Strategies for prenatal and preimplantation genetic diagnosis in Marfan syndrome (MFS)

Marfan syndrome (MFS) is an autosomal dominant disorder with a prevalence of 2–3 per 10 000 individuals. Symptoms range from skeletal overgrowth, cutaneous striae to ectopia lentis and aortic dilatation leading to dissection. Prenatal diagnosis was until recently mainly performed in familial cases by linkage analysis. However, mutation detection has become available with thorough screening methods. The phenotypic variability observed in MFS makes reproductive options difficult, as molecular diagnosis cannot predict clinical severity of the disease. Data are presented on 15 prenatal and/or preimplantation genetic diagnoses (PGD) in nine families, originating from Belgium, the Netherlands, Spain and France. In four families data from linkage analysis were used, whereas in five other families the causative FBN1 mutation was characterised. Four PGD cycles in two couples led to one ongoing pregnancy. In addition, two amniocenteses and nine chorionic villus (CV) samplings were performed. In five pregnancies an affected fetus was diagnosed. In one of them, the couple chose to continue the pregnancy and an affected child was born, whereas the other four couples decided to terminate the pregnancy. It is expected that the greater availability of mutation testing of the FBN1 gene will increase requests for prenatal diagnosis. PGD appears to be an acceptable alternative for couples facing ethical reproductive dilemmas. Copyright © 2002 John Wiley & Sons, Ltd.     

Single-cell sequencing and mini-sequencing for preimplantation genetic diagnosis

There is increasing interest in the use of preimplantation genetic diagnosis (PGD) as an alternative to routine prenatal diagnosis. However, the costs associated with development and testing of new PGD protocols have forced some PGD centres to limit the number of diseases for which PGD is offered. One of the main factors in the design of new protocols, which affects cost and accuracy, is the choice of the mutation-detection technique. We have assessed the reliability of DNA sequencing and mini-sequencing for clinical diagnosis at the single-cell level and have found them to be rapid and accurate. Extensive optimisation for individual mutations is not usually necessary when employing these versatile techniques and consequently a smaller investment of time and resources should be required during development of new protocols. Additionally, we report single-cell protocols for the diagnoses of cystic fibrosis, sickle cell anaemia and β-thalassaemia, which utilise mini-sequencing. Unlike most mutation-detection techniques, mini-sequencing permits analysis of very small DNA fragments. Small amplicons experience low allele dropout (ADO) rates, and consequently this approach could potentially improve the reliability of PGD. Copyright © 2003 John Wiley & Sons, Ltd.     

Aspects of biopsy procedures prior to preimplantation genetic diagnosis

Today, preimplantation genetic diagnosis (PGD) is offered in over 40 centres worldwide for an expanded range of genetic defects causing disease. This very early form of prenatal diagnosis involves the detection of affected embryos by fluorescent in situ hybridization (FISH) (sex determination or chromosomal defects) or by polymerase chain reaction (PCR) (monogenic diseases) prior to implantation. Genetic analysis of the embryos involves the removal of some cellular mass from the embryos (one or two blastomeres at cleavage-stage or some extra-embryonic trophectoderm cells at the blastocyst stage) by means of an embryo biopsy procedure. Genetic analysis can also be performed preconceptionally by removal of the first polar body. However, additional information is then often gained by removal of the second polar body and/or a blastomere from the embryo. Removal of polar bodies or cellular material from embryos requires an opening in the zona pellucida, which can be created in a mechanical way (partial zona dissection) or chemical way (acidic Tyrode's solution). However, the more recent introduction of laser technology has facilitated this step enormously. Different biopsy procedures at different preimplantation stages are reviewed here, including their pros and cons and their clinical applications. The following aspects will also be discussed: safety of zona drilling by laser, use of Ca²⁺/Mg²⁺-free medium for decompaction, and removal of one or two cells from cleavage-stage embryos. Copyright © 2001 John Wiley & Sons, Ltd.     

Huntington disease–unaffected fetus diagnosed from maternal plasma using QF-PCR

The discovery of fetal DNA in maternal plasma from early pregnancies has led to new opportunities for clinical application. In the last few years there have been numerous reported applications, mainly fetal gender and RhD genotyping. The prenatal diagnosis of some inherited genetic diseases such as Huntington disease is also very frequently required in the prenatal diagnosis routine. We have successfully diagnosed, with a non-invasive procedure, an unaffected HD fetus at the 13th week of gestation using fetal DNA from maternal plasma and the quantitative fluorescent PCR method, which is one of the most sensitive ways to detect fetal DNA in maternal plasma at such an early time of gestation. Copyright © 2003 John Wiley & Sons, Ltd.     

Personal desires of patients and social obligations of geneticists: applying preimplantation genetic diagnosis for non-medical sex selection

The arguments against the use of preimplantation genetic diagnosis (PGD) for non-medical sex selection are analysed. It is concluded that the distinction between medical and non-medical reasons is difficult to maintain, that the disproportionality of means and end is not a decisive counterargument and that the fear of damage to the reputation of PGD does not justify the refusal of controversial applications. Moreover, since non-medical sex selection does not belong to basic health care, it should not be equally accessible to all. The position defended in this article is founded on two basic principles: (1) medical reasons have priority on non-medical reasons, and (2) personal reasons do not qualify for public funding. In order to respect both principles, it is proposed that restrictions should be installed to control the number of requests for social sexing and that a tax should be imposed on these elective services. The tax should compensate the society for the investment it made in the training and education of the physician. Copyright © 2002 John Wiley & Sons, Ltd.     

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.     

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.