Preimplantation Diagnosis for Early-Onset Alzheimer Disease Caused by V717L Mutation

PRELIMINARY COMMUNICATION

Yury Verlinsky, PhD Context Indications for preimplantation genetic diagnosis (PGD) have recently been Svetlana Rechitsky, PhD expanded to include disorders with genetic predisposition to allow only embryos free Oleg Verlinsky, MS of predisposing genes to be preselected for transfer back to patients, with no potential for pregnancy termination. Christina Masciangelo, MS Objective To perform PGD for early-onset Alzheimer disease (AD), determined by Kevin Lederer, MD nearly completely penetrant autosomal dominant mutation in the amyloid precursor Anver Kuliev, MD, PhD protein (APP) gene. Design Analysis undertaken in 1999-2000 of DNA for the V717L mutation (valine CCORDING TO THE MOST RE- to leucine substitution at codon 717) in the APP gene in the first and second polar cent review,1 preimplanta- bodies, obtained by sequential sampling of oocytes following in vitro fertilization, to tion genetic diagnosis (PGD) preselect and transfer back to the patient only the embryos that resulted from mutation- has been applied to at least 50 free oocytes. differentA genetic conditions in more than Setting An in vitro fertilization center in Chicago, Ill. 3000 clinical cycles. In addition to tra- Patients A 30-year-old AD-asymptomatic woman with a V717L mutation that was ditional indications, similar to those in identified by predictive testing of a family with a history of early-onset AD. prenatal diagnosis, PGD was per- Main Outcome Measures Results of mutation analysis; pregnancy outcome. formed for an increasing number of new indications, such as late-onset disor- Results Four of 15 embryos tested for maternal mutation in 2 PGD cycles, originating from V717L mutation–free oocytes, were preselected for embryo transfer, yield- ders with genetic predisposition and ing a clinical pregnancy and birth of a healthy child free of predisposing gene muta- HLA testing combined with PGD for pre- tion according to chorionic villus sampling and testing of the neonate’s blood. existing single-gene disorders.2,3 These Conclusion This is the first known PGD procedure for inherited early-onset AD re- conditions have never been an indica- sulting in a clinical pregnancy and birth of a child free of inherited predisposition to tion for prenatal diagnosis because of po- early-onset AD. tential pregnancy termination, which is JAMA. 2002;287:1018-1021 www.jama.com highly controversial if performed for genetic predisposition alone. With the in- troduction of PGD, it has become pos- early-onset Alzheimer disease (AD), early-onset dementias associated with sible to avoid the transfer of the embryos representing a rare autosomal domi- APP mutations are nearly completely carrying the genes that predispose a per- nant familial predisposition to the pre- penetrant and, therefore, are potential son to common disorders, thereby es- senile form of dementia. Three differ- candidates for not only predictive test- tablishing only potentially healthy preg- ent genes have been found to be ing but also PGD. Of the 10 APP mu- nancies and overcoming important involved in this form of AD, including tations currently described, muta- ethical issues in connection with selec- presenilin 1 located on chromosome tions in exons 16 and 17 have been tive abortions. 14,4 presenilin 2 on chromosome 1,5 To our knowledge, this article pre- and amyloid precursor protein (APP) Author Affiliations: Reproductive Genetics Institute 6 (Drs Verlinsky, Rechitsky, and Kuliev, Mr Verlinsky, sents the first experience of PGD for on chromosome 21, which is well and Ms Masciangelo) and IVF Illinois (Dr Lederer), known for its role in the formation of Chicago. amyloid deposits found in the charac- Corresponding Author and Reprints: Yury Verlin- See also p 1038. sky, PhD, 2825 N Halsted St, Chicago, IL 60657 (e-mail: teristic plaques of patients with AD. The [email protected]).

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reported in the familial cases with the the linked polymorphic marker, rep- Figure 1. Pedigree of Family With earliest onset. One of these mutations, resenting the short tandem repeat in in- Early-Onset Alzheimer Disease (AD) 10 with onset as early as the mid or late tron 1 ([GA]n . . . [GT]n).

