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.     

Pregnancy after preimplantation genetic diagnosis for Sanjad–Sakati syndrome

Sanjad–Sakati syndrome (SSS) is an autosomal recessive disorder characterized by congenital hypoparathyroidism, growth and mental retardation. In Saudi Arabia, the disease is caused by a deletion of 12 bp (155-166nt) in the tubulin-specific chaperone E gene. In a family with two affected siblings with SSS, preimplantation genetic diagnosis (PGD) was performed. Fluorescent PCR (F-PCR) was utilized to check the heterozygosity and the homozygosity status of the parents and the affected children, respectively. F-PCR was then optimized for single-cell analysis by using peripheral blood lymphocytes. The patient underwent a cycle with intra-cytoplasmic sperm injection. A total of 11 embryos were obtained and biopsied. There were five heterozygous, three homozygous affected and three normal embryos. One heterozygous and one normal embryo were transferred because of their very good quality (morula). A singleton pregnancy was obtained, and amniosynthesis confirmed the presence of the heterozygous fetus. These results show for the first time, the feasibility of PGD for SSS. Copyright © 2004 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.     

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.     

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.     

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.