Download Preimplantation genetic testing for Marfan syndrome

Molecular Human Reproduction vol.2 no.9 pp. 713-715, 1996
Preimplantation genetic testing for Marfan syndrome
G.LHarton 1 - 5 , P.Tsipouras2, M.E.Sisson 1 , K.M.Starr 1 , B.S.Mahoney 1 , E.F.Fugger1-3, J.D.Schulman 1 - 3 ' 4 ,
M.W.Kilpatrick 2 , G.Levinson 14 and S.H.Black 13
Genetics & IVF Institute, 3020 Javier Road, Fairfax, VA 22031, department of Pediatrics, The School of Medicine of The
University of Connecticut Health Center, Farmington, CT 06030, 3Department of Obstetrics and Gynecology, Medical
College of Virginia, Richmond, VA, department of Human Genetics, Medical College of Virginia, Richmond, VA, USA
5
To whom correspondence should be addressed
Marfan syndrome (MFS) is an autosomal dominant disease that affects the skeletal, ocular and cardiovascular
systems. Defects in the gene that codes for fibrillin (FBN-1) are responsible for MFS. Here we report the
world's first use of preimplantation genetic testing (PGT) to achieve a clinical pregnancy and live birth of a
baby free of a Marfan mutation. One or two blastomeres from each embryo were tested for a CA repeat
within the FBN-1 gene. The prospective mother is homozygous for the CA repeat (2/2) and has two normal
copies of the FBN-1 gene, while the prospective father is heterozygous for the CA repeat (1/2), and is affected
with the Marfan syndrome. In the father's family, allele 2 segregates with the mutated FBN-1 gene. For PGT,
any embryo diagnosed as heterozygous for the CA repeat (1/2) would be presumed to have inherited normal
FBN-1 genes from the father and the mother and be unaffected. One in-vitro fertilization (IVF) cycle yielded
12 embryos for preimplantation testing; six of the embryos were heterozygous for the CA repeat (1/2) and
presumed to be free of the Marfan mutation. Five of the six embryos were subsequently transferred into the
uterus. The fetus was tested by chorionic villus sampling and found to be free of the Marfan mutation by the
same linkage analysis, had a normal fetal echocardiogram, and was normal at birth.
Key words: fibrillin-1/linkage analysis/Marfan syndrome/polymerase chain reaction (PCR)/preimplantation
genetic testing (PGT)
Introduction
Materials and methods
Marfan syndrome (MFS) is a disease of the connective tissues,
and is inherited in an autosomal dominant fashion. MFS
affects approximately 1 in 10 000 people (Dietz et al, 1993).
Manifestations of MFS include pectus deformity, arachnodactyly, scoliosis, high and narrow palate, ectopia lentis,
myopia, dilation of the ascending aorta, aortic dissection,
mitral valve abnormality, and dural ectasia (Pyeritz, 1993).
The clinical features of MFS arise from a lack of normal
fibrillin, a structural constituent of connective tissue. The
underlying defect in MFS is due to a defective fibrillin gene
(FBN-1) which has been localized to chromosome 15q 15-21
Patient history
The prospective mother was 35 years old, gravida 1, parity 0. Her
husband was affected with the Marfan syndrome. His sister had a
sudden aortic dissection at age 32, and his father, who was also
affected, died at 34 years of age. The patient's previous pregnancy
was terminated after prenatal linkage analysis revealed that the fetus
had a mutated FBN-1 allele.
The prospective mother and father had a distinguishable complement of alleles at the CA repeat within FBN-1 (Figure 1). The
prospective mother was homozygous (2/2) at this locus, while the
prospective father was heterozygous (1/2). Allele 2 segregated with
MFS in the father's family. Any embryo that exhibited a heterozygous
complement at this locus (1/2) would be free of the MFS mutation.
(Leeetal., 1991).
Preimplantation genetic testing (PGT) of in-vitro fertilized
(IVF) embryos offers prospective parents who carry genetic
disorders the chance to conceive children of their own with a
greatly reduced risk of transmission of the disease (Harper
and Handyside, 1994; Levinson et al, 1995). Until now,
patients affected with MFS have not had this opportunity.
Their only option was prenatal genetic testing (analysis of
fetal cells after amniocentesis or chorionic villus sampling),
followed by possible termination of the pregnancy if the fetus
was carrying the mutation. In this report, we describe a
preimplantation genetic testing cycle to obtain embryos free
of the Marfan syndrome mutation.
© European Society for Human Reproduction and Embryology
IVF and embryo biopsy
IVF stimulation and oocyte retrieval were as previously described
(Levinson et al., 1995). A total of 17 eggs were retrieved, of which
16 were viable. Each viable egg was inseminated in a microdroplet
under oil with ~2500 spermatozoa 6 h after retrieval. In all, 12
dividing embryos developed, and were biopsied as previously
described (Levinson et al, 1992).
Nested polymerase chain reaction
Reagents
Lysis mix I was as follows: 1.29b Tween 20, 1.2% Triton X-100,
3.4 mM EDTA, 2.7 mM dithiothreitol, 50 mM Tris-HCl, 4 IU/ml
713
G.L.Harton et al.
