins after preimplantation genetic diagnosis of cystic fibrosis combined with gender determination 总被引:2,自引:0,他引:2
Ray PF Frydman N Attié T Hamamah S Kerbrat V Tachdjian G Romana S Vekemans M Frydman R Munnich A 《Molecular human reproduction》2002,8(7):688-694
Two healthy sisters with a familial history of mental retardation were referred to our centre for preimplantation genetic diagnosis (PGD). Their two brothers showed severe mental retardation. The molecular basis for their disorder could not be identified, but one of the sisters and the mother presented a highly skewed pattern of X-inactivation reinforcing the likelihood of an X-linked mode of inheritance. Both sisters requested PGD to avoid the abortion of potentially affected male fetuses. PGD for sex by fluorescent in-situ hybridization was carried out for the first sister and resulted in the birth of a female child. The second sister and her partner, whose niece had cystic fibrosis (CF), were tested for CF mutations, and were both found to be deltaF508 heterozygous. We developed an efficient single cell PCR protocol for the simultaneous amplification of the CF (deltadeltaF508) locus as well as the X-linked amelogenin gene and its highly homologous pseudogene on the Y chromosome. Two PGD cycles were carried out to screen against male and deltaF508 homozygous deleted embryos. In each case several embryos could be selected for transfer and the second cycle resulted in a twin pregnancy followed by the birth of two healthy female infants. 相似文献
11.
De Vos A Sermon K De Rijcke M Goossens V Henderix P Van Ranst N Platteau P Lissens W Devroey P Van Steirteghem A Liebaers I 《Molecular human reproduction》2003,9(7):429-435
Charcot-Marie-Tooth (CMT) disease is the 'common' name for a range of hereditary peripheral neuropathies. CMT1 is the most common form and is transmitted in an autosomal dominant manner. CMT1A maps to chromosome 17p11.2 and is caused, in the majority of cases, by a 1.5 Mb DNA duplication, that includes the peripheral myelin protein 22 (PMP) gene. This paper reports on preimplantation genetic diagnosis (PGD) for CMT1A in five couples. The CMT1A duplication was detected by fluorescent PCR analysis using polymorphic (CA)n markers localized within the duplication. Single-cell PCR on blastomeres allowed genetic analysis of embryos obtained after ICSI. Only healthy unaffected embryos were transferred to the uterus. PCR experiments with single EBV-transformed lymphoblasts or with research blastomeres allowed the evaluation of amplification efficiencies, as well as contamination and allele drop-out (ADO) rates for each PCR protocol. Three simplex PCR protocols (using one primer pair) and two duplex PCR protocols (using two primer pairs) were developed for CMT1A. Additionally, a protocol using all three primer pairs in triplex was also established. Thirteen clinical ICSI-PGD cycles were performed for five couples (12 simplex PCR cycles and one duplex PCR cycle), resulting in seven embryo transfers. Three singleton pregnancies ensued in two couples and three healthy babies were delivered. This report describes different fluorescent PCR-based tests which allow efficient and accurate single-cell level detection of the CMT1A duplication. On the basis of the presence of the healthy allele of the affected parent-to-be (and/or absence of the affected one), healthy embryos can be selected for transfer. The assays are suitable for PGD for other couples who present with the same CMT1A duplication [depending on their informativity for the (CA)n markers available] as described here. 相似文献
12.
Sánchez-García JF Benet J Gutiérrez-Mateo C Luís Séculi J Monrós E Navarro J 《Molecular human reproduction》2005,11(6):463-468
PGD is becoming an alternative to prenatal diagnosis. The combination of IVF techniques with the PCR technology allows for the detection of genetic abnormalities in first polar bodies from oocytes and blastomeres from cleavage-stage embryos. Dealing with a genetic disease with a heterogeneous spectrum of mutations like cystic fibrosis, one of the objectives of centres offering PGD is the application of simple and efficient protocols that allow for the detection of a wide range of mutations with a single procedure. In the present work, 29 normal loci and the 31 most frequent cystic fibrosis transmembrane conductance regulator (CFTR) mutations in Southern Europe could be detected at the same time in single cells applying a modified and improved primer extension preamplification-PCR. Two different Taq polymerases were tested in isolated buccal cells heterozygous for several mutations. The protocol that gave statistically significant better results was also successful in oocytes and their first polar bodies. 相似文献
13.
