Pamela Renwick

Preimplantation genetic diagnosis for monogenic diseases: overview and emerging issues

Preimplantation genetic diagnosis (PGD) is an established reproductive option for couples at risk of conceiving a pregnancy affected with a known genetic disease, who wish to avoid an (additional) affected child, termination of pregnancy or recurrent miscarriages. Early technologies concentrated on different approaches to direct mutation testing for monogenic diseases using single cell PCR protocols, or sex selection by fluorescent in situ hybridization for X-linked monogenic disease. Development of multiplex fluorescent PCR allowed simultaneously testing of linked markers alongside the mutation test, increasing the accuracy by controlling for contamination and identifying allele drop-out. The advent of highly effective whole genome amplification methods has opened the way for new technologies such as preimplantation genetic haplotyping and microarrays, thus increasing the number of genetic defects that can be detected in preimplantation embryos; the number of cases carried out and the new indications tested increases each year. Different countries have taken very different approaches to legislating and regulating PGD, giving rise to the phenomenon of reproductive tourism. PGD is now being performed for scenarios previously not undertaken using prenatal diagnosis, some of which raise significant ethical concerns. While PGD has benefited many couples aiming to have healthy children, ethical concerns remain over inappropriate use of this technology.

First use of preimplantation genotyping in prevention of recurrent diandric complete hydatidiform mole

Complete hydatidiform moles have a diploid chromosome constitution, generally with only paternal genetic material present (diandry). Diandric complete moles are thought to arise either by fertilization of an anucleate oocyte by two spermatozoa or, more commonly, doubling of a single sperm genotype. Molar pregnancies are usually sporadic, and may be accompanied by malignant transformation; however, recurrence is associated with increased risk of further affected pregnancies and of persistent trophoblastic neoplasia or choriocarcinoma. This study presents the first use of preimplantation genotyping to ensure biparental inheritance in a woman presenting with recurrent diandric complete hydatidiform mole. Following an IVF cycle, a single cell from each of 11 embryos was tested by whole genome amplification and genotyping at 16 different simple tandem repeat loci. All embryos showed normal biparental inheritance; one blastocyst was transferred, resulting in the delivery of healthy monozygotic twin girls.

Preimplantation genetic diagnosis for the prevention of sickle cell disease: Current trends and barriers to uptake in a London teaching hospital

Introduction.u2003Sickle cell disease (SCD) is a clinically significant hemoglobinopathy with increasing global incidence. We describe our experience of using pre-implantation genetic diagnosis (PGD) for the prevention of SCD at a tertiary referral centre in London. Methods.u2003Between January 2002 and December 2007, of 78 at-risk couples referred for PGD treatment, 12 couples (15%) underwent 16 PGD cycles for the prevention of SCD. Results.u2003The live birth rate was 13% per initiated cycle, 18% per embryo transfer and 17% per couple. Conclusions.u2003Although PGD for prevention of the birth of a child affected by SCD is a viable treatment option for couples at risk of having an affected child, potential barriers to uptake of this service need to be fully addressed to ensure its availability to all couples seeking to avoid having a child affected with SCD.

PGD Analysis of Embryos for Monogenic Disorders

The term monogenic disorder describes inherited disease caused by a defect in a single gene and encompasses those inherited in an autosomal recessive pattern, autosomal dominant, and sex-linked. DNA from single cells provides an acceptable PGD starter material but requires a lot of initial and ongoing test optimisation. PGD tests using whole gene amplification (WGA) allows diagnosis with standard DNA-based tests. Haplotyping has the advantage over the disease-specific tests being applicable to all couples known to have a mutation in the disease gene. Developing PGD tests is now faster and more efficient, allowing more equity of access for couples with rare disorders.

New Developments in PGD

PGD is a fast-changing landscape of reproductive medicine and constantly adopts new developments to improve its availability, accuracy and safety. These developments have been made possible because of rapid advances in assisted conception techniques and genetic testing tools. This chapter will cover some of those new developments, which are gradually being incorporated into the day-to-day PGD service.

Oral 5, Expanding choice for prenatal testing in couples at reproductive risk of Herlitz junctional epidermolysis bullosa

Following the early demise of their first child with Herlitz junctional epidermolysis bullosa (HJEB) a couple wished to consider prenatal testing. An immediate option would be fetal skin biopsy (FSB), a technique available for over 25 years, based on transmission electron microscopy and immunohistochemical analysis of laminin-332 expression, looking for similar changes to those in the deceased childs skin. However, this test could only be conducted after 15 weeks’ gestation and was not acceptable to this family. Mutation analysis was therefore performed on the laminin-332 genes by heteroduplex analysis and direct nucleotide sequencing and the deceased child was shown to be a compound heterozygote for the LAMB3 nonsense mutation p.R635X (maternal) and splice site mutation c.3228GA (paternal). With this information, chorionic villus sampling (CVS) with DNA sequencing could be conducted at an earlier gestation (10–11 weeks). However, both FSB and CVS involve termination of an affected pregnancy. An alternative option is preimplantation genetic diagnosis (PGD), based on DNA analysis of single blastomeres extracted from late cleavage stage embryos following in vitro fertilization. We have therefore developed a novel generic PGD test for HJEB based on whole genome amplification of DNA from single blastomeres, followed by multiplex polymerase chain reactions analysing 12 linkage markers within and flanking the LAMB3 gene. This assay is now licensed for clinical use in the U.K. and its suitability for this couple is being assessed. The development of PGD broadens personal choice for couples at reproductive risk of HJEB and represents a useful translational advance from basic research into epidermolysis bullosa.