Preimplantation HLA Testing

ORIGINAL CONTRIBUTION

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 theP 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]).

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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,

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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. This allowed Marker avoidance of misdiagnosis due to preferential amplification and allele drop out Father 100.78 bp exceeding 10% in PCR analysis of single 5000 12,14 blastomeres, potential recombina- 4000 119.87 bp 3000 tion within the HLA region, and a pos- 2000 sible aneuploidy or uniparental disomy 1000 of chromosome 6, which may also affect Fluorescence 0 the diagnostic accuracy of HLA typing of the embryo. The multiplex nature of Mother the first round of PCR analysis required 106.42 bp 4000 a similar annealing temperature for the 125.65 bp outside primers. Thirty cycles of PCR 3000 2000 were performed with a denaturation step 1000 Fluorescence at 95°C for 20 seconds, annealing at 62°C 0 to 50°C for a minute, and elongation at 72°C for 30 seconds. Twenty minutes of Affected Child 119.79 bp incubation at 96°C were performed be- 125.65 bp fore starting cycling. After cycling, 10 2000 minutes of elongation were performed 1500 1000 at 72°C. Annealing temperature for the 500 second round was programmed at 55°C Fluorescence 0 (details of the method have been 8,12 published ).The applied strategy pro- HLA-Matched Child 119.77 bp vided a 100% HLA match, because the 125.67 bp selected embryos had the same pater- 1200 900 nal and maternal chromosome 6 as the 600 affected siblings. 300

Fluorescence 0 Two to 3 embryos determined to be 90 95 100 105 110 115 120 125 130 HLA compatible with the affected sib- Size, bp ling were transferred; the other HLA-

Table 1. Preimplantation HLA Typing Resulting in Birth of a Child HLA Matched to a Sibling With Acute Lymphoid Leukemiaa Embryo No. HLA Genes Affected and STRs 12345678910Father Mother Child D6S276* 130/118 144/139 130/118 130/118 130/139 144/139 130/118 144/139 130/139 130/139 144/130 118/139 144/139 HLA-A 3/2 1/32 3/2 3/ADO 3/32 1/32 FA 1/32 3/32 3/32 1/3 2/32 1/32 D6S510 163/167 148/163 163/167 ADO/167 163/163 148/163 148/ADO 148/163 163/163 163/163 148/163 167/163 148/163 HLA-B ADO/27 27/18 ADO/27 FA FA 27/18 ADO/27 27/18 ADO/18 ADO/18 27/57 27/18 27/18 MIB 118/114 114/124 118/114 ADO/114 118/124 114/124 118/114 FA 118/124 118/124 114/118 114/124 114/124 TNF a 94/102 110/102 94/102 94/102 94/102 110/102 94/102 110/102 94/102 94/102 110/94 102/102 110/102 D6S273* 275/273 273/271 275/273 275/273 275/271 273/271 275/273 273/271 275/271 275/271 273/275 273/271 273/271 HLA-DRB1 ADO/1 11/10 ADO/1 ADO/1 ADO/10 11/10 ADO/1 11/10 ADO/10 FA 11/7 1/10 11/10 G51152 181/208 181/191 181/208 ADO/208 181/191 181/191 181/208 ADO/191 181/191 181/191 181/181 208/191 181/191 Ring 3CA 162/162 160/155 162/162 162/162 162/155 160/155 162/162 FA 162/155 162/155 160/162 162/155 160/155 D6S426 144/130 140/144 144/130 140/130 144/144 140/144 144/130 140/144 144/130 144/144 140/144 130/144 140/144 Predicted Nonmatch Match Nonmatch Nonmatch Nonmatch Match Nonmatch Match Nonmatch Nonmatch NA NA NA genotype Recombinantb Recombinantc Recombinantb Abbreviations: ADO, allele drop out; FA, failed amplification; NA, not applicable; STR, short tandem repeat. aPaternally derived markers in each cell are shown on the left and maternally derived markers on the right (not applicable to father and mother). Paternally and maternally derived matched markers are shown in boldface. The numbers represent the size of the polymerase chain reaction products in base pairs, corresponding to the number of STR repeats or an HLA allele name. Of note, for ADO/27 entries for HLA-B, and for the ADO/114 entry for MIB, for which both parents share the same repeat sequence, the other genetic information points to a genotype of maternal origin, with ADO involving the paternal marker. bRecombination detected between D6S426 and Ring 3CA. cRecombination detected between D6S276* and D6S510.