The minisequencing method: an alternative strategy for preimplantation genetic diagnosis of single gene disorders 总被引:11,自引:0,他引:11
Fiorentino F Magli MC Podini D Ferraretti AP Nuccitelli A Vitale N Baldi M Gianaroli L 《Molecular human reproduction》2003,9(7):399-410
We have applied a new method of genetic analysis, called 'minisequencing', to preimplantation genetic diagnosis (PGD) of monogenic disorders from single cells. This method involves computer-assisted mutation analysis, which allows exact base identity determination and computer-assisted visualization of the specific mutation(s), and thus facilitates data interpretation and management. Sequencing of the entire PCR product is unnecessary, yet the same qualitative characteristics of sequence analysis are maintained. The main benefit of the minisequencing strategy is the use of a mutation analysis protocol based on a common procedure, irrespective of the mutations involved. To evaluate the reliability of this method for subsequent application to PGD, we analysed PCR products from 887 blastomeres including 55 PGD cases of different genetic diseases, such as cystic fibrosis, beta-thalassaemia, sickle cell anaemia, haemophilia A, retinoblastoma, and spinal muscular atrophy. Minisequencing was found to be a useful technique in PGD analysis, due to its elevated sensitivity, automation, and easy data interpretation. The method was also efficient, providing interpretable results in 96.5% (856/887) of the blastomeres tested. Fifteen clinical pregnancies resulted from these PGD cases; conventional prenatal diagnosis confirmed all the PGD results, and 10 healthy babies have already been born. Its applicability to PGD could be helpful, particularly in cases in which the mutation(s) involved are difficult to assess by restriction analysis or other commonly used methods. 相似文献
14.
Sermon K; De Vos A; Van de Velde H; Seneca S; Lissens W; Joris H; Vandervorst M; Van Steirteghem A; Liebaers I 《Molecular human reproduction》1998,4(8):791-796
Myotonic dystrophy (DM), or Steinert's disease, is an autosomal dominant
disease characterized by myotonia, muscular weakness and atrophy, as well
as lens opacities, cardiomyopathy and mild endocrine changes. The gene for
DM located on 19q contains a triplet repeat at the 3' end of the gene. In
DM patients, this repeat is found to be expanded. We have previously
described a preimplantation genetic diagnosis (PGD) for DM using polymerase
chain reaction (PCR) followed by conventional analysis on ethidium
bromide-stained gels. The major drawback of this system was that allelic
dropout occurred in >20% of the cells, leading to the loss of healthy
embryos for transfer. To resolve this problem, we developed a PGD for DM
using fluorescent PCR followed by fragment analysis on an automated DNA
sequencer and made a comparison between the conventional PCR described
earlier and fluorescent PCR, which turned out to be superior in accuracy
and efficiency. Three PGD cycles were performed using fluorescent PCR and
are described here.
相似文献
15.
Preimplantation genetic diagnosis for achondroplasia: genetics and gynaecological limits and difficulties 总被引:1,自引:0,他引:1
Moutou C Rongieres C Bettahar-Lebugle K Gardes N Philippe C Viville S 《Human reproduction (Oxford, England)》2003,18(3):509-514
BACKGROUND: We report the first attempts at preimplantation genetic diagnosis (PGD) and IVF and their accompanying difficulties for achondroplasia (ACH) patients. METHODS: A PGD test was developed using fluorescent single cell PCR on lymphoblasts from patients and controls and from blastomeres from surplus IVF embryos. A specific digestion control based on the use of two fluorochromes was elaborated. Ovarian stimulation and oocyte retrieval were carried out using conventional protocols. RESULTS: We performed 88 single cell tests for which amplification was obtained in 86 (97.7%) single lymphoblasts. Allele drop out (ADO) was observed in two out of 53 (3.7%) heterozygous lymphoblasts. If we combine the results from the blastomere testing from surplus embryos with those from PGD cycles and re-analysis after PGD, we obtained a PCR signal in 84% of cases of which 91% were correctly diagnosed at the G380 locus. A total of six cycles were performed resulting in three embryo transfers. We observed difficulties in ovarian stimulation and oocyte retrieval with affected female patients. No pregnancy was obtained. CONCLUSION: A PGD test for ACH is now available at our centre but our initial practice raises questions on the feasibility of such a test, specially with affected female patients. 相似文献
16.
Findlay lan; Ray Pierre; Quirke Phil; Rutherford Anthony; Lilford Richard 《Molecular human reproduction》1995,1(4):209-218
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. allelic drop-out/cystic fibrosis/preferential amplification/preimplantation diagnosis/sexing 相似文献
17.
