2005 © Oxford University Press
Preimplantation Genetic Diagnosis (PGD) for Heritable Neoplasia
Affiliations of authors: Department of Obstetrics and Gynecology (JLS, SAC, PC) and Department of Molecular and Human Genetics (JLS), Baylor College of Medicine, Houston, TX
Correspondence to: Joe Leigh Simpson, MD, Department of Obstetrics and Gynecology, Baylor College of Medicine, 6550 Fannin, Ste. 901A, Houston, TX 77030 (e-mail: jsimpson{at}bcm.tmc.edu).
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ABSTRACT |
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 Top Abstract IntroductionOBTAINING PREIMPLANTATION CELLS...TECHNICAL CONSIDERATIONS IN...PGD FOR MENDELIAN DISORDERS...NOVEL INDICATIONS FOR PGDIS PGD SAFE?References |
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Especially applicable for heritable neoplasia, preimplantation genetic diagnosis (PGD) is possible for any Mendelian disorder whose gene has been localized, whether the molecular basis is known or not. Methods and Results: PGD requires DNA from gametes (oocytes) or embryos before 6 days postconception, when implantation occurs. Approaches include 1) polar body biopsy, 2) blastomere biopsy (aspiration of one or two cells from the six- to eight-cell embryos at 2 or 3 days), and 3) trophectoderm biopsy, which allows recovery of 20 or more cells (20–50) from 125- to 150-cell, 5- to 6-day blastocysts. Of some 6000 PGD cycles worldwide, approximately 1500 have been performed for Mendelian indications. The approximately 25% live birth rates following PGD parallel the general U.S. experience for assisted reproductive technology. PGD has been accomplished for both cancer-specific disorders like adenomatous polyposis coli (APC), BRCA1, retinoblastoma, Li-Fraumeni syndrome, and von Hippel-Lindau syndrome (VHL), as well as disorders predisposing to neoplasia (Fanconi anemia, Wiskott-Aldrich syndrome). PGD also makes possible the identification and, hence, transfer of embryos of specific HLA genotypes. This allows cord blood harvesting for stem cell implantation into a moribund child, often an older sibling of the fetus. Conclusions: PGD is a complex, but achievable, approach especially applicable to Mendelian forms of neoplasia. PGD is an attractive addition to the prenatal diagnostic armamentarium, especially relevant to heritable neoplasia. PGD also makes possible novel indications having special relevance to heritable neoplasia.
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INTRODUCTION |
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TopAbstractIntroductionOBTAINING PREIMPLANTATION CELLS...TECHNICAL CONSIDERATIONS IN...PGD FOR MENDELIAN DISORDERS...NOVEL INDICATIONS FOR PGDIS PGD SAFE?References |
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A preimplantation genetics diagnostic (PGD) program requires 1) assisted reproductive technology (ART), 2) micromanipulation skills sufficient to obtain a polar body or blastomere needed for analysis, and 3) single-cell molecular technology prowess more sophisticated than that required for traditional prenatal diagnosis. PGD requires close collaboration among clinical geneticists, laboratory investigators, and ART providers (2,3).
In this contribution we briefly consider the current status of PGD as it relates to heritable neoplasia.
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OBTAINING PREIMPLANTATION CELLS FOR PGD |
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TopAbstractIntroductionOBTAINING PREIMPLANTATION CELLS...TECHNICAL CONSIDERATIONS IN...PGD FOR MENDELIAN DISORDERS...NOVEL INDICATIONS FOR PGDIS PGD SAFE?References |
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PGD requires gametes (oocytes) or embryos before 6 days postconception, the time when implantation occurs. Approaches include 1) polar body biopsy (PBB), 2) blastomere biopsy (aspiration) of one or two cells from the six- to eight-cell embryo (2 to 3 days), and 3) trophectoderm biopsy, which yields approximately 20 cells from the 125- to 150-cell, 5- to 6-day blastocyst.
Blastomere Biopsy
Blastomere biopsy was the first technique utilized for PGD and is still the most widely applicable. This technique remains our preference here at Baylor College of Medicine. One or two of the six to eight cells within the zona pellucida are aspirated. One first dissociates a portion of the zona pellucida by mechanical (razor), chemical (pronase, ethylene diamine tetra-acetic acid [EDTA]), or laser (4). A second pipette then aspirates the blastomere (Fig. 1).
