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Research Updates

Preimplantation Genetic Diagnosis for Inherited Skin Disorders: From Bench to Bedside to Birth

Hiva Fassihi, John McGrath

Tuesday, November 07, 2006

One of the major recent successes of the molecular era has been the discovery of novel gene mutations that underlie a large number of inherited skin disorders. This new information has been useful clinically, improving diagnostic accuracy, establishing better genetic counseling, and furthering disease understanding through genotype-phenotype correlations. For affected individuals, however, the new discoveries have yet to lead to substantial advances in treatment through gene, protein, or cell therapy.1

Nevertheless, for couples at reproductive risk of recurrence of a severe life-threatening skin disorder, prevention of disease through DNA-based prenatal diagnosis represents one of the key direct translational benefits of the new gene discoveries.2 One particular molecular option for couples at risk is preimplantation genetic diagnosis (PGD).

What Is PGD?

PGD is a highly specialized procedure (for a detailed review, see reference 3). The method is based on testing cellular material from oocytes, or early human embryos, for specific genetic abnormalities before pregnancy has begun. PGD involves stimulation of the ovaries with exogenous gonadotrophins. When there are appropriate numbers of adequately sized follicles, oocyte maturation is hormonally triggered. The oocytes are then collected by transvaginal ultrasound-guided aspiration of the follicular fluid. The individual oocytes are transferred to suitable culture medium and are fertilized by intracytoplasmic sperm injection (ICSI), a procedure whereby a single spermatozoan is injected directly into a mature oocyte.4 The following day, the embryos are examined for the presence of two pronuclei (the haploid nuclei of the oocyte and the spermatozoan), which indicates successful fertilization.

The embryos can then be sampled at various stages of development. A cleavage-stage biopsy is the preferred option for many PGD centers and has been used successfully in numerous clinical procedures worldwide.5 This is performed at the 8-12-cell stage (about 72 hours after fertilization) when the individual cells of the embryo, referred to as blastomeres, are still totipotent.6 The biopsy procedure involves breaching the zona pellucida, either by a laser beam or by a jet of acidified Tyrodes solution. Following this, a sampling pipette is introduced into the embryo and a single nucleated blastomere is removed by suction for analysis (see Figure 1). After genetic diagnosis on DNA from the single cell, suitable embryos can be transferred to the uterus on day 4 or 5 of development. Because PGD is performed before pregnancy is established, it obviates the need for termination of an affected pregnancy, which can be associated with significant psychological and physical morbidity for couples undergoing conventional prenatal diagnosis.

Cleavage stage biopsy
Figure 1: Cleavage-stage biopsy

The first successful clinical applications of PGD were performed in 1990 and involved biopsying embryos from couples who were at risk of transmitting two different X-linked disorders (adrenoleukodystrophy and X-linked mental retardation). The cells removed were sex-typed by polymerase chain reaction (PCR) amplification of a Y chromosome-specific repeat sequence and only female embryos were then selected for implantation.7 For autosomal Mendelian disorders, the first live birth following PGD occurred in 1992 in a couple at reproductive risk of cystic fibrosis.8 Since then, several thousand cycles have been performed worldwide, resulting in the births of hundreds of healthy children. No significant long-term clinical adverse effects have been reported in individuals born following PGD intervention.9

PGD for Severe Inherited Skin Diseases

Until recently, however, there had been no successful cases of PGD for severe inherited skin disorders. Two cases of PGD for Herlitz junctional EB had been described, although pregnancy (beyond initial biochemical tests) was not established in either case.10

Nonetheless, the first case of successful PGD for a severe inherited skin disease, skin fragility-ectodermal dysplasia syndrome (OMIM 604536), was reported in 2006.11 This is a rare autosomal recessive disorder that results from loss-of-function mutations in plakophilin 1 (PKP1), a component of desmosome cell-cell junctions. For PGD in this case, the molecular screening involved a nested PCR protocol using DNA from a single cell with primers specific for the PKP1 mutations in this family.12 Pregnancy was established and progressed to term with delivery of an unaffected baby girl.

Although clinically successful, the laboratory work was very labor-intensive to set up and took 9 months to optimize. For PGD to be more widely clinically applicable, therefore, simpler and more generic protocols are needed.

Developing PGD Protocols

To develop a more generic test, i.e. a standardized PGD protocol that could be used for most couples at risk for a particular disorder, focusing on mutation detection is clearly inappropriate as most mutations are family-specific. Instead, an alternative approach is to assess linkage markers, either microsatellites (variable polymorphic repeats of DNA) or single nucleotide polymorphisms that are close to or within the disease gene locus. Provided that there is no genetic heterogeneity for a particular disease (as is the case for dystrophic epidermolysis bullosa and the type VII collagen gene, COL7A1) or that the candidate gene harboring the mutations in a genetically heterogeneous disorder is known (e.g. mutations in either LAMA3, LAMB3 or LAMC2, the three laminin-332 genes, in junctional epidermolysis bullosa), then PGD by linkage analysis is, at least on paper, a means to develop a more widely applicable test. Indeed, a protocol for linkage-based PGD for the COL7A1 gene has been optimized and published, although this has yet to be utilized clinically.13

In designing PGD protocols, however, there are two factors that make the development of clinical tests technically difficult: First, there is only a small amount of template DNA available from a single cell (just 6 pg). Second, because there are only two copies of each chromosome in a single cell, there may be a failure to amplify one or both alleles of interest. When only one allele is amplified, this is known as allele dropout (ADO).

One useful approach, therefore, is to try to increase the amount of template DNA by opting for a technique that amplifies the whole genome before any disease markers are assessed. A recently developed and suitable method is that of multiple displacement amplification (MDA).14 This is an isothermal whole-genome amplification using the bacteriophage φ29 DNA polymerase and results in a one million-fold amplification, thus increasing the template DNA from a single cell to about 6 µg. Then to counter the risk of PCR failure or ADO, a sensible option is to assess more than one gene marker. As such, using multiple polymorphic linkage markers within and flanking the disease gene represents a more robust strategic approach.

A Major Advance: Preimplantation Genetic Haplotyping (PGH)

PGD using this new approach is now referred to as preimplantation genetic haplotyping (PGH).15 PGH represents a major advance in reproductive technology applied to the prevention of inherited diseases. It will reduce the time taken to develop assays for other genetic disorders and will widen the scope and availability of preimplantation genetic testing, making it a reality for many more couples at risk of a variety of severe inherited disorders. Indeed, PGH protocols for the dystrophic and junctional forms of epidermolysis bullosa have recently been optimized.16

The combination of increasing knowledge about the molecular basis of single gene disorders and technological advances in defining genetic markers within single cells is leading to new possibilities for preimplantation testing for dermatological as well as many other inherited diseases. Laboratory protocols are becoming quicker, more reliable, and technically easier. Therefore, counseling of couples at risk for recurrence of a specific disease should include mention of the significant and clinically relevant advances that are occurring in this field.


Support for the authors' own studies on PGD has been provided by the Dystrophic Epidermolysis Bullosa Research Association (DebRA, UK).


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