Polymerase chain reaction analysis of the cystic fibrosis ΔF508 mutation in human blastomeres following oocyte injection of a single sperm from a carrier

The efficiency of the polymerase chain reaction (PCR) in detecting the cystic fibrosis (CF) ΔF508 mutation (which is the most common mutation of CF) was assessed in single human blastomeres. Twenty-one human immature oocytes (germinal-vesicle-stage oocytes) that had been donated for research were matured in vitro and a single spermatozoon from a carrier of the CF ΔF508 mutation was injected into the ooplasm. Fourteen embryos were obtained after intracytoplasmic sperm injection (ICSI). PCR analysis was carried out on 70 single blastomeres isolated from these 14 embryos. The results showed that the efficiency of DNA amplification by PCR in single nucleate blastomeres was 94 per cent (59/63). There were no false-positive results since none of the blank samples or the blastomeres without a nucleus showed an amplified signal. We found that nine embryos were homozygous for the unaffected genotype and that four embryos were heterozygous since they contained both the unaffected and the ΔF508 genotype. In a four-cell embryo, we observed the homozygous unaffected genotype in one blastomere and a heterozygous ΔF508/unaffected genotype in the other three blastomeres.     

Preimplantation genetic diagnosis for single gene disorders: experience with five single gene disorders

We report our experience of 14 preimplantation genetic diagnosis (PGD) cycles in eight couples carrying five different single gene disorders, during the last 18 months. Diagnoses were performed for myotonic dystrophy (DM), cystic fibrosis (CF) [ΔF508 and exon 4 (621+1 G>T)], fragile X and CF simultaneously, and two disorders for which PGD had not been previously attempted, namely neurofibromatosis type 2 (NF2) and Crouzon syndrome. Diagnoses for single gene disorders were carried out on ideally two blastomeres biopsied from Day 3 embryos. A highly polymorphic marker was included in each diagnosis to control against contamination. For the dominant disorders, where possible, linked polymorphisms provided an additional means of determining the genotype of the embryo hence reducing the risk of misdiagnosis due to allele dropout (ADO). Multiplex fluorescent polymerase chain reaction (F-PCR) was used in all cases, followed by fragment analysis and/or single-stranded conformation polymorphism (SSCP) for genotyping. Embryo transfer was performed in 13 cycles resulting in one biochemical pregnancy for CF, three normal deliveries (a twin and a singleton) and one early miscarriage for DM and a singleton for Crouzon syndrome. In each case the untransferred embryos were used to confirm the diagnoses performed on the biopsied cells. The results were concordant in all cases. The inclusion of a polymorphic marker allowed the detection of extraneous DNA contamination in two cells from one case. Knowing the genotype of the contaminating DNA allowed its origin to be traced. All five pregnancies were obtained from embryos in which two blastomeres were biopsied for the diagnosis. Our data demonstrate the successful strategy of using multiplex PCR to simultaneously amplify the mutation site and a polymorphic locus, fluorescent PCR technology to achieve greater sensitivity, and two-cell biopsy to increase the efficiency and success of diagnoses. Copyright © 2002 John Wiley & Sons, Ltd.     

Allele-specific amplification for preimplantation genetic diagnosis (PGD) of spinal muscular atrophy

We have developed a new allele-specific amplification method for the preimplantation genetic diagnosis (PGD) of spinal muscular atrophy (SMA; Werdnig-Hoffmann disease) from a single cell. This method is based on the detection of the deletion of exon 7 of the telomeric copy of the survival motor neurone (SMN^t) gene. An oligonucleotide was designed to be specific to the SMN^t nucleotidic sequence with exonic mismatch G (for SMN^t)→A (for SMN^c) at its 3′ end. This test produces reliable PCR products in 95% of single lymphoblasts (85/88) tested as well as in 16/16 blastomeres from normal controls. Specificity analysis showed that we were able to detect homozygous deletion of the SMN^t gene in 99% of single lymphoblasts (103/104) from a SMA patient. No contamination was detected in 68 blanks tested. Multiple cell and DNA dilution analysis revealed that the test is accurate and specific up to 100 pg DNA and should thus also be suitable for PGD at the blastocyst stage. This rapid procedure requires a single round of fluorescent PCR and no restriction digestion, while previously described single cell methods include nested PCR followed by restriction enzyme digestion. Two PGD cycles for SMA using this procedure were performed in our centre. Copyright © 2001 John Wiley & Sons, Ltd.     