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matched embryos were frozen for fu- cycle on the average), of which 45 (23%) phoid leukemia. Of 10 embryos tested ture availability. As per the parents’ were determined to be HLA matched simultaneously for 11 alleles within the written informed consent, some of the to the affected siblings. The HLA- relevant HLA region in this family, re- HLA-incompatible embryos not cho- matched embryos were available for combination between the alleles for the sen for freezing, and those with incon- transfer in all but 1 cycle, for which there D6S426 and Ring 3CA markers was ob- clusive results, underwent PCR analy- were no matched embryos. Overall, 28 served in embryos 4 and 9, and be- sis of the whole embryo to corroborate HLA-matched embryos were trans- tween D6S276* and D6S510 in em- the diagnosis based on blastomere analy- ferred in 12 clinical cycles, resulting in bryo 7. Of the remaining 7 embryos, 3 sis. A follow-up assessment of the HLA 5 singleton pregnancies (42%) and births were fully matched (embryos 2, 6, and match was conducted in the estab- of 5 HLA-matched children. Of course, 8), while the other 4 were HLA incom- lished pregnancies using chorionic vil- some couples require multiple cycles of patible with the affected sibling, as seen lus sampling or amniocentesis. Since the IVF to achieve pregnancy. These re- from the haplotypes of the mother, fa- leukemias and the DBA in the affected sults suggest that testing of approxi- ther, and affected child. siblings were sporadic and not associ- mately 10 embryos per cycle allows for FIGURE 3 presents the results of pre- ated with chromosome 6, there was no selection of a sufficient number of the implantation HLA matching for DBA. reason for concern that the HLA- HLA-matched embryos for transfer to One embryo with maternal recombi- matched children would be at risk for the achieve a pregnancy and birth of an nation (embryo 8) and another with same disease as the affected siblings. HLA-matched progeny. both paternal and maternal recombi- The usefulness of detection of re- nation (embryo 16) (1 in the allele for RESULTS combination within the HLA region is the Ring 3CA marker and the other in A total of 199 embryos were tested for demonstrated in TABLE 1, describing the the allele for D6S426) were detected in the 9 couples in 13 clinical cycles per- results of HLA typing of 1 of the cycles testing of 16 embryos (examples of dif- formed by the time the data were sub- resulting in the birth of a child HLA ferent HLA typing results are shown). mitted for publication (15 embryos per matched to a sibling with acute lym- In addition, there was another em-

Figure 3. Preimplantation HLA Typing for Diamond-Blackfan Anemia, Resulting in Birth of an HLA-Matched Child

STR or Gene STR or Gene Telomere Telomere ∗ ∗ D6S276 118144 135 144 D6S276 D6S258147133 137 135 D6S258 HLA-AA 2 A 2 A 24 A 3 HLA-A D6S265179 181 176 172 D6S265 HLA-C C 4 C 4 C 4 C 4 HLA-C HLA-B B 51 B 51 B 57 B 35 HLA-B TNF a 110 94 94 100 TNF a ∗ HLA-DRB1 1∗11 1∗01 1∗07 1 01 HLA-DRB1 Ring 3CA 159 155 161 157 Ring 3CA D6S426 147 143 129 139 D6S426 Centromere Centromere

Affected Child Embryo 1 Embryo 5 Embryo 8 Embryo 16

118 144 118 144 118 135 144 118 135 144 144 147 135 147 135 147 137 135 147 137 133 135 A 2 A 3 A 2 A 3 A 2 A 24 A 3 A 2 A 24 A 2 A 3 179 172 179 172 179 176 172 179 176 181 172 C 4 C 4 C 4 C 4 C 4 C 4 C 4 C 4 C 4 C 4 C 4 B 51 B 35 B 51 B 35 B 51 B 57 B 35 B 51 B 57 B 51 B 35 110 100 110 100 110 94 100 110 94 94 100 1∗11 1∗01 1∗11 1∗01 1∗11 1∗07 1∗01 1∗11 1∗07 1∗01 1∗01 159 157 159 157 159 161 157 159 157 159 157 147 139 147 139 147 129 139 147 139 147 129

HLA Match Trisomy Recombinant Double Recombinant

Short tandem repeat (STR) and gene order for haplotypes of mother, father, and affected child. Examples of different results of HLA typing of biopsied blastomeres are shown. Embryo 1 is HLA matched to the affected sibling. Embryo 8 shows maternal recombination and embryo 16 shows double recombination involving both the paternal and maternal alleles. Embryo 5 shows an extra maternal chromosome consistent with trisomy 6. Markers with asterisks indicate possible ambiguity in localization. Genes with asterisks indicate allelic variants.

