Published online September 1, 2008
PEDIATRICS Vol. 122 No. 3 September 2008, pp. e778-e782 (doi:10.1542/peds.2008-0123)
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EXPERIENCE & REASON

First Successful Bone Marrow Transplantation for X-linked Chronic Granulomatous Disease by Using Preimplantation Female Gender Typing and HLA Matching

Janine Reichenbach, MDa, Hilde Van de Velde, PhDb, Martine De Rycke, PhDc, Cathérine Staessen, PhDc, Peter Platteau, MDb, Patricia Baetens, MAb, Tayfun Güngör, MDa, Hulya Ozsahin, MDd, Franziska Scherer, MDa, Ulrich Siler, PhDa, Reinhard A. Seger, MDa and Inge Liebaers, MD, PhDc

a Division of Immunology/Hematology/Bone Marrow Transplantation, University Children's Hospital Zurich, Zurich, Switzerland
b Centre for Reproductive Medicine
c Centre for Medical Genetics, UZ Brussels, Brussels, Belgium
d Division of Hematology/Oncology, Department of Pediatrics, University Hospital of Geneva, Geneva, Switzerland

ABSTRACT

Allogeneic hematopoietic stem cell transplantation from an human leukocyte antigen (HLA)-identical donor is currently the only proven curative treatment for chronic granulomatous disease. Hematopoietic stem cell transplantation with alternative donors is associated with higher morbidity and mortality. Therefore, we performed in vitro fertilization and preimplantation HLA matching combined with female sexing for hematopoietic stem cell transplantation in chronic granulomatous disease. Ethical and psychological issues were considered carefully. We used in vitro fertilization with X-enriched spermatozoa followed by preimplantation genetic diagnosis to identify female HLA-genoidentical embryos in a family in need of a suitable donor for their boy affected with severe X-linked chronic granulomatous disease. Two preimplantation genetic diagnosis cycles were performed in the family. In the second cycle, 2 HLA-genoidentical female embryos were transferred and a singleton pregnancy was obtained, resulting in the birth of an unaffected girl at term. Because of insufficient cell numbers in the cord-blood source, conventional hematopoietic stem cell transplantation had to be performed at 12 months of age of the donor and 5 years of age of the recipient and resulted in complete stable donor chimerism and immunologic reconstitution up to 25 months post–hematopoietic stem cell transplantation. Hematopoietic stem cell transplantation after in vitro fertilization and combined female sexing and HLA matching offers a new and relatively rapid therapeutic option for patients with X-linked primary immunodeficiency such as chronic granulomatous disease who need hematopoietic stem cell transplantation but lack an HLA-genoidentical donor.

Key Words: preimplantation genetic diagnosis • hematopoietic stem cell transplantation • chronic granulomatous disease • female sexing • HLA matching

Abbreviations: CGD, chronic granulomatous disease • HSCT, hematopoietic stem cell transplantation • PGD, preimplantation genetic diagnosis • FISH, fluorescence in situ hybridization analysis • PCR, polymerase chain reaction • X-CGD, X-linked CGD • ICSI, intracytoplasmic sperm injection • STR, short tandem repeat • DHR, dihydrorhodamine • IVF, in vitro fertilization

Chronic granulomatous disease (CGD) is a primary immunodeficiency that affects the oxidative mechanism of microbial killing of phagocytic cells, as a result of absent or severely impaired activity of the nicotinamide adenine dinucleotide phosphate oxidase of phagocytes. CGD occurs with an estimated frequency of 1 in 200000 live births.1 Morbidity is considerable with early recurrent and life-threatening bacterial and fungal infections and development of chronic inflammatory granulomas. Despite antibiotic and antifungal prophylaxis, the annual mortality rate is 2% to 5%.1 Allogeneic hematopoietic stem cell transplantation (HSCT) is the only curative treatment for CGD, with excellent outcome after HLA-genoidentical sibling HSCT.2

Preimplantation genetic diagnosis (PGD) is an established method for the diagnosis of genetic diseases at the early embryonic stage so that implantation of affected embryos can be avoided and the potential need for termination of pregnancy is eliminated.3,4 We combined gender selection by fluorescence in situ hybridization (FISH) analysis on 1 cell and indirect HLA typing by multiplex polymerase chain reaction (PCR) on a second cell from a couple's preimplantation embryos to obtain a healthy girl who could possibly be an HLA-matched donor for her sibling affected with X-linked CGD (X-CGD). This strategy may offer a new therapeutic perspective for patients who have X-CGD and lack a suitable HLA-genoidentical sibling donor.