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PBB
Verlinsky and colleague (5) prefer removal of the first and, often, second polar biopsy. Let us consider the strategy. Suppose a woman and her partner are both heterozygous for the same autosomal recessive disorder. If the first polar body shows the mutant allele, it should be complemented by a primary oocyte having the normal allele. This normal oocyte could then be fertilized in vitro and transferred for implantation. Conversely, if the polar body were normal genetically, fertilization would not be allowed to proceed because the oocyte presumptively contains the mutant allele.
PBB has the theoretical advantages of no reduction in cell number, and contamination with sperm is less likely. However, ability to assess the paternal genotype is limited, precluding polar body utilization if a father has an autosomal dominant disorder. Detecting a mutant autosomal recessive allele in a polar body precludes allowing that oocyte to be fertilized, whereas the resulting embryo would be clinically abnormal only if the sperm had inherited the father's mutant allele.
In PBB, one must take into account recombination, which occurs regularly between homologous chromosomes. If crossover involves the region containing the heterozygous alleles in question, a single chromosome in the primary oocyte will contain DNA sequences encoding both mutant and normal (wild-type) alleles. Crossing over occurs frequently. For example, the frequency within the HLA locus is 4.3%, based on 330 embryos (6). If the two chromatids of a single chromosome differ in genotype (heterozygosity), the status of the secondary oocyte can be predicted only following biopsy of the second polar body or biopsy of the embryo per se (blastomere or blastocyst).
Blastocyst (Trophectoderm) Biopsy
If one biopsies the 5- to 6-day blastocyst, more cells would be available. This technique has been utilized less often than PBB or blastomere biopsy because fewer ART programs transfer embryos at this stage. However, a recent trend toward routine blastocyst culture could lead to more biopsies at this stage.
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TECHNICAL CONSIDERATIONS IN DIAGNOSING SINGLE-GENE DISORDERS FROM A SINGLE CELL |
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TopAbstractIntroductionOBTAINING PREIMPLANTATION CELLS...TECHNICAL CONSIDERATIONS IN...PGD FOR MENDELIAN DISORDERS...NOVEL INDICATIONS FOR PGDIS PGD SAFE?References |
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Discussed elsewhere (3,5,7), technical pitfalls are unavoidable when arriving at a diagnosis on only a single cell.
Unreliability of Enzyme Analysis
Diagnosing Mendelian disorders through chorionic villus or amniotic fluid cells is possible with either DNA or the (protein) gene product. In PGD, however, enzymatic analysis is hazardous because embryonic mRNA is not ordinarily transcribed until the eight-cell stage. Prior to that, only maternally derived mRNA is translated. Demonstrating a given gene product (enzyme protein) in a blastomere thus need not necessarily connote a genetically unaffected embryo.
PCR Failure and Allele Dropout
In PGD, small amounts of DNA (6 picograms per cell) must be amplified. Despite use of nested DNA primers (one primer to amplify a larger sequence of the gene locus and one to bind to DNA specifically within the locus) and heteroduplex analysis (a technique to discriminate between a mutant allele and a "wild-type" allele), PCR fails in 5% to 10% of cells. Most labs accept this standard of accuracy, assuming that the primer sometimes fails to anneal to the target area of DNA. Sometimes the cell nucleus or chromosome is actually lost. Overall, failure to find the target area of DNA is called allele dropout (ADO). This is a particular hazard when the cell has compound heterozygosity, i.e., when each of the two alleles for the gene being analyzed carries a different mutant. The few known errors in PGD for Mendelian disorders have occurred in this situation (8), although techniques such as use of fluorescent PCR can reduce the chance of error.
For disorders caused by autosomal dominant mutations, such as inherited breast and ovarian cancer syndrome, ADO can be especially troublesome because the mutated allele must be identified in order to exclude it. The best solution is to transfer only embryos in which a normal allele for the target gene has been identified (9). Accuracy can be confirmed using linked DNA markers.
Although a 5% error rate per cell (embryo) may seem unacceptable, most cycles of IVF yield 8 to 10 embryos; thus, it is usually possible to select one or more embryos that clearly carry the desired allele and thereby avoid an autosomal dominant disorder. Even in recessive disorders, ADO at worst may result in transferring embryos that are heterozygous for the mutation (10). If ADO occurs in the normal allele, the result is failure to transfer a phenotypically normal yet actually heterozygous embryo. The resulting child would still be clinically normal though a carrier of the problematic allele. If the normal allele fails to amplify, a heterozygous embryo would be erroneously diagnosed as affected. No transfer would occur; an opportunity for pregnancies would be lost but without consequence for live-born infants.