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bryo with trisomy 6 (embryo 5) with embryo 7, which was also unacceptable approximately 1 year) are healthy and an extra maternal chromosome 6, mak- for transfer. Of the remaining 7 em- are comparable to more than 1000 chil- ing this and the other 2 above also un- bryos, only 2 (only embryo 4 is shown) dren born after preimplantation ge- acceptable for transfer. However, 5 em- were HLA matched to the affected sib- netic diagnosis procedures, including bryos appeared to be HLA matched, of ling and transferred, resulting in the birth more than 400 children born after pre- which 2 were transferred, resulting in of an HLA-matched child. implantation genetic diagnosis proce- the birth of an HLA-matched child. Of only 7 embryos available for test- dures at our center. The mean birth The relevance of aneuploidy testing for ing in another case of DBA, 4 were HLA weight percentile was 47% and the chromosome 6 is seen from the results nonmatched, including the one with mean birth length percentile was 57%. of HLA typing in another cycle, result- recombination between the alleles for the The 5 children born after preimplan- ing in birth of an infant who was HLA TAP1 and Ring 3CA markers (embryo tation genetic diagnosis procedures in matched to the sibling with acute lym- 8). The remaining 3 HLA-matched the current study were confirmed to be phoid leukemia (FIGURE 4). Two of 10 embryos (embryos 2, 3, and 9) were HLA matched (per HLA typing of embryos tested in this case (examples of transferred, resulting in a singleton preg- blood) to their affected siblings. One different HLA typing results are shown) nancy and birth of an HLA-matched sibling with DBA received transplan- appeared to have only maternally de- child (TABLE 2). In the remaining cycle tation and is no longer red blood cell rived chromosomes 6; 1 had only 1 ma- performed for acute lymphoid leuke- transfusion dependent, whereas the oth- ternal chromosome (embryo 1), and the mia and resulting in the birth of an HLA- ers are in preparation for transplanta- other had 2 maternal chromosomes, rep- matched child, 6 HLA-matched embryos tion or are in remission. resenting uniparental maternal disomy were selected from 11 tested, with no evi- of chromosome 6 (embryo 2). In addi- dence of recombination or aneuploidy. COMMENT tion, recombination between D6S291 The 5 children born as a result of pre- The data herein show the potential fea- and class II HLA alleles was evident in implantation HLA matching (mean age, sibility of preimplantation HLA match-

Figure 4. Preimplantation HLA Typing for Acute Lymphoid Leukemia, Resulting in Birth of an HLA-Matched Child

STR STR Telomere Telomere D6S306∗ 104 106 106 108 D6S306∗ D6S1624∗ 176 178 178 184 D6S1624∗ MOG a 174 167 186 178 MOG a D6S510148 163 148 167 D6S510 TNF a 103 99 93 97 TNF a 62 200 197 189 195 62 D6S273∗ 270 272 279 268 D6S273∗ LH1 158 171 151 149 LH1 G51152 181 181 183 187 G51152 TAP1 207 207 207 218 TAP1 D6S291 114 124 122 116 D6S291 Centromere Centromere

Affected Child Embryo 1 Embryo 2 Embryo 4 Embryo 5 Embryo 6 Embryo 7

104 108 106 106 108 104 108 106 108 104 106 106 108 176 184 178 178 184 176 184 178 184 176 178 178 184 174 178 186 186 178 174 178 167 178 174 186 167 178 148 167 148 148 167 148 167 163 167 148 148 163 167 103 97 93 93 97 103 97 99 97 103 93 99 97 200 195 189 189 195 200 195 197 195 200 189 197 195 270 268 279 279 268 270 268 272 268 270 279 272 268 158 149 151 151 149 158 149 171 149 158 151 171 149 181 187 183 183 187 181 187 181 187 181 183 181 187 207 218 207 207 218 207 218 207 218 207 207 207 218 114 116 122 122 116 114 116 124 116 114 122 124 122 Monosomy 6 Uniparental Disomy HLA Match Maternal Match Paternal Match Recombinant