Case report: birth after preimplantation genetic diagnosis of a subtle mutation in SMN1 gene

Spinal muscular atrophy (SMA) preimplantation genetic diagnosis (PGD) has been available since 1998. Protocols are based on the detection of the homozygous deletion of exon 7, which are present in 90–98% of SMA patients. A couple where the woman was a heterozygous carrier of the usual SMN1 Del7 mutation and the man was a heterozygous carrier of pMet263Arg substitution in exon 6 of SMN1 gene was referred for PGD. The usual PGD test being unsuitable for this couple, we developed a novel duplex polymerase chain reaction (PCR)-based PGD test for the detection of the mutation pMet263Arg by allele specific amplification, combined with the amplification of D5S641 extragenic polymorphic marker. PCR conditions were established using single control lymphoblasts and lymphocytes from the pMet263Arg substitution carrier. Amplification was obtained in 100% of the 86 single cells tested, amplification refractory mutation system (ARMS) PCR was specific in 100% of single cells tested and a complete genotype (mutation plus D5S641) was achieved in 88% of them. A PGD cycle was performed successfully and a pregnancy was obtained. An unaffected girl was born and postnatal diagnosis confirmed PGD results. This is the first PGD described for SMA because of another mutation than the major homozygous exon 7 deletion of SMN1. In the future, a similar strategy could be adopted for other subtle mutations of this gene. Copyright © 2006 John Wiley & Sons, Ltd.     

A comparison of different lysis buffers to assess allele dropout from single cells for preimplantation genetic diagnosis

Single cell polymerase chain reaction (PCR) for preimplantation genetic diagnosis (PGD) requires high efficiency and accuracy. Allele dropout (ADO), the random amplification failure of one of the two parental alleles, remains the most significant problem in PCR-based PGD testing since it can result in serious misdiagnosis for compound heterozygous or autosomal dominant conditions. A number of different strategies (including the use of lysis buffers to break down the cell and make the DNA accessible) have been employed to combat ADO with varying degrees of success, yet there is still no consensus among PGD centres over which lysis buffer should be used (ESHRE PGD Consortium, 1999 ). To address this issue, PCR amplification of three genes (CFTR, LAMA3 and PKP1) at different chromosomal loci was investigated. Single lymphocytes from individuals heterozygous for mutations within each of the three genes were collected and lysed in either alkaline lysis buffer (ALB) or proteinase K/SDS lysis buffer (PK). PCR amplification efficiencies were comparable between alkaline lysis and proteinase K lysis for PCR products spanning each of the three mutated loci (ΔF508 in CFTR 90% vs 88%; R650X in LAMA3 82% vs 78%; and Y71X in PKP1 91% vs 87%). While there was no appreciable difference between ADO rates between the two lysis buffers for the LAMA3 PCR product (25% vs 26%), there were significant differences in ADO rates between ALB and PK for the CFTR PCR product (0% vs 23%) and the PKP1 PCR product (8% vs 56%). Based on these results, we are currently using ALB in preference to PK/SDS buffer for the lysis of cells in clinical PGD. Copyright © 2001 John Wiley & Sons, Ltd.     

Early prenatal direct gene diagnosis of cystic fibrosis in a twin pregnancy and subsequent selective termination

We present a case of prenatal diagnosis of cystic fibrosis (CF) in one twin at 11–12 weeks of gestation. The parents had previously had two children, one of whom is alive and healthy and one who died of CF at the age of 2½ months. The parents were both known to be carriers of the ΔF508 mutation. Chorionic villus sampling (CVS) was performed and direct gene analysis showed that one fetus was homozygous for the ΔF508 mutation, while the other fetus did not have the mutation at all. Both fetuses had normal karyotypes. Selective termination was subsequently performed. The pregnancy continued without complications except for mild pre-eclampsia at term. The woman had a Caesarean section. The genetic diagnosis was confirmed after birth.     