30s, is due to a single G-to-C nucleo- The first-round amplification cock- I tide substitution in exon 17, resulting tail for the multiplex nested PCR sys- in a valine-to-leucine amino acid change tem contained outer primers for both the 7 at codon 717 (V717L). This mutation APP gene and linked marker, whereas the II was identified in 3 of 5 family mem- second-round PCR used inner primers 1 2 3 4∗ 5 6

bers (siblings) tested, 1 of whom pre- for each gene. We designed the outer (10) (6) (7) (6) sented for PGD. primers APP-1 (5Ј-GTGTTCTTTG- CAGAAGATG-3Ј) and APP-102 (5Ј- Preimplantation III Genetic METHODS CATGGAAGCACACTGATTC-3Ј)for Diagnosis The patient who presented for PGD was performing the first-round amplifica- (6) (6) a 30-year-old woman with no signs of tion and the inner primers APP-101 (5Ј- Ј (10) Mutant Affected AD who carried the V717L mutation. GTTCAAACAAAGGTGCAATC-3 ) and Allele Ј The patient had been tested because her APP-103 (5 -TCTTAGCAAAAAGC- (6) (6) (7) Normal sister developed symptoms of AD at age TAAGCC-3Ј) for the second round of Alleles Unaffected 38 years and was found to be carrying PCR. As shown in FIGURE 2, second- Male Presumptively Affected this mutation.7 This sister is still alive, round PCR produces a 115–base pair Female but her cognitive problems pro- (bp) product, undigested by MnlI restric- Proband Deceased gressed to the point where she was tion enzyme, corresponding to the nor- placed in an assisted living facility. The mal allele, and 2 restriction fragments of Numbers in parentheses indicate number of repeats. patient’s father had died at age 42 years 72 and 43 bp, corresponding to the Preimplantation genetic diagnosis for asymptomatic carrier (II: 6) of the mutant gene linked to the 10 re- and had a history of psychological dif- mutant allele. There was also an invari- peats (10) (normal gene [N] is linked to 6 repeats [6]), ficulties and marked memory prob- ant fragment of 84 bp produced in both resulting in the birth of an unaffected child (III). Pa- ternal genotype is also shown, with the normal alle- lems. The V717L mutation was also de- normal and mutant alleles, which was les (N) linked to 6 repeats (6) and 7 repeats (7). Hap- tected in one of her brothers, who used as a control. lotype analysis shows that the child inherited normal maternal allele (N) linked to the 6 repeats (6). The pa- experienced mild short-term memory To perform nested PCR for specific tient’s sister (II: 1), brother (II: 3), and father (I) were problems as early as age 35 years, with amplification of the linked marker affected by early-onset AD. No predictive testing was a moderate decline in memory, new (GA) . . . (GT) in intron 1, we designed performed in her asymptomatic brother, indicated by n n an asterisk. learning, and sequential tracking in the the outer primers In1-1 (5Ј-CCT- next 2 to 3 years. Other family mem- TATTTCAAATTCCCTAC-3Ј) and In1-2 bers, including 1 brother and 2 sis- (5Ј-GATTGGAGGTTAAGTTTCTG- patient was also informed about the ex- ters, were asymptomatic,7 although pre- 3Ј) for the first round and the inner prim- pected number of embryos to be trans- dictive testing was done only in the ers In1-3 (5Ј-CAGCATCTGTCACT- ferred to achieve a pregnancy and the sisters, who appeared to be free of the CAAG-3Ј) and In1-4 (5Ј-AATATT- risks of multiple gestation, the misdi- APP gene mutation (FIGURE 1). TGTTACATTCCTCTC-3Ј) for the agnosis rates depending on the avail- Two PGD cycles were performed, in- second round of amplification. The hap- ability of the marker information in ad- volving 2 standard in vitro fertiliza- lotype analysis, based on the PB geno- dition to mutation analysis, and the tion cycles, coupled with microma- typing, demonstrated that the affected need for confirmation of PGD by pre- nipulation procedures, including allele was linked to the 10 and the nor- natal diagnosis. removal of polar body 1 (PB1) and po- mal one to the 6 repeats. lar body 2 (PB2) and intracytoplasmic The patient was counseled and gave RESULTS sperm injection, for which the patient consent for unaffected embryos that re- In the first in vitro fertilization cycle, 8 gave informed consent. The study was sulted from oocytes determined to be oocytes were available for testing, of approved by the institutional review mutation-free, based on both muta- which 2 were tested by both PB1 and board of the Illinois Masonic Medical tion and short tandem repeat analysis, PB2; both were affected. In the second Center, Chicago. Testing for the ma- to be preselected for transfer back to her in vitro fertilization cycle, 15 oocytes ternal mutation was done by DNA and those predicted to be mutant to be were available for testing, of which 13 analysis of PB1 and PB2, which were re- exposed to the confirmatory analysis us- were tested by both PB1 and PB2. The moved sequentially following matura- ing the genomic DNA from these em- mutation and linked marker analysis in tion and fertilization of oocytes.8 A mul- bryos to evaluate the accuracy of the intron 1 revealed 6 normal and 7 af- tiplex nested polymerase chain reaction single cell–based PGD. (We did not fected oocytes. The results of the sec- (PCR) was performed,9 involving the counsel the patient about her decision ond cycle, resulting in the embryo trans- mutation testing simultaneously with to undergo the PGD testing itself.) The fer, are presented in Figure 2. As shown