Short tandem repeat (STR) order for haplotypes of mother, father, and affected child. Examples of different results of HLA typing of biopsied blastomeres are shown. Embryo 4 is HLA matched to the affected sibling. Embryo 1 has no paternal chromosome present (monosomy 6). Embryo 2 shows only maternal chromosomes being present (uniparental disomy). Embryo 7 is both a paternal and maternal nonmatch, the latter being due to maternal recombination. The other 2 embryos are non- matches, embryo 5 being a maternal match only and embryo 6 being a paternal match only. Markers with asterisks indicate possible ambiguity in localization.

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ing for couples having a child affected diagnosis due to allele drop out, aneu- countries, this appeared to be a reason- with a bone marrow disorder, who may ploidy, or recombination of HLA alleles. able option for couples, because only a wish to have another child as a poten- Recombination in the HLA region was limited number of the embryos result- tial HLA-matched donor of stem cells observed in 4.3% of blastomeres tested, ing from a hormonal hyperstimulation for transplantation to the affected sib- suggesting the existence of a few areas in IVF are actually selected for transfer ling. As our data show, HLA-matched in the HLA region that are prone to a anyway. Therefore, instead of selecting embryos were selected and trans- higher recombination rate, which embryos for transfer based on morpho- ferred in all but 1 cycle, resulting in should be detected in order to avoid the logic criteria, those unaffected embryos pregnancies and birth of HLA- risk for misdiagnosis (Verlinsky et al, representing an HLA match are selected. matched children in 42% of the trans- unpublished data, September 2003). On There are important ethical issues involv- ferred cycles, much higher than the the other hand, aneuploidy of chromo- ing the selection of embryos with dif- pregnancy rate observed in women un- some 6 was detected in 6.4% of blas- ferent normal parameters such as HLA dergoing IVF.15 Despite the relatively tomeres, including trisomy (2.2%) and type; however, preimplantation HLA typ- high rate of preferential amplification monosomy 6 (4.2%) (Verlinsky et al, ing allows for the avoidance of the ethi- and allele drop out observed in PCR unpublished data, September 2003). cal dilemma of therapeutic cloning.17,18 analysis of single blastomeres and po- As mentioned, preimplantation HLA The decision about preimplantation HLA tential for recombination within the matching has previously been used typing is presently left with individuals HLA region described in our material, together with PGD for FA to select and and clinicians, as it is still expensive (ap- and a high rate of mosaicism for aneu- transfer unaffected embryos, avoiding proximately $3000 on average for pre- ploidies at the cleavage stage,16 the in- affected pregnancy and also producing implantation HLA typing alone [the cost troduced approach appeared to be a potential donor progeny for stem cell of IVF can exceed $10000 in the United highly accurate in the selection of HLA- transplantation for the affected sibling States and is covered by insurance in only matched embryos for transfer. This ap- with FA.8 In contrast, no testing for a some states; HLA testing is not cov- proach involves a multiplex PCR analy- causative gene was performed in the pres- ered]) and not obligatorily covered by sis involving testing for HLA alleles ent series of couples, the sole objective health insurers. together with short tandem repeat being to identify the HLA-matched Our results also demonstrate the markers within HLA and flanking re- embryos. Although still highly contro- prospect for the application of this ap- gions, allowing the avoidance of mis- versial and even not allowed in some proach to other conditions requiring an