First application of preimplantation genetic diagnosis to neurofibromatosis type 2 (NF2)

Neurofibromatosis type 2 (NF2) is a dominantly inherited cancer predisposition syndrome that is caused bymutations in the NF2 gene. We report here the first clinical preimplantation genetic diagnosis (PGD) forNF2. A protocol was developed to simultaneously amplify the mutation and a single nucleotide polymorphism (SNP) located within the gene. The mutation and polymorphism were analysed by simultaneous fluorescent single-strand conformation polymorphism (SSCP) on an automated DNA sequencer. The mutation, carried by the male partner, was a single base pair substitution affecting a splice site in intron 4 of the gene. The female partner was infertile due to polycystic ovary syndrome and would require IVF to conceive. The couple was found to be informative at a linked intragenic SNP situated in the 5′ untranslated region of the gene. The SNP was included in the assay to reduce the risk of misdiagnosis due to allele dropout (ADO). The couple underwent three cycles of treatment during which a total of 43 blastomeres were biopsied from 31 embryos. Amplification at both loci was obtained in 35 cells (81%). A total of five embryos were transferred, two in the first cycle, two in the second and one in the third. No pregnancy ensued. The results of the diagnoses indicated that, in this couple, the inheritance of the mutation may be non-Mendelian. Out of a total of 32 embryos tested only four were found not to carry the mutation. The reasons for this apparent skew remain unknown. Copyright © 2002 John Wiley & Sons, Ltd.     

Preimplantation genetic diagnosis for familial amyloidotic polyneuropathy (FAP)

Preimplantation genetic diagnosis (PGD) was developed more than a decade ago to offer an alternative to prenatal diagnosis for couples at risk of transmitting an inherited disease to their offspring. Portuguese-type familial amyloidotic polyneuropathy (FAP type I), is an autosomal dominant disease presenting an inherited mutation in the gene encoding the plasma protein transthyretin (TTR). We here report the first protocol for single-cell detection of the Met30 mutation in FAP type I and its application to PGD. A nested PCR reaction for exon 2 of the TTR gene was developed. The PCR product was then analysed by restriction enzyme analysis and SSCP allowing the detection of the point mutation. Ten clinical cycles were performed in seven couples. From the 93 metaphase II (MII) injected oocytes, 82 were normally fertilized and 78 were biopsied. A positive signal in the nested PCR reaction was obtained in 61 blastomeres, corresponding to a DNA amplification efficiency of 78.2%. No allele dropout (ADO) or contamination were detected. A biochemical pregnancy was obtained in three cases and a clinical pregnancy in one couple is actually in normal evolution. Copyright © 2001 John Wiley & Sons, Ltd.     

Prenatal diagnosis of neurofibromatosis type 1: From flanking rflps to intragenic microsatellite markers

Even though the neurofibromatosis type 1 (NF1) gene was cloned more than 3 years ago, the process of identifying mutations has not been fruitful, and genetic counselling is mainly based on the use of linked markers. Since 1990, we have analysed 130 NF1 families and have performed six prenatal diagnoses. In each case, genetic counselling has relied on linked markers and informativity was achieved in all of them. The use of intragenic microsatellite polymorphisms (IVS27AAAT2.1, IVS27AC28.4, IVS27AC33.1, and IVS38GT53.0) has increased the informativeness in our series of NF1 families to an average of 90 per cent, providing accurate diagnosis and confirmation of the disease status.     

Birth of healthy children after preimplantation diagnosis of β-thalassemia by whole-genome amplification

Preimplantation genetic diagnosis (PGD) offers couples at risk for transmitting an inherited disorder the possibility to avoid the need to terminate affected pregnancies. PGD for monogenic diseases is most commonly accomplished by blastomere biopsy from cleavage-stage embryos, followed by PCR-based DNA analysis. However, the molecular heterogeneity of many monogenic diseases requires a diagnostic strategy capable of detecting a range of mutations and compound genotypes. With the above considerations, we developed an accurate and reliable strategy for analysis of β-globin gene mutations, applicable for PGD for the wide spectrum of β-thalassemia major mutations in the Chinese population. The strategy involves primer-extension preamplification (PEP), followed by nested PCR and reverse dot blot (RDB) for mutation detection since it facilitates simultaneous analysis of more than one mutation in a single cell. This report describes the application of the strategy in two clinical IVF/PGD cycles at risk for transmitting β-thalassemia major, which resulted in the first thalassemia-free children born after PGD in China. Copyright © 2003 John Wiley & Sons, Ltd.     

