Caroline Mackie Ogilvie

PREIMPLANTATION GENETIC DIAGNOSIS

Preimplantation genetic diagnosis (PGD) is an evolving technique that provides a practical alternative to prenatal diagnosis and termination of pregnancy for couples who are at substantial risk of transmitting a serious genetic disorder to their offspring. Samples for genetic testing are obtained from oocytes or cleaving embryos after in vitro fertilization. Only embryos that are shown to be free of the genetic disorders are made available for replacement in the uterus, in the hope of establishing a pregnancy. PGD has provided unique insights into aspects of reproductive genetics and early human development, but has also raised important new ethical issues about assisted human reproduction.

Strategies for the rapid prenatal diagnosis of chromosome aneuploidy

Rapid diagnosis of common chromosome aneuploidies in raised risk pregnancies, usually prior to full karyotype analysis, is now carried out in a number of European genetic centres; several techniques for detecting genomic copy number changes have been described. Prenatal diagnosis of genetic disease requires accurate and robust assays; the invasive procedures are associated with a risk of pregnancy loss and an abnormal result may lead to termination of the pregnancy. The testing of prenatal material (amniotic fluid, chorionic villi or, more rarely, fetal blood) is associated with specific problems, including the quality and quantity of the tissue and difficulties of interpretation due to phenomena such as maternal cell contamination and mosaicism. In addition, there are 24-h, high-throughput demands on centres offering such a service. The extent to which existing and proposed strategies, including different PCR-based assays, a multiplex ligation-dependent probe amplification approach, and microarrays, fulfil the requirements of rapid prenatal testing is discussed. In the past 3 years, we have tested 7720 prenatal samples for trisomies 13, 18 and 21 using a quantitative fluorescence-PCR (QF-PCR) approach. The abnormality rate was 5.7%. There were no misdiagnoses for nonmosaic trisomy, the amplification failure rate was 0.09% of samples, and 97% of samples received a report on the working day following sample receipt. Maternal cell contamination and mosaicism were also detected. Our data recommend a QF-PCR approach as the current method of choice for rapid aneuploidy testing.

Rare deletions at the neurexin 3 locus in autism spectrum disorder.

The three members of the human neurexin gene family, neurexin 1 (NRXN1), neurexin 2 (NRXN2), and neurexin 3 (NRXN3), encode neuronal adhesion proteins that have important roles in synapse development and function. In autism spectrum disorder (ASD), as well as in other neurodevelopmental conditions, rare exonic copy-number variants and/or point mutations have been identified in the NRXN1 and NRXN2 loci. We present clinical characterization of four index cases who have been diagnosed with ASD and who possess rare inherited or de novo microdeletions at 14q24.3-31.1, a region that overlaps exons of the alpha and/or beta isoforms of NRXN3. NRXN3 deletions were found in one father with subclinical autism and in a carrier mother and father without formal ASD diagnoses, indicating issues of penetrance and expressivity at this locus. Notwithstanding these clinical complexities, this report on ASD-affected individuals who harbor NRXN3 exonic deletions advances the understanding of the genetic etiology of autism, further enabling molecular diagnoses.

Proof of principle and first cases using preimplantation genetic haplotyping--a paradigm shift for embryo diagnosis.

Preimplantation genetic haplotyping (PGH) proof-of-principle was demonstrated by multiple displacement amplification (MDA) of single buccal cells from a female donor and genotyping using 12 polymorphic markers within the dystrophin gene; the known paternal genotype enabled identification of the paternal haplotype in the MDA products despite 27% allele dropout. MDA amplified DNA from 49 single human blastomeres with 100% success. The MDA products were genotyped using a total of 57 polymorphic markers for chromosomes 1, 7, 13, 18, 21, X and Y; 72% of alleles amplified providing results at 90% of the loci tested. A PGH cycle was carried out for Duchenne muscular dystrophy. One embryo was biopsied: PGH showed a non-carrier female, which was transferred with no resulting pregnancy. A PGH cycle was carried out for cystic fibrosis. Seven embryos were biopsied and PGH allowed the exclusion of 2 affected embryos; a carrier and a non-carrier embryo were transferred resulting in an on-going twin pregnancy. PGH represents a paradigm shift in embryo diagnosis, as one panel of markers can be used for all carriers of the same monogenic disease, bypassing the need for development of mutation-specific tests, and widening the scope and availability of preimplantation genetic testing.