Table 2. Preimplantation HLA Typing for Diamond-Blackfan Anemiaa Short Embryo No. Tandem Affected Repeats 23 7 8 91011Father Mother Child D6S461 225/227 225/227 229/227 229/227 229b/227 229/229 229/229 225/229 229/227 225/229b D6S276* 143/143 143/143 141/143 141/ADO 143/143 141/117 141/117 143/141 117/143 143/143 D6S258 144/132 144/132 135/132 135/132 144/132 135/135 135/135 144/135 135/132 144/132 D6S248* 253/278 253/278 276/278 276/278 253/278 276/278 276/278 253/276 278/278 253/278 MOG a 159/169 159/169 167/169 167/169 159/169 167/169 167/169 159/167 169/169 159/169 RF 275/269 275/ADO 257/269 257/269 275/269 257/263 257/263 275/257 263/269 275/269 9N-2 129/133 129/133 131/133 131/127/133 129/133 131/127 131/127 129/131 127/133 129/133 D6S273* 276/270 276/270 274/270 274/270 276/270 274/270 274/270 276/274 270/270 276/270 LH1 163/168 163/168 163/168 163/168 163/168 163/179 163/179 163/163 179/168 163/168 D6S2447 147/159 147/159 151/159 151/159 147/159 151/151 151/151 147/151 151/159 147/159 TAP1 205/220 205/220 205/220 205/220 205/220 205/207 205/207 205/205 207/220 205/220 Ring 3CA 159/155 159/155 155/155 155/159 159/155 155/159 155/159 159/155 159/155 159/155 D6S439* 125/125 125/125 123/125 123/125 125/125 123/125 123/125 125/123 125/125 125/125 D6S291 114/123 114/123 116/123 116/114 114/123 116/114 116/114 114/116 114/123 114/123 D6S426 129/129 129/129 144/129 FA 129/142b 144/142 144/142 129/144 142/129 129/129 Predicted Match Match Nonmatch Nonmatch Match Nonmatch Nonmatch NA NA NA genotype Recombinantc Abbreviations: ADO, allele drop out; FA, failed amplification; NA, not applicable. aPaternally derived markers in each cell are shown on the left and maternally derived markers on the right (not applicable to father and mother). Paternally and maternally derived matched markers are shown in boldface. The numbers represent the size of the polymerase chain reaction products in base pairs, corresponding to the number of STR repeats. Of note, for marker 9N-2 the 3 numbers suggest the possibility of genetic material from 2 maternal chromosomes, but this cannot be so because other markers indicate the presence of only 1 maternal chromosome 6. Thus, the genotype is considered to be of an unknown nature. bOf note, there is recombination between alleles for the D6S461 and D6S276* markers in the affected child, as well as in embryo 9 (which has paternal recombination between these markers vs the child, which has maternal recombination); embryo 9 also has recombination between alleles for D6S291 and D6S426 markers. However, because these markers are outside the HLA region, and other markers are in agreement with the match, the embryo was considered to be HLA matched to the affected child. cRecombination detected between TAP1 and Ring 3CA.

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HLA-compatible donor for stem cell nor for a child with other types of can- of the data and the accuracy of the data analyses. Study concept and design: Verlinsky, Kuliev. transplantation. This provides a real- cer. These expanding indications make Acquisition of data: Rechitsky, Sharapova, Morris, istic option for those couples who are preimplantation testing a comple- Taranissi. Analysis and interpretation of data: Rechitsky, Kuliev. parents of an affected child, who de- ment to traditional prenatal diagnosis, Drafting of the manuscript: Kuliev. sire to have another child, and who—as allowing parents to avoid inherited con- Critical revision of the manuscript for important in- a wholly separate issue—hope that the ditions and pregnancy termination. At tellectual content: Verlinsky, Rechitsky, Sharapova, Morris, Taranissi. subsequent child might serve as a do- the same time, the evidence suggests Obtained funding: Verlinsky. nor of stem cells for the affected sib- that it may now be possible for a preg- Administrative, technical, or material support: Verlinsky, Sharapova, Morris, Taranissi. ling. In addition to sporadic forms of nancy to have genetic characteristics Study Supervision: Verlinsky, Rechitsky, Kuliev. DBA, as well as leukemia, the method that may be beneficial for affected in- Funding/Support: This study was funded by the Re- productive Genetics Institute. may potentially be applied to other con- dividuals in the family. Role of the Sponsor: The Reproductive Genetics In- ditions; for example, the method might stitute sponsored the design and conduct of the study; Author Contributions: Dr Kuliev, as principal inves- the collection, management, analysis, and interpre- be used by parents who have unsuc- tigator of this study, had full access to all of the data tation of the data; and the preparation, review, and cessfully sought an HLA-compatible do- in the study and takes responsibility for the integrity approval of the manuscript.

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