Misdiagnoses have been observed in live-born infants, but an established rate cannot be cited. Most have occurred during a center's early experience or through use of now-abandoned techniques. In the third report of the ESHRE PGD Consortium (11), the rate of misdiagnoses was 2% (8 of 451). More experienced centers are likely to have lower rates, but a finite error rate can be expected.
Sequential Analysis of First and Second PBBs
The recombination inherent in polar body analysis (8,12) can be used to diagnostic advantage. If both mutant and normal alleles are amplified from the first polar body, ADO will be known not to have occurred. If the second PBB reveals the mutant allele, the complementary secondary oocyte can confidently be presumed to be genetically normal. If the converse obtains, the secondary oocyte is abnormal.
Linkage Analysis
Even if the DNA sequence is not known, PGD is possible if the location of a mutant gene is known. Similarly, the gene may have been sequenced, but the exact molecular perturbation remains unknown. PGD can be accomplished through linkage analysis with polymorphic DNA alleles flanking the gene. If both mutant and its linked polymorphic variants (cis) are present, ADO is excluded. Verlinsky and Kuliev (5) provide illustrative examples.
Cryopreservation of Biopsied Embryos
Until recently, pregnancy rates have been essentially zero after thawing a cryopreserved biopsied embryo. Presumably, this finding is related to the freezing or thawing technique (13). However, successful pregnancies are increasingly reported using thawed cryopreserved biopsied embryos (14–16).
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PGD FOR MENDELIAN DISORDERS OF NEOPLASTIC PREDISPOSITION |
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TopAbstractIntroductionOBTAINING PREIMPLANTATION CELLS...TECHNICAL CONSIDERATIONS IN...PGD FOR MENDELIAN DISORDERS...NOVEL INDICATIONS FOR PGDIS PGD SAFE?References |
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Sex Determination for X-Linked Recessive Disorders
If a neoplastic disorder is X-linked recessive (e.g., Wiskott-Aldrich syndrome), one could determine sex (46,XY or 46,XX) in the six- to eight-cell embryos or blastocyst. Polar body analysis is not applicable, providing no information concerning transmission of paternal genes. Sex determination is based on fluorescent in situ hybridization (FISH) using chromosome-specific probes.
Performing PGD for sex determination in couples at risk for X-linked recessive disorders carries the obvious limitation that one cannot transfer those 50% of male embryos that have inherited the normal maternal X and, hence, are clinically normal. Only if the specific mutation is sought could unaffected male embryos be transferred. Sometimes the specific mutation is not known, and sometimes it is not practical to analyze an X-linked mutation unique to a single family.
Autosomal Disorders
About 35 Mendelian disorders can be detected with PGD and are listed in Table 1.
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Of some 6000–7000 PGD cycles worldwide, perhaps 1500 have been performed for Mendelian indications (17). The approximately 25% pregnancy rate for Mendelian PGD parallels the IVF/ICSI experience in general, which among 2001 U.S. cycles was 27% (18).
Only a few reports have focused on PGD for heritable neoplasias. Rechitsky et al. (9) reported 20 PGDs in 10 couples (FAP, VHL, p53 for Li-Fraumeni, neurofibromatosis I and II, and familial posterior fossa brain tumor [hSNF2]). In this series, a total of 40 genetically normal embryos were transferred. The result was five clinical pregnancies, yielding four healthy children and no diagnostic errors.
Pragmatic Impediments to PGD
ART costs $6000 to $12 000 per cycle and is only infrequently reimbursed by third-party carriers in North America. Molecular evaluation to identify the mutant allele and linked polymorphic markers generally costs another $3000, and analysis during the actual PGD cycle costs a further $2000 to $3000. Cumulative costs total perhaps $12 000 to $15 000. Still, PGD is cost-effective compared to the financial burden incurred by an affected child.
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NOVEL INDICATIONS FOR PGD |
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TopAbstractIntroductionOBTAINING PREIMPLANTATION CELLS...TECHNICAL CONSIDERATIONS IN...PGD FOR MENDELIAN DISORDERS...NOVEL INDICATIONS FOR PGDIS PGD SAFE?References |
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PGD makes possible novel management regimens not feasible with traditional prenatal genetic diagnosis.