Multiplex ligation-dependent probe amplification using a completely synthetic probe set

The recent development of multiplex ligation-dependent probe amplification (MLPA) has provided an efficient and reliable assay for dosage screening of multiple loci in a single reaction. However, a drawback to this method is the time-consuming process of generating a probe set by cloning in single-stranded bacteriophage vectors. We have developed a synthetic probe set to screen for deletions in a region spanning 18.5 Mb within chromosome 3q. In a pilot study, we tested 15 synthetic probes on 4 control samples and on 2 patients previously found to possess a heterozygous deletion in the region 3q26-q28. These synthetic probes detected deletions at all previously known deleted loci. Furthermore, using synthetic probes, the variability of results within samples was similar to that reported for commercially available M13-derived probes. Our results demonstrate that this novel approach to MLPA provides a generic solution to the difficulties of probe development by cloning; such synthetically generated probes may be used to screen a large number of loci in a single reaction. We conclude that the use of synthetic probes for MLPA is a rapid, robust, and efficient alternative for research (and potentially diagnostic) deletion and duplication screening of multiple genomic loci.

Preimplantation Genetic Diagnosis—An Overview

Since the early 1990s, preimplantation genetic diagnosis (PGD) has been expanding in scope and applications. Selection of female embryos to avoid X-linked disease was carried out first by polymerase chain reaction, then by fluorescence in situ hybridization (FISH), and an ever-increasing number of tests for monogenic diseases have been developed. Couples with chromosome rearrangements such as Robertsonian and reciprocal translocations form a large referral group for most PGD centers and present a special challenge, due to the large number of genetically unbalanced embryos generated by meiotic segregation. Early protocols used blastomeres biopsied from cleavage-stage embryos; testing of first and second polar bodies is now a routine alternative, and blastocyst biopsy can also be used. More recently, the technology has been harnessed to provide PGD-AS, or aneuploidy screening. FISH probes specific for chromosomes commonly found to be aneuploid in early pregnancy loss are used to test blastomeres for aneuploidy, with the aim of replacing euploid embryos and increasing pregnancy rates in groups of women who have poor IVF success rates. More recent application of PGD to areas such as HLA typing and social sex selection have stoked public controversy and concern, while provoking interesting ethical debates and keeping PGD firmly in the public eye.

Derivation and feeder-free propagation of human embryonic stem cells under xeno-free conditions

BACKGROUND AIMS Human embryonic stem (hES) cells hold great potential for cell therapy and regenerative medicine because of their pluripotency and capacity for self-renewal. The conditions used to derive and culture hES cells vary between and within laboratories depending on the desired use of the cells. Until recently, stem cell culture has been carried out using feeder cells, and culture media, that contain animal products. Recent advances in technology have opened up the possibility of both xeno-free and feeder-free culture of stem cells, essential conditions for the use of stem cells for clinical purposes. To date, however, there has been limited success in achieving this aim. METHODS, RESULTS AND CONCLUSIONS Protocols were developed for the successful derivation of two normal and three specific mutation-carrying (SMC) (Huntingtons disease and myotonic dystrophy 1) genomically stable hES cell lines, and their adaptation to feeder-free culture, all under xeno-free conditions.

The future of prenatal diagnosis: rapid testing or full karyotype? An audit of chromosome abnormalities and pregnancy outcomes for women referred for Down's Syndrome testing

Objective  To assess the implications of a change in prenatal diagnosis policy from full karyotype analysis to rapid trisomy testing for women referred primarily for increased risk of Downs Syndrome.

Chromosomal Mosaicism in Cleavage-Stage Human Embryos and the Accuracy of Single-Cell Genetic Analysis

Purpose:Our purpose was to assess the effect of chromosomal mosaicism in cleavage-stage human embryos on the accuracy of single-cell analysis for preimplantation genetic diagnosis.Methods:Multicolor fluorescence in situ hybridization with X, Y, and 7 or X, Y, 7, and 18 chromosome-specific probes was used to detect aneuploidy in cleavage-stage human embryos.Results:Most nuclei were diploid for the chromosomes tested but there was extensive mosaicism including monosomic, double-monosomic, nullisomic, chaotic, and haploid nuclei.Conclusions:Identification of sex by analysis of a single cleavage-stage nucleus is accurate but 7% of females are not identified. One or both parental chromosomes 7 were absent in at least 6.5% of the nuclei. With autosomal recessive conditions such as cystic fibrosis, carriers would be misdiagnosed as normal or affected. With autosomal dominant conditions, failure to analyze the affected parents allele (1.6–2.5%) would cause a serious misdiagnosis and analysis of at least two nuclei is necessary to reduce errors.

Rapid Prenatal Diagnosis of Aneuploidy Using Quantitative Fluorescence-PCR (QF-PCR)

Molecular cytogenetic aneuploidy testing for pregnant women at increased risk of chromosome abnormality leads to rapid reassurance for those with normal results and earlier decisions on pregnancy management in the case of abnormality. We tested 9080 prenatal samples using a one-tube QF-PCR test for trisomies 13, 18, and 21; the abnormality rate was 5.9%. There were no misdiagnoses for non-mosaic trisomy. A sex chromosome multiplex was developed that detects structural sex chromosome abnormalities as well as aneuploidies. The sex chromosome test was targeted at pregnancies (272) with specific abnormalities suggestive of Turner syndrome; 13.2% showed 45,X, confirmed by follow-up analysis.