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in this figure, oocytes 4, 9, 14, and 15 marker was available for testing, a .05 study of the embryos that resulted from were clearly normal because both mu- probability of allele dropout of the nor- these oocytes confirmed their affected tant and normal genes were present in mal gene in the corresponding PB1 could status in all but 1 (oocyte 7). The latter their PB1, with the mutant gene further not be excluded, as established in our may be explained by allele dropout of extruded with the corresponding PB2, previous observations.11 the mutant allele in the apparently het- leaving only the normal gene in the re- The remaining oocytes were pre- erozygous PB1, which was left undetec- sulting oocyte. In addition, oocytes 3 and dicted to be mutant, based on hetero- ted because of the amplification failure 13 were also normal because their cor- zygous PB1 and normal PB2 in 4 of them of the linked marker in this case. responding PB1s were homozygous mu- (oocytes 1, 8, 10, and 11; the heterozy- To exclude any probability of mis- tant, suggesting that the resulting oo- gous status of PBI in oocyte 10 was based diagnosis, the priority in the embryo cytes should have been normal, as further on the presence of markers linked to transfer was given to 4 of the 6 normal confirmed by the presence of the nor- both normal and mutant alleles, which embryos, resulting from the oocytes mal gene in the extruded PB2s, also in is not shown in Figure 2) and homozy- with heterozygous PB2 and mutant PB2. agreement with the linked markers gous normal PB1 and mutant PB2 in 3 However, only 3 of these embryos de- analysis. However, because only 1 linked (oocytes 2, 6, and 7). The follow-up veloped into the cleavage stage and

Figure 2. Preimplantation Diagnosis for V717L Mutation in the Amyloid Precursor Protein (APP) Gene

A APP Gene

(GA)n(GT)n V717L (G C) Mutation In1-1 In1-3 APP1 APP101 5'Intron 1Exon 17 Intron 17 3'