1/2
2/2
1/2
1/2
2/2
Figure 1. Family pedigree. • = unaffected male; O = unaffected
female; • = male affected with Marfan syndrome; • = female
affected with Marfan syndrome. Numbers show CA repeat alleles 1
and 2. Allele 2 is associated with Marfan syndrome mutation in
the affected family.
freshly added proteinase K (Boehringer Mannheim, Indianapolis, IN,
USA), pH 7.5, plus first round PCR primers FBN1-10: 5' GAT GTC
CCT ATT GCC ATC A 3' (2 u:M), FBN1-20: 5' CCT GTG CAG
GGT AAG AC A A 3' (2 ujvl), AMXY1: 5' CCC CTT TGA AGT
GGT ACC AGA G 3' (0.6 uM), AMXY2: 5' ACG GGG ATG ATT
TGG TGG TG 3' (0.6 uM), DYZ yl.l: 5' TCC ACT TTA TTC
CAG GCC TGT CC 3' (0.06 nM), and DYZ yl.2: 5' TTG AAT
GGA ATG GGA ACG AAT GG 3' (0.06 u;M).
PCR mix I was as follows: 10 mM Tris-HCl, 50 mM KC1, 0.13 mM
KH2PO4, 12 mM NaCI, 4.3 mM MgCl2, 0.7 mM Na2HPO4, 200 uJvl
each of the four dNTPs (Pharmacia, Piscataway, NJ, USA), 5 |ig/ml
acetylated bovine serum albumin (Sigma), and 0.03 IU/|il of freshly
added Taq DNA polymerase (Boehringer Mannheim), pH 8.3.
PCR Mix O was as follows: 10 mM Tris-HCl, pH 8.3, 50 mM
KC1, 1.5 mM MgCl2, 200 uM each of the four dNTPs, 0.03 IU/u.1
freshly added Taq DNA polymerase, and second round PCR primers
FBN1-1: 5' GAT GTC CCT ATT GCC ATC AC 3', and end-labelled
FBN1-2: 5' CCT GTG CAG GGT AAG ACA AG 3' (y*2P ATP,
6000 Ci/mmol, NEN Dupont, Boston, MA, USA), each at 0.2 U.M
final concentration (P.Tsipouras, personal communication).
Procedure
12
3 4 5 6 7 allele
.. • 11
Figure 2. Autoradiograph of polymerase chain reaction (PCR)
products from a CA repeat within the FBN-l gene from clinical
preimplantation genetic testing for Marfan syndrome. Lane I,
maternal DNA (10 000 copies); lane 2, paternal DNA (10 000
copies); lanes 3-7, single blastomeres from each of the five
embryos that were transferred.
Table I. Summary of individual blastomere data
Embryo No.
1
2
3
*
Blastomere No. FBN-l Status Gender
2/2
2/2
2/2
2/2
1/2
1/2
1/2
1/2
•S
1/2
1/2
&
1/2
1/2
2/2
2/2
2/2
1/2
No Signal
1/2
No Signal
1/2
1/2
2/2
2/2
7
8
11
13
15
16
714
Male
Male
Male
Male
Female
Ambiguous
Male
Male
Male
No signals
Female
Female
Male
Male
No signals
Female
No signals
Female
No signals
Female
Female
Male
Female
Transferred
No.
No
No
Yes
Yes
Yes
No
No
Yes
No
Yes
No
Each individual blastomere was removed from the surrounding
medium using a silanized 20 fj.1 Unopette (Fisher Scientific, Pittsburgh,
PA, USA), and washed two times in 5% fetal bovine serum/phosphatebuffered saline (FBS/PBS) (Life Technologies Inc., Gaithersburg,
MD, USA) at room temperature. After two washes, the blastomere
was transferred in 1-2 (il of 5% FBS/PBS to a labelled 0.5 ml
silanized polymerase chain reaction (PCR) tube containing 5 (0.1 of
Lysis Mix I using a different silanized Unopette. Samples were
covered with two drops of mineral oil (Sigma Chemical Co., St.
Louis, MO, USA), and incubated at 65°C for 10 min, followed by
immediate incubation at 95°C for 10 min. Each sample was then
allowed to cool to room temperature while all other samples were
being processed. PCR Mix I (44 (j.1) was then added to each sample,
under the oil. The samples were held at 85°C prior to the next step
(-10 min). Samples were then thermocycled (Perkin Elmer Cetus,
Foster City, CA, USA) as follows: initial denaturation, 94°C, 4 min,
followed by four cycles of denaturation at 94°C, 2 min, annealing at
56°C, 1 min, and extension at 72°C, 1 min 30 s, followed by 31
cycles as above but with denaturation shortened to 1 min, followed
by a final extension of 72°C for 5 min.
Second round PCR for FBN-l was performed by adding 1 (il of
first round PCR product to 49 |il PCR Mix II, covering with two
drops of mineral oil and thermocycling with initial denaturation of
94°C, 4 min, followed by 35 cycles of denaturation at 94°C, 1 min,
annealing at 60°C, 1 min, and extension at 72°C, 1 min 30 s, followed
by final extension of 72°C for 5 min.
