Context Indications for preimplantation genetic diagnosis (PGD) have recently
been expanded to include disorders with genetic predisposition to allow only
embryos free of predisposing genes to be preselected for transfer back to
patients, with no potential for pregnancy termination.
Objective To perform PGD for early-onset Alzheimer disease (AD), determined by
nearly completely penetrant autosomal dominant mutation in the amyloid precursor
protein (APP) gene.
Design Analysis undertaken in 1999-2000 of DNA for the V717L mutation (valine
to leucine substitution at codon 717) in the APP
gene in the first and second polar bodies, obtained by sequential sampling
of oocytes following in vitro fertilization, to preselect and transfer back
to the patient only the embryos that resulted from mutation-free oocytes.
Setting An in vitro fertilization center in Chicago, Ill.
Patients A 30-year-old AD-asymptomatic woman with a V717L mutation that was identified
by predictive testing of a family with a history of early-onset AD.
Main Outcome Measures Results of mutation analysis; pregnancy outcome.
Results Four of 15 embryos tested for maternal mutation in 2 PGD cycles, originating
from V717L mutation–free oocytes, were preselected for embryo transfer,
yielding a clinical pregnancy and birth of a healthy child free of predisposing
gene mutation according to chorionic villus sampling and testing of the neonate's
blood.
Conclusion This is the first known PGD procedure for inherited early-onset AD resulting
in a clinical pregnancy and birth of a child free of inherited predisposition
to early-onset AD.
According to the most recent review,1
preimplantation genetic diagnosis (PGD) has been applied to at least 50 different
genetic conditions in more than 3000 clinical cycles. In addition to traditional
indications, similar to those in prenatal diagnosis, PGD was performed for
an increasing number of new indications, such as late-onset disorders with
genetic predisposition and HLA testing combined with PGD for preexisting single-gene
disorders.2,3 These conditions
have never been an indication for prenatal diagnosis because of potential
pregnancy termination, which is highly controversial if performed for genetic
predisposition alone. With the introduction of PGD, it has become possible
to avoid the transfer of the embryos carrying the genes that predispose a
person to common disorders, thereby establishing only potentially healthy
pregnancies and overcoming important ethical issues in connection with selective
abortions.
To our knowledge, this article presents the first experience of PGD
for early-onset Alzheimer disease (AD), representing a rare autosomal dominant
familial predisposition to the presenile form of dementia. Three different
genes have been found to be involved in this form of AD, including presenilin
1 located on chromosome 14,4 presenilin 2 on
chromosome 1,5 and amyloid precursor protein
(APP) on chromosome 21,6
which is well known for its role in the formation of amyloid deposits found
in the characteristic plaques of patients with AD. The early-onset dementias
associated with APP mutations are nearly completely
penetrant and, therefore, are potential candidates for not only predictive
testing but also PGD. Of the 10 APP mutations currently
described, mutations in exons 16 and 17 have been reported in the familial
cases with the earliest onset. One of these mutations, with onset as early
as the mid or late 30s, is due to a single G-to-C nucleotide substitution
in exon 17, resulting in a valine-to-leucine amino acid change at codon 717
(V717L).7 This mutation was identified in 3
of 5 family members (siblings) tested, 1 of whom presented for PGD.
The patient who presented for PGD was a 30-year-old woman with no signs
of AD who carried the V717L mutation. The patient had been tested because
her sister developed symptoms of AD at age 38 years and was found to be carrying
this mutation.7 This sister is still alive,
but her cognitive problems progressed to the point where she was placed in
an assisted living facility. The patient's father had died at age 42 years
and had a history of psychological difficulties and marked memory problems.
The V717L mutation was also detected in one of her brothers, who experienced
mild short-term memory problems as early as age 35 years, with a moderate
decline in memory, new learning, and sequential tracking in the next 2 to
3 years. Other family members, including 1 brother and 2 sisters, were asymptomatic,7 although predictive testing was done only in the sisters,
who appeared to be free of the APP gene mutation
(Figure 1).
Two PGD cycles were performed, involving 2 standard in vitro fertilization
cycles, coupled with micromanipulation procedures, including removal of polar
body 1 (PB1) and polar body 2 (PB2) and intracytoplasmic sperm injection,
for which the patient gave informed consent. The study was approved by the
institutional review board of the Illinois Masonic Medical Center, Chicago.
Testing for the maternal mutation was done by DNA analysis of PB1 and PB2,
which were removed sequentially following maturation and fertilization of
oocytes.8 A multiplex nested polymerase chain
reaction (PCR) was performed,9 involving the
mutation testing simultaneously with the linked polymorphic marker, representing
the short tandem repeat in intron 1 ([GA]n . . . [GT]n).10
The first-round amplification cocktail for the multiplex nested PCR
system contained outer primers for both the APP gene
and linked marker, whereas the second-round PCR used inner primers for each
gene. We designed the outer primers APP-1 (5′-GTGTTCTTTGCAGAAGATG-3′)
and APP-102 (5′-CATGGAAGCACACTGATTC-3′) for performing the first-round
amplification and the inner primers APP-101 (5′-GTTCAAACAAAGGTGCAATC-3′)
and APP-103 (5′-TCTTAGCAAAAAGCTAAGCC-3′) for the second round
of PCR. As shown in Figure 2, second-round
PCR produces a 115–base pair (bp) product, undigested by MnlI restriction enzyme, corresponding to the normal allele, and 2
restriction fragments of 72 and 43 bp, corresponding to the mutant allele.