Prenatal Diagnosis Without Disclosure of Parental Genotype
Suppose a clinically normal partner is known to be at risk for an autosomal dominant disorder of adult onset. This couple may wish to avoid transmitting a mutant gene to offspring yet still not know its own genotype. The prototype is Huntington disease, an adult onset disorder manifested after the age of reproduction. If a patient's parent has Huntington disease, the patient himself or herself has a 50% risk of inheriting the mutant allele and being affected; the risk for any given fetus is thus 25% (
x ).
If treatment were possible, early diagnosis would be beneficial and should be sought. If treatment is not possible, as is true for Huntington disease, draconian implications arise when an asymptomatic individual learns he/she has the Huntington disease gene. This scenario is also applicable to heritable neoplasias, such as Li-Fraumeni syndrome, in which multiple malignancies are common and the risk is 50% for having cancer before the age of 30. Traditional prenatal genetic diagnosis without disclosure is available, but in the long term it is impractical to expect the at-risk parent not to learn if he/she has the mutant gene. With PGD it becomes practical for at-risk adults to avoid transmitting the potential mutant gene to offspring yet still remain unaware of their own status. All embryos would be screened, but only those that are unaffected would be actually transferred. Given expectations for several unaffected embryos per cycle, success is realistic. A pitfall is that this subterfuge must persist in subsequent pregnancies even if testing proves the at-risk parent to be genetically normal.
Transfer of HLA-Antigen Identical Embryos ("Savior Siblings")
Although theoretically possible through traditional prenatal genetic diagnosis, PGD pragmatically allows identification and, hence, transfer of embryos of a specific HLA genotype. This approach was first performed by Verlinsky and colleagues (19), on a couple whose older daughter was dying of Fanconi anemia. The molecular basis of this autosomal recessive disorder is known (20,21). Treating the bone marrow failure and nearly inevitable leukemia requires stem cell transplantation. Stem cell transplantation with cord blood is highly successful if the cord blood is HLA compatible; success is far less in cases of HLA-IA compatibility (22,23).
If the couple desires another child, a reasonable strategy is to exclude Fanconi anemia as well as concurrently identify HLA-compatible embryos. The likelihood of an embryo being genetically normal (for an autosomal recessive trait) is 3 in 4; the likelihood of being HLA compatible is 1 in 4. Thus, the probability of the embryo fulfilling both requirements is 3 in 16. Success is rare but achievable. Using the same approach with CVS or amniocentesis would be daunting, likely resulting in multiple terminations with a still-low likelihood of success.
PGD for HLA testing was reported in 2001 (19), and by 2002 Rechitsky et al. (24) had summarized 18 PGD cycles: five 
-thalessemia, six Fanconi anemia, one Wiskott-Aldrich syndrome, and six leukemia. Experience is increasing especially with Fanconi anemia (25) and with -thalessemia. Overall, 45 cycles for HLA typing have been performed by the Reproductive Genetics Institute (6); 17.5% of embryos were genetically suitable for transfer, very near the expected 18.7% (3/16) (7).
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IS PGD SAFE? |
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TopAbstractIntroductionOBTAINING PREIMPLANTATION CELLS...TECHNICAL CONSIDERATIONS IN...PGD FOR MENDELIAN DISORDERS...NOVEL INDICATIONS FOR PGDIS PGD SAFE?References |
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Experience is limited in PGD, since only about 1000 PGD babies have been born (17). Systematic studies are lacking, but anomaly rates do not seem increased.
No increase in anomalies was observed in PGD neonates followed by the ESHRE Preimplantation Genetic Diagnosis Consortium (11) or in detailed descriptive analysis in one center (26). A caveat of relevance to the current communication is that most cases have been studied for chromosomal indications, rather than for Mendelian disorders or specifically for heritable neoplasia. However, there is little reason to believe safety is affected by indication. Mean birth weight seems normal (26).
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REFERENCES |
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TopAbstractIntroductionOBTAINING PREIMPLANTATION CELLS...TECHNICAL CONSIDERATIONS IN...PGD FOR MENDELIAN DISORDERS...NOVEL INDICATIONS FOR PGDIS PGD SAFE?References |
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