Preimplantation HLA Testing

ORIGINAL CONTRIBUTION Preimplantation HLA Testing Yury Verlinsky, PhD Context Preimplantation genetic diagnosis (PGD) has become an option for couples Svetlana Rechitsky, PhD for whom termination of an affected pregnancy identified by traditional prenatal diagnosis is unacceptable and is applicable to indications beyond those of prenatal di- Tatyana Sharapova, MS agnosis, such as HLA matching to affected siblings to provide stem cell transplanta- Randy Morris, MD tion. Mohammed Taranissi, MD Objective To describe preimplantation HLA typing, not involving identification of a causative gene, for couples who had children with bone marrow disorders at need for Anver Kuliev, MD, PhD HLA-matched stem cell transplantation. REIMPLANTATION GENETIC DIAG- Design, Setting, and Participants HLA matching procedures conducted at a single nosis (PGD) has become avail- site during 2002-2003 in an in vitro fertilization program for 9 couples with children able as an alternative to prena- affected by acute lymphoid leukemia, acute myeloid leukemia, or Diamond-Blackfan anemia requiring HLA-matched stem cell transplantation. In 13 clinical cycles, DNA in tal diagnosis in order to avoid single blastomeres removed from 8-cell embryos following in vitro fertilization was Pthe risk for pregnancy termination, be- analyzed for HLA genes simultaneously with analysis for short tandem repeats in the cause PGD allows selection of unaf- HLA region to select and transfer only those embryos that were HLA matched to fected embryos before a pregnancy is es- affected siblings. tablished. Despite the need for ovarian Main Outcome Measures Results of HLA matching and pregnancy outcome. stimulation and in vitro fertilization (IVF) to be part of the procedure, PGD Results As a result of testing a total of 199 embryos, 45 (23%) HLA-matched embryos were selected, of which 28 were transferred in 12 clinical cycles, resulting in 5 has become an acceptable method for singleton pregnancies and birth of 5 HLA-matched healthy children. avoiding the birth of children with genetic disorders.1-3 Introduced more than Conclusion This is the first known experience of preimplantation HLA typing performed without PGD for a causative gene, providing couples with a realistic option of a decade ago, PGD has been applied in 4 having HLA-matched offspring to serve as potential donors of stem cells for their af- thousands of clinical cycles. fected siblings. At present, more than 100 different JAMA. 2004;291:2079-2085 www.jama.com genetic conditions are indications for PGD, including a few novel indications for which traditional prenatal di- sulted in the birth of an HLA-matched METHODS agnosis has never been used.5-9 This in- child free of FA whose cord blood stem Setting cludes preimplantation HLA matching cells were transplanted to the affected The preimplantation testing proce- combined with PGD, not only to al- sibling with FA, resulting in a success- dures were conducted at the Reproduc- low couples to have an unaffected child, ful hematopoietic reconstitution. HLA tive Genetics Institute (RGI), Chi- but also to select a potential donor prog- matching would not be considered ap- cago, Ill. The RGI was established in eny for stem cell transplantation.8 The propriate for prenatal diagnosis be- 1989 and in 1994 was designated as a approach has previously been applied cause of the potential for termination Pan American Health Organization/ to avoid the birth of a child with of pregnancy, which could not be jus- World Health Organization Col- Fanconi anemia (FA), which is a se- tified for the reason of HLA incompat- laborating Center for Prevention of vere autosomal recessive disorder char- ibility. Genetic Disorders. The study was ap- acterized by inherited bone marrow fail- We describe the first clinical expe- ure requiring bone marrow or cord rience of preimplantation HLA match- Author Affiliations: Reproductive Genetics Insti- blood transplantation from an HLA- ing without PGD of a causative gene, tute, Chicago, Ill (Drs Verlinsky, Rechitsky, Morris, and 8 Kuliev and Ms Sharapova); and Assisted Reproduc- matched sibling. This approach re- demonstrating the feasibility of this tion and Gynaecology Center, London, England (Dr novel approach for stem cell trans- Taranissi). plantation in siblings with bone mar- Corresponding Author: Anver Kuliev, MD, PhD, Re- For editorial comment see p 2125. productive Genetics Institute, 2825 N Halsted St, Chi- row failure. cago, IL 60657 ([email protected]). ©2004 American Medical Association. All rights reserved. (Reprinted) JAMA, May 5, 2004—Vol 291, No. 17 2079 Downloaded From: https://jamanetwork.com/ on 09/24/2021 PREIMPLANTATION HLA TESTING proved by the RGI institutional re- the mutation was performed elsewhere). Figure 1. Set of Polymorphic STR Markers in HLA Region of Chromosome 6 Used for view board, which is composed of in- Each of the couples desired to have an- Preimplantation