In1-4 In1-2 APP103 APP102

B Mnl Ι Restriction Digestion

115 bp 84 bp Normal

72 bp 43 bp 84 bp Mutant

Mnl Ι Restriction Site

C Sequential Polar Body Analysis for V717L Mutation in APP Gene

Polar Body Polar Body

L 1 2 1 2 1 2 1 2 1 2 1 2 Uncut 1 L 2 1 21 2 1 21 21 21 2

ET ET ET ET Normal 115 bp

Invariant 84 bp Mutant 72 bp

Mutant 43 bp

Oocyte No. 12 3 4 6 78131415 9 10 11 ∗ ∗ Oocyte Genotype M M N N MM M N MMN NN

ET indicates embryo transfer; L, ladder (size standard); bp, base pair; PB, polar body; and uncut, the undigested polymerase chain reaction product. Arrows indicate fully nested primer sets. A, Map of human APP gene, showing sites and location of V717L G-to-C mutation and polymorphic markers. B, Restriction map for normal and abnormal alleles. C, Polyacrylamide gel analysis of MnlI restriction digestion, showing 6 unaffected (N) oocytes (3, 4, 9, 13, 14, and 15) and 7 mutant (M) oocytes (1, 2, 6, 7, 8, 10, and 11). Unaffected oocytes are identified by the gel showing normal (top band) and affected (lowest 2 bands) genes in PB1. After extrusion of the affected genes in PB2, only normal genes remain. Four of these oocytes (4, 9, 14, and 15) had heterozygous PB1 and mutant PB2, and only 2 (3, 13) had homozygous mutant PB1 and normal PB2, leaving a .05 probability for misdiagnosis, noted as N*. Oocyte 10 had heterozygous PB1 detected by marker analysis (not shown).

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could be transferred (4, 14, and 15), so to AD from the onset. Because the dis- herein, together with previously de- an additional embryo (3) was prese- ease never presents at birth or early scribed cases of PGD for late-onset lected, originating from the oocyte with childhood and even later may not be ex- disorders with genetic predisposition homozygous mutant PB1 and the nor- pressed in 100% of cases, the applica- and HLA typing, demonstrate the mal PB2, since these results were also tion of PGD for AD is still controver- extended practical implications of confirmed by the linked marker analy- sial. However, because there is currently PGD, such as providing prospective sis. These 4 embryos were transferred no treatment for AD, which may arise couples at genetic risk with more re- back to the patient, yielding a single- despite presymptomatic diagnosis and productive options for having unaf- ton clinical pregnancy, confirmed to be follow-up, PGD seems to be the only fected children. unaffected by chorionic villus sam- relief for at-risk couples, such as the pre- pling and birth of a mutation-free child sented case and the previously re- Author Contributions: Study concept and design: confirmed after birth by a blood test. ported cases of PGD for p53 tumor sup- Y. Verlinsky, Rechitsky, Kuliev. pressor gene mutations.2 Acquisition of data: Rechitsky, O. Verlinsky, COMMENT Masciangelo, Lederer. Therefore, prospective parents who Analysis and interpretation of data: Rechitsky, The results presented herein demon- are determined by strong genetic pre- Kuliev. Drafting of the manuscript: Kuliev. strate the feasibility of PGD for early- disposition to be at risk for producing Critical revision of the manuscript for important in- onset AD, providing a nontraditional progeny with severe disorders should tellectual content: Y. Verlinsky, Rechitsky, O. Verlin- option for patients who wish to avoid be informed about this emerging tech- sky, Masciangelo, Lederer. Obtained funding: Y. Verlinsky. the transmission of the mutant gene that nology so they can make a choice about Administrative, technical, or material support: O. Ver- predisposes their potential children to reproduction.12,13 This seems to be ethi- linsky, Masciangelo, Lederer. Study supervision: Y. Verlinsky, Rechitsky, Kuliev. early-onset AD. For some patients, this cally more acceptable than suppress- Funding/Support: Funding for this study was pro- may be the only reason for undertak- ing information on the availability of vided by the Reproductive Genetics Institute, Chicago, Ill. ing pregnancy, since the pregnancy may PGD. Despite raising important ethi- Acknowledgment: We thank the patient for her per- be free of an inherited predisposition cal issues,14,15 the results presented mission to publish this information.

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