PCR primers for gender were multiplexed with FBN-l primers as
an internal control for PCR of single cells. Separate second rounds
were performed for amelogenin X and amelogenin Y, as well as for
DYZ1, as previously described (Levinson el al., 1992, 1995).
PCR products from FBN-l primer sets were visualized by denaturing polyacrylamide gel electrophoresis using 5% Long Ranger™
modified acrylamide (FMC Bioproducts, Rockland, ME, USA) prepared with urea (Life Technologies) per manufacturer's instructions.
One volume of second round PCR product was mixed with two
volumes of formamide/EDTA Stop Solution (US Biochemicals, Cleveland, OH, USA), heated to 95°C for 5 min and then held at 72°C
prior to loading on the gel. The gel was prewarmed at 80 W constant
power for 30 min prior to loading 6 |il/ lane. To aid in determining
the size of PCR products, a G lane from an M13mpl8 sequencing
reaction (US Biochemical) labelled with [33S]-dATP (NEN Dupont)
was run, as well as second round PCR products from maternal and
Preimplantation genetic testing for Marfan syndrome
paternal DNAs as gel controls. Gels were run at 80 W constant power
for 2000 volt-hours, vacuum dried at 80°C without fixation, and
autoradiographed with XAR-5 film (Kodak, Rochester, NY, USA) for
30 min to 1 h at room temperature.
Results
By ~72 h after retrieval, 12 embryos had divided and were
biopsied at the 3-10-cell stage. For added reliability, two
blastomeres were removed from all embryos except the 3-cell
embryo (from which only one blastomere was removed), and
analysed in separate PCR reaction tubes. Six embryos were
determined to be heterozygous (1/2) for the CA repeat (Table I).
The patients elected to transfer five of the six presumably
unaffected embryos (Figure 2). A healthy 8 pound, 11 ounce
male was delivered vaginally at term. His physical examination
was normal. Testing by chorionic villus sampling had confirmed
that the fetus did not inherit the MFS allele, and a fetal
echocardiogram had also been normal. All other embryos were
cryopreserved.
References
Dietz, H.C., Mclntosh, I., Sakai, L.Y. et al. (1993) Four novel FBN-1
mutations: significance for mutant transcript level and EGF-like domain
calcium binding in the pathogenesis of Marfan syndrome. Cenomics, 17,
468-^*75.
Harper, J.C. and Handyside, A.H., (1994) The current status of preimplantation
diagnosis. Curr. Obstel. Gynaecol., 4, 143-149.
Lee, B., Godfrey. M., Vitale, E. et al. (1991) Linkage of Marfan syndrome
and a phenolypically related disorder to two different fibrillin genes. Nature,
352, 330-334.
Levinson, G., Fields, R.A., Harton, G.L. et al. (1992) Reliable gender screening
for human preimplantation embryos, using multiple DNA target-sequences.
Hum. Reprod., 7, 1304-1313.
Levinson, G., Keyvanfar, K., Wu, J. et al. (1995) DNA-based X-enriched
sperm separation as an adjunct to preimplantation genetic testing for the
prevention of X-linked disease. Mol. Hum. Reprod., 1, see Hum. Reprod.,
10, 979-982.
Pyeritz, R.E. (1993) The Marfan syndrome. In Steinmann B. (ed.), Connective
Tissue and its Heritable Disorders. Wiley-Liss, New York, pp. 437—468.
Received on May 20. 1995; accepted on July 15. 1996
Discussion
We report the first clinical use of PGT to avoid a child affected
with the Marfan syndrome. The method of linkage analysis
should be applicable to this and other diseases for which a
direct test is not available, providing that: (i) linkage phase
can be rigorously determined from the DNA of relatives who
are known carriers; (ii) sufficient polymorphism exists to allow
the investigator to clearly distinguish maternal and paternal
markers such as CA repeats; and (iii) the linkage markers
allow robust nested amplification from a single cell.
The importance of internal PCR controls in each reaction
and the use of more than one blastomere from each embryo,
whenever possible, can be seen in Table I. Our laboratory uses
both of these steps to try to eliminate any possibility of
contamination from exogenous DNA or cellular material in
our PCR reactions for PGT. Any contamination from outside
the PCR tube could result in a misdiagnosis of an affected
embryo as a normal embryo and lead to an affected baby
being bom. Embryos that have conflicting PCR results are not
recommended for transfer.
Allelic dropout (failure to detect an allele which may result
from competition between two alleles for reagents in the PCR
tube) is another important problem for PGT of autosomal
dominant diseases. The diagnostic system must be tested
rigorously before a patient is cycled to determine the frequency
of allelic dropout for single cell assays. In the present case,
allelic dropout of the normal allele would lead to nonreplacement of an unaffected embryo, but not to the misdiagnosis of an affected embryo as normal.
Because of phenotypic variability, lack of a genetic or
biochemical test for the disease, and a high rate of sporadic
disease, the actual incidence of the Marfan syndrome may be
considerably higher than the reported 1 in 10 000 (Dietz et al.,
1993). With growing awareness of and research on the Marfan
syndrome and the present case experience, the clinical demand
for a reliable PGT is likely to increase.
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