Co-amplification of the cystic fibrosis ΔF508 mutation with the HLA DQA1 sequence in single cell PCR: Implications for improved assessment of polar bodies and blastomeres in preimplantation diagnosis

We have developed a heminested PCR (polymerase chain reaction) method, performed on single cells, for the analysis of the most common cystic fibrosis (CF) mutation (AF508). As a quality control, the polymorphic exon 2 of the HLA DQA1 locus was co-amplified from the same cell. With a non-radioactive reverse dot-blot assay, the genotype of these two loci could be determined. Experiments on 98 single fibroblasts, heterozygous for the CFTR and the DQA1 locus, showed that amplification of either locus could be obtained in 97 per cent of the cases, but only 90 per cent showed heterozygosity for CF, 75 per cent showed heterozygosity for DQA1, and 74 per cent showed heterozygosity for both CF and DQA1. Contaminations detected only after DQA1 typing occurred in 3 per cent of our samples. Error rate calculations based on our experimental PCR data indicate that single blastomere diagnosis would lead to unacceptable errors, i.e., an affected fetus, in less than 1 per cent of the cases. The risk of undetected crossing-over or the dubious results that crossing-over could generate, would make isolated polar body diagnosis at the present time very difficult. The combined approach of PCR on polar bodies followed by confirmation of the diagnosis on blastomeres, however, should give a solid base for preimplantation diagnosis of monogenic disorders.     

Duplex PCR for preimplantation genetic diagnosis (PGD) of spinal muscular atrophy

The main difficulty in developing a molecular diagnosis of spinal muscular atrophy (SMA) resides in the specific genomic structure of the locus. Indeed, two highly homologous survival motor neurone genes, SMN1 and SMN2, are present at the locus. The detection of the homozygous deletion of exons 7 and 8 of the SMN1 gene, which is present in 90 to 98% of the patients, is based on methods highlighting 1 of the 8 nucleotidic mismatches existing between these 2 genes. In order to offer preimplantation genetic diagnosis (PGD) for SMA, we developed a new allele-specific amplification method. The main disadvantage of our previously described strategy resided in the possibility of diagnosing, in case of amplification failure, an unaffected embryo as affected. We present here a new PGD-SMA method. We established the conditions for three different duplex PCRs, allowing the specific detection of the SMN1 gene and one polymorphic marker, either D5S629, D5S1977, or D5S641. Of the 60 to 90 single cells tested, the PCR efficiency varied from 98 to 100% with a complete genotype obtained in a range between 81 and 87% with a global allele drop-out rate of 9%. Such a test was used to perform 1 PGD cycle for which 7 embryos could be analysed. All the embryos were fully diagnosed, six as unaffected and one as affected. Four embryos were transferred, but no pregnancy ensued. Copyright © 2003 John Wiley & Sons, Ltd.     

Double locus analysis of chromosome 21 for preimplantation genetic diagnosis of aneuploidy

Preimplantation genetic diagnosis (PGD) of numerical chromosome abnormalities significantly reduces spontaneous abortions and may increase pregnancy rates in women of advanced maternal age undergoing in vitro fertilization. However, the technique has an error rate of around 10% and trisomy 21 conceptions have occurred after PGD. To further reduce the risk of transferring trisomy 21 embryos to the patient, we designed a protocol that analyzes chromosome 21 twice by targeting two different loci. This protocol was applied to 388 embryos from 60 cycles of PGD of aneuploidy. The scoring criterion used was based on giving equal importance to both probe results. Of the 242 embryos diagnosed as abnormal, 125 were re-biopsied to assess the rate of false positives and false negatives of the protocol and their clinical relevance. The results of the present study showed no reduction in the overall fluorescent in situ hybridization (FISH) error rate for single cells. However, by using a different scoring criterion, the incidence of false negative can be reduced to 1.6% without missing any trisomy 21. In addition, the present study suggests that if two or more loci from the same chromosome could be simultaneously analyzed in single cells, errors caused by false monosomies could be reduced. Copyright © 2001 John Wiley & Sons, Ltd.     

Extraction of DNA from amniotic fluid cells for the early prenatal diagnosis of genetic disease

Ten-ml samples of amniotic fluid were taken from pregnancies being terminated at 8–14 weeks' gestation. DNA was extracted from the amniotic cells by sequential centrifugation and analysed using the polymerase chain reaction (PCR). Fifteen samples were analysed for evidence of maternal contamination using Mfd5 oligo-nucleotide primers for repeat polymorphisms. Ten amniotic fluid samples were tested for the Delta-F₅₀₈ deletion characteristic of cystic fibrosis to demonstrate a diagnostic application for the technique. In each case, DNA extracted from fetal tissue from the same pregnancy was included in the controls. In 14 of the 15 cases tested with the Mfd5 primers, both the amniotic fluid DNA and the fetal DNA showed no evidence of contaminating DNA. In one case, neither the amniotic fluid cells nor the fetal cells yielded results. In nine of the ten cases tested with the Delta-F₅₀₈ primers, the amniotic fluid cell DNA provided accurate information about the genetic status of the fetus; in the tenth, the fetal DNA failed to amplify. The results indicate that adequate DNA can be extracted from amniotic fluid from 8 weeks' gestation onward and these samples are suitable for prenatal diagnosis using PCR.     