CFTR‐France,a national relational patient database for sharing genetic and phenotypic data associated with rare CFTR variants
下载免费PDF全文
![点击此处可从《Human mutation》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Mireille Claustres Corinne Thèze Marie des Georges David Baux Emmanuelle Girodon Thierry Bienvenu Marie‐Pierre Audrezet Ingrid Dugueperoux Claude Férec Guy Lalau Adrien Pagin Alain Kitzis Vincent Thoreau Véronique Gaston Eric Bieth Marie‐Claire Malinge Marie‐Pierre Reboul Patricia Fergelot Lydie Lemonnier Chadia Mekki Pascale Fanen Anne Bergougnoux Souphatta Sasorith Caroline Raynal Corinne Bareil 《Human mutation》2017,38(10):1297-1315
Most of the 2,000 variants identified in the CFTR (cystic fibrosis transmembrane regulator) gene are rare or private. Their interpretation is hampered by the lack of available data and resources, making patient care and genetic counseling challenging. We developed a patient‐based database dedicated to the annotations of rare CFTR variants in the context of their cis‐ and trans‐allelic combinations. Based on almost 30 years of experience of CFTR testing, CFTR‐France ( https://cftr.iurc.montp.inserm.fr/cftr ) currently compiles 16,819 variant records from 4,615 individuals with cystic fibrosis (CF) or CFTR‐RD (related disorders), fetuses with ultrasound bowel anomalies, newborns awaiting clinical diagnosis, and asymptomatic compound heterozygotes. For each of the 736 different variants reported in the database, patient characteristics and genetic information (other variations in cis or in trans) have been thoroughly checked by a dedicated curator. Combining updated clinical, epidemiological, in silico, or in vitro functional data helps to the interpretation of unclassified and the reassessment of misclassified variants. This comprehensive CFTR database is now an invaluable tool for diagnostic laboratories gathering information on rare variants, especially in the context of genetic counseling, prenatal and preimplantation genetic diagnosis. CFTR‐France is thus highly complementary to the international database CFTR2 focused so far on the most common CF‐causing alleles. 相似文献
18.
PGD for autosomal dominant polycystic kidney disease type 1 总被引:7,自引:0,他引:7
De Rycke M Georgiou I Sermon K Lissens W Henderix P Joris H Platteau P Van Steirteghem A Liebaers I 《Molecular human reproduction》2005,11(1):65-71
Autosomal dominant polycystic kidney disease (ADPKD) is primarily characterized by renal cysts and progression to renal failure. It is a genetically heterogeneous disease, with mutations in the PKD1 gene accounting for the majority of cases. Direct mutation detection for PKD1-linked ADPKD or type 1 is complicated by the large size and complex genomic structure of PKD1. This paper describes a microsatellite marker-based assay for PGD in couples at risk of transmitting ADPKD type 1. During PGD, genetic analysis is carried out on single blastomeres biopsied from preimplantation embryos obtained after IVF, and only embryos unaffected by the disease under investigation are selected for transfer. Single-cell genetic analysis relied on a fluorescent duplex-PCR of linked polymorphic markers followed by fragment length determination on an automated sequencer. The co-amplification of the intragenic KG8 and the extragenic D16S291 marker at the single-cell level was evaluated in pre-clinical tests on lymphoblasts and research blastomeres. The developed assay proved to be efficient (96.1% amplification) and accurate (1.4% allele drop-out and 4.3% contamination), and can be applied in all informative ADPKD type 1 couples. From five clinical cycles carried out for three couples, two pregnancies ensued, resulting in the birth of two healthy children. 相似文献
19.
Keymolen K Goossens V De Rycke M Sermon K Boelaert K Bonduelle M Van Steirteghem A Liebaers I 《European journal of human genetics : EJHG》2007,15(7):752-758
Preimplantation genetic diagnosis is an alternative for prenatal diagnosis that makes it possible to perform the diagnosis of a chromosomal or monogenic disorder at the preimplantation embryo level. Cystic fibrosis is one of the monogenic diseases for which PGD can be performed. In this study, we looked at the requests and PGD cycles for this particular disorder over an 11-year period. Sixty-eight percent of the requests eventually led to at least one complete PGD cycle. In 80% of the cycles, an embryo transfer was performed and an ongoing pregnancy was obtained in 22.2% of the cycles with oocyte retrieval. After embryo transfer, a couple had 27.8% chance of giving birth to a liveborn child. No misdiagnosis was recorded. The rate of perinatal deaths/stillborn children was relatively high, but no excess of major congenital anomalies was observed in the surviving children. 相似文献
20.
Ben-Chetrit A Antenos M Jurisicova A Pasyk EA Chitayat D Foskett JK Casper RF 《Molecular human reproduction》2002,8(8):758-764
Formation of the blastocyst is one of the first morphological changes in early embryonic development. Ion transport has been shown to be crucial for blastocoele cavity formation and expansion, although the mechanisms that underlie this process are presently unknown. As a transmembrane Cl(-) channel, the cystic fibrosis transmembrane conductance regulator (CFTR) may participate in ion transport and early blastocoele formation. CFTR mRNA was detected throughout preimplantation embryo development and in the unfertilized oocyte. Immunocytochemistry disclosed the presence of CFTR protein from the 8-cell stage, reaching maximum immunoreactivity at early blastocyst stage embryos. Patch clamp electrophysiology of morulae and blastocysts demonstrated typical CFTR Cl(-) channel activities in the apical membrane of trophectoderm cells. Thus CFTR is expressed both at mRNA and protein levels in human morulae and blastocysts, and functions as a cAMP-regulated apical membrane Cl(-) channel. These data suggest that CFTR may contribute to blastocoele formation in the early human embryo. 相似文献