HLA Typing dividuals who are independent of the other child—a desire separate from the RGI and which includes 2 obstetri- hope of having another child who could cians; 1 clinical geneticist physician; 1 potentially serve as a donor of stem cells Telomere internist; 1 anesthesiologist; 1 indi- for the affected sibling, for whom an ac- STRs vidual with a master’s degree in ge- ceptable HLA match had not been found. D6S461 netic counseling; 1 medical laboratory As there is no institutional setting for D6S276∗ technologist; 2 individuals with doc- identification of couples at need for pre- D6S299 torate degrees in public health and psy- implantation HLA testing, all couples re- chology, respectively; 3 individuals with quested the test on their own, having master’s degrees in public relations, in- learned of the availability of the tech- ∗ D6S464 dustrial relations, and nursing, respec- nique through their physicians or the D6S105 tively; and a church pastor. Of these, 6 Internet. D6S306∗ are women. The institutional review A standard protocol for IVF, which D6S1624∗ board approval covers protocol and includes a psychological evaluation per- D6S258 consent forms, which allows publica- formed by a psychologist at the RGI, D6S248∗ tion of the preimplantation genetic di- was used in combination with a micro- MOG a,b,c,d agnosis data presented herein. All sets manipulation procedure to remove of parents provided written informed single blastomeres from the cleaving RF Genes consent for preimplantation genetic di- embryos at day 3, as described else- HLA-F agnosis testing. where.12 HLA genes from the blasto- HLA-A meres were tested simultaneously with D6S265 IVF Protocol short tandem repeats in the HLA re- D6S510 Thirteen IVF cycles (initial cycles did not gion13 using a multiplex heminested HLA-E have the desired outcome for 4 couples) polymerase chain reaction (PCR) sys- 12,14 HLA-C were initiated in 2002-2003 at the RGI tem involving only closely linked HLA-B for 9 couples from the United States and polymorphic short tandem repeat mark- MIB England who had a child affected with ers located throughout the HLA re- acute lymphoid leukemia or acute my- gion, as shown in FIGURE 1 (HLA genes: Chr 6 MICA eloid leukemia (11 cycles), or Diamond- HLA-A1, -A2, -A3, -A24; -A32; HLA- TNF a,b,c,d Blackfan anemia (DBA) (2 cycles). These C4, -C6; HLA-B27, -B18, -B35, -B51, p HLA Region 62 conditions require bone marrow trans- -B57; HLA-DRB1*1, -DRB1*7, -DRB1*10, 6p21.3 82-1 plantation and also have been success- and -DRB1*11. Short tandem repeats: 9N-2 fully treated by cord blood transplanta- D6S461; D6S276*; D6S299; D6S464*; 10 * * D6S273∗ tion. Although mutation testing also D6S105; D6S306 ; D6S1624 ; D6S258; q * HLA-DR may be required in combination with D6S248 ; MOG a,b,c,d; RF; D6S265; DN HLA typing in DBA, which can be caused D6S510; MIB; MICA; TNF a,b,c,d; 62; * LH1 by mutations in the gene encoding ri- 82-1; 9N-2; D6S273 ; DN; LH1; DQ- bosomal protein S19 on chromosome 19 CAR II; DQ-CAR; G51152; D6S2447; HLA-DQ (19q13.2) or in another gene mapped to TAP1; Ring 3CA; D6S439*; D6S291; DQ-CAR II chromosome 8 (8p23.3-p22), the ma- and D6S426). Figure 1 presents posi- DQ-CAR Class IIjority Class III of DBA cases are sporadic Class I with no tions of closely linked short tandem G51152 mutation detected,11 such as in both repeats throughout the HLA region D6S2447 cases included in this study (testing for ordered from telomere (top) to centro- TAP1 mere (bottom), allowing accurate HLA Ring 3CA Graphic representation of short tandem repeats (STRs) typing and identification of possible re- in HLA classes I, II, and III on chromosome 6. All STR combinations, which may lead to mis- markers are dinucleotide repeats (AC)n, except for DQ- diagnosis. An example of typing for a ∗ CARII, which is (CTG)n; TNF b,c,d—(CT)n; 62—(TC- D6S439 CA)n; MICA—(GCT)n; D6S510—(CA-GA)n; MOG d— marker is given in FIGURE 2. For each D6S291 (CTC)n. The STRs needed for identification of matching family we selected heterozygous alle- are shown in relation to genes in the HLA region, or- D6S426 dered from telomere (top) to centromere (bottom), al- les and markers not shared by the par- lowing accurate HLA typing and identification of pos- ents. Such markers provide the infor- Centromere sible recombinations, which may lead to misdiagnosis. Markers with asterisks indicate possible ambiguity in mation about the origin of chromosome localization. 6. A haplotype analysis for father, 2080 JAMA, May 5, 2004—Vol 291, No. 17 (Reprinted) ©2004 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/24/2021 PREIMPLANTATION HLA TESTING mother, and the affected child was per- Figure 2. Example of Typing for Short Tandem Repeat Markers Showing a Capillary formed for each family prior to preim- Electrophoregram of Fluorescently Labeled Polymerase Chain Reaction Products of the MIB plantation HLA typing.

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