Asymptomatic carrier of two CFTR mutations: Consequences for prenatal diagnosis?

The cystic fibrosis (CF) gene has been observed to have the highest frequency of mutations in the Caucasian population. Prenatal diagnosis can now be performed with a high degree of accuracy since the identification of most of the gene's mutations, as well as the characterization of intragenic markers. However, the observation of a distribution of clinical phenotypes increases the need to identify a mild phenotype and avoid false-negative diagnosis. By screening most of the exons of the CFTR gene, we showed that a supposed obligate carrier of CF was in fact an asymptomatic affected woman.     

Mid–trimester hyperechogenic bowel in a fetus of Japanese origin carrying a new mutation of CFTR gene (L548Q)

We present a case of a fetus with hyperechogenic bowel, in which the L548Q mutation was detected in the mother of Japanese origin and the ΔF508 mutation in the father of Caucasian origin. The fetus proved to be compound heterozygous. Research into cystic fibrosis transmembrane conductance regulator (CFTR) mutations in this case was triggered by the fact that the fetus had a characteristic hyperechogenic bowel image with normal karyotype and no indications of intrauterine infections. Hyperechogenic bowel is highly indicative of a CFTR gene mutation. The incidence of cystic fibrosis (CF) in fetuses with mid-trimester hyperechogenic bowel is 5%, but once the most frequent mutations have been accounted for, rarer mutations must be investigated. Copyright © 2006 John Wiley & Sons, Ltd.     

Preimplantation genetic diagnostic protocols for α- and β-thalassaemias using multiplex fluorescent PCR

Haemoglobinopathies including α- and β-thalassaemia are the world's most common class of single gene disorder. Prenatal diagnosis (PND) for β-thalassaemia has been proven to be an effective strategy for controlling the incidence of new cases and is widely used in several countries where the disease is common. Successful preimplantation genetic diagnosis (PGD) protocols for β-thalassaemia have been introduced using restriction fragment length polymorphism (RFLP), single-stranded conformation polymorphism (SSCP) and denaturing gradient gel electrophoresis (DGGE). However, contamination and allele dropout (ADO) remain an important concern for all of these strategies. In the present study two PGD protocols for detecting β-thalassaemia mutations (codon 41-42 and IVSI-110) and one for α-thalassaemia (SEA mutation) have been designed and tested. These methods contain failsafe mechanisms to reduce the risk of misdiagnosis due to ADO or contamination and utilise multiplex fluorescent PCR (F-PCR). Interestingly, amplification efficiency and ADO were significantly affected by the choice of DNA polymerase and the freshness of the single cells used. The close similarity between the DNA sequences of β-globin and δ-globin was also found to be an important issue that necessitated careful design of primers for the β-globin gene. Copyright © 2001 John Wiley & Sons, Ltd.     

Five years' experience of prenatal diagnosis of cystic fibrosis in the former U.S.S.R.

From a total of 490 cystic fibrosis (CF) high-risk families under supervision (mostly Russian Slavs from the European part of the country), DNA data including both direct screening for some CF gene(CFTR)mutations(deIF508, G551D and 1677delTA) and allelic polymorphism studies with tightly CF linked DNA markers were collected from 261 families. All full families (129) and 86 CF families with a deceased index child were found to be either fully (42 per cent) or partially (40 per cent) informative for DNA analysis. Prenatal diagnosis (PD) was carried out in 161 CF families. Microvillar enzyme (MVE) assay was applied to all 140 PD at the second trimester either as a single test (88) or in conjunction with DNA analysis (52). The frequency of false-negative results of the MVE assay was 1.3 percent and that of false-positive results, as judged by the albumin meconium test, was 5.0 per cent. Ambiguous results of MVE analysis were found in 30 cases, 12 of which were verified by DNA analysis. Molecular diagnosis of CF at the first trimester was carried out in 21 cases and four pregnancies were terminated. Altogether, 39 pregnancies with a predicted high risk of CF fetuses were terminated. The low average frequency of delF508 in CF chromosomes of Russian Slavs (50 per cent), its remarkable inter-population variation, and the significant proportion of at-risk families without an affected child determine the necessity of combined molecular and biochemical (MVE assay) approaches for efficient prenatal diagnosis of CF in the former U.S.S.R.