There was also an invariant fragment of 84 bp produced in both normal and
mutant alleles, which was used as a control.
To perform nested PCR for specific amplification of the linked marker
(GA)n . . . (GT)n in intron 1, we designed the outer
primers In1-1 (5′-CCTTATTTCAAATTCCCTAC-3′) and In1-2 (5′-GATTGGAGGTTAAGTTTCTG-3′)
for the first round and the inner primers In1-3 (5′-CAGCATCTGTCACTCAAG-3′)
and In1-4 (5′-AATATT-TGTTACATTCCTCTC-3′) for the second round
of amplification. The haplotype analysis, based on the PB genotyping, demonstrated
that the affected allele was linked to the 10 and the normal one to the 6
repeats.
The patient was counseled and gave consent for unaffected embryos that
resulted from oocytes determined to be mutation-free, based on both mutation
and short tandem repeat analysis, to be preselected for transfer back to her
and those predicted to be mutant to be exposed to the confirmatory analysis
using the genomic DNA from these embryos to evaluate the accuracy of the single
cell–based PGD. (We did not counsel the patient about her decision to
undergo the PGD testing itself.) The patient was also informed about the expected
number of embryos to be transferred to achieve a pregnancy and the risks of
multiple gestation, the misdiagnosis rates depending on the availability of
the marker information in addition to mutation analysis, and the need for
confirmation of PGD by prenatal diagnosis.
In the first in vitro fertilization cycle, 8 oocytes were available
for testing, of which 2 were tested by both PB1 and PB2; both were affected.
In the second in vitro fertilization cycle, 15 oocytes were available for
testing, of which 13 were tested by both PB1 and PB2. The mutation and linked
marker analysis in intron 1 revealed 6 normal and 7 affected oocytes. The
results of the second cycle, resulting in the embryo transfer, are presented
in Figure 2. As shown in this figure,
oocytes 4, 9, 14, and 15 were clearly normal because both mutant and normal
genes were present in their PB1, with the mutant gene further extruded with
the corresponding PB2, leaving only the normal gene in the resulting oocyte.
In addition, oocytes 3 and 13 were also normal because their corresponding
PB1s were homozygous mutant, suggesting that the resulting oocytes should
have been normal, as further confirmed by the presence of the normal gene
in the extruded PB2s, also in agreement with the linked markers analysis.
However, because only 1 linked marker was available for testing, a .05 probability
of allele dropout of the normal gene in the corresponding PB1 could not be
excluded, as established in our previous observations.11
The remaining oocytes were predicted to be mutant, based on heterozygous
PB1 and normal PB2 in 4 of them (oocytes 1, 8, 10, and 11; the heterozygous
status of PBI in oocyte 10 was based on the presence of markers linked to
both normal and mutant alleles, which is not shown in Figure 2) and homozygous normal PB1 and mutant PB2 in 3 (oocytes
2, 6, and 7). The follow-up study of the embryos that resulted from these
oocytes confirmed their affected status in all but 1 (oocyte 7). The latter
may be explained by allele dropout of the mutant allele in the apparently
heterozygous PB1, which was left undetected because of the amplification failure
of the linked marker in this case.
To exclude any probability of misdiagnosis, the priority in the embryo
transfer was given to 4 of the 6 normal embryos, resulting from the oocytes
with heterozygous PB2 and mutant PB2. However, only 3 of these embryos developed
into the cleavage stage and could be transferred (4, 14, and 15), so an additional
embryo (3) was preselected, originating from the oocyte with homozygous mutant
PB1 and the normal PB2, since these results were also confirmed by the linked
marker analysis. These 4 embryos were transferred back to the patient, yielding
a singleton clinical pregnancy, confirmed to be unaffected by chorionic villus
sampling and birth of a mutation-free child confirmed after birth by a blood
test.
The results presented herein demonstrate the feasibility of PGD for
early-onset AD, providing a nontraditional option for patients who wish to
avoid the transmission of the mutant gene that predisposes their potential
children to early-onset AD. For some patients, this may be the only reason
for undertaking pregnancy, since the pregnancy may be free of an inherited
predisposition to AD from the onset. Because the disease never presents at
birth or early childhood and even later may not be expressed in 100% of cases,
the application of PGD for AD is still controversial. However, because there
is currently no treatment for AD, which may arise despite presymptomatic diagnosis
and follow-up, PGD seems to be the only relief for at-risk couples, such as
the presented case and the previously reported cases of PGD for p53 tumor
suppressor gene mutations.2
Therefore, prospective parents who are determined by strong genetic
predisposition to be at risk for producing progeny with severe disorders should
be informed about this emerging technology so they can make a choice about
reproduction.12,13 This seems
to be ethically more acceptable than suppressing information on the availability
of PGD. Despite raising important ethical issues,14,15
the results presented herein, together with previously described cases of
PGD for late-onset disorders with genetic predisposition and HLA typing, demonstrate
the extended practical implications of PGD, such as providing prospective
couples at genetic risk with more reproductive options for having unaffected
children.
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