METHODS

X-CGD was diagnosed in the patient at age 3 weeks by a positive family history, negative nitroblue-tetrazolium and O2– production tests, and DNA CYBB gene sequencing. He has sustained severe neonatal bilateral pulmonary infection with Aspergillus fumigatus, recurrent perirectal abscesses with Klebsiella oxytoca from 7 months of age onward, and severe granulomatous colitis from age 1.58 years onward. Prophylactic treatment with oral cotrimoxazole and itraconazole was started at diagnosis. Onset, severity, and recurrence of infections qualified the boy for HSCT. The indication for HSCT was established at University Children's Hospital Zurich. The child had no suitable HLA-genoidentical sibling donor, and PGD is currently banned in Switzerland5; however, PGD for HLA typing is explicitly allowed by Belgian law. The patient's parents presented to the Centre for Medical Genetics in Brussels by their own initiative. The PGD procedure was explained to them by an experienced geneticist. A discussion concerning the fate of the unaffected but HLA-nonidentical embryos took place, the options being (1) cryopreservation for future transfer, (2) research to confirm the diagnosis in the nontransferred embryos for estimation of the reliability of the HLA-typing test, or (3) destruction. Informed consent concerning the PGD procedure as well as the use of nontransferred embryos for research was given. An experienced psychologist evaluated the parents' motivation for having another child to become a donor for their affected son.6

We used a PGD approach that saved time combining female gender typing by FISH with indirect HLA typing. Additional search of the disease-specific mutation in multiplex single-cell PCR would have required more time. For increasing the chance of having female embryos, the husband's sperm was enriched for X-bearing spermatozoa.7 Two clinical PGD cycles were performed for the selection of healthy female HLA-matched embryos in the 34-year-old mother. Ovarian stimulation, oocyte retrieval, removal of cumulus and corona cells, and intracytoplasmic sperm injection (ICSI) were conducted as described previously.8 Two blastomeres were isolated from each 6- to 8-cell stage embryo 72 hours after fertilization by laser biopsy. HLA typing by multiplex PCR was performed using short tandem repeats (STR) in the HLA locus as described previously.9 Of a panel of STR markers, 4 were selected on the basis of their localization within the locus and on their informativity in the CGD family (ie, the possibility to distinguish the 4 parental HLA haplotypes through these markers; Table 1). Informativity was ensured by using 1 telomeric STR (D6S1571), 1 STR very close to class I-A (MOG3), 1 STR within class II-DQ (D6S2443), and 1 centromeric STR (D6S1610). Both analyzed embryos were diagnosed to be female HLA-genoidentical and were transferred into the uterus on day 5, resulting in a successful singleton pregnancy. A healthy girl with normal nicotinamide adenine dinucleotide phosphate oxidase function, as reflected in normal superoxide production by dihydrorhodamine (DHR) test, was born at term.


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  		TABLE 1 Segregation Analysis of the Informative STR Markers Within the HLA Loci in the X-CGD Family With the Affected Child P1 and His Healthy HLA-Nongenoidentical Sibling P2

 
Unluckily, the stem cell number in the cord blood was poor (0.4 x 106 CD34+ cells), so HSCT had to be planned from bone marrow, and cord blood was frozen for possible later use. Harvest of 200 mL of bone marrow was done at donor age 12 months and patient age 5.667 years and yielded 15 x 109/L total nucleated cells, with 6.8 x 106/kg recipient weight CD34+ cells. The patient underwent myeloablative conditioning with busulfan (Busilvex, Pierre Fabre Medicament, Boulogne, France) 16 mg/kg body weight intravenously (2 x 2 mg/kg per day, days –9 to –6), cyclophosphamide 200 mg/kg intravenously (50 mg/kg per day, days –5 to –2), and alemtuzumab (anti-CD52; Campath-1H, Genzyme Corporation, Cambridge, MA), once daily intravenously for 3 consecutive days (0.1 mg/kg on day –4, 0.2 mg/kg on days –3 to –2). Graft-versus-host disease prophylaxis consisted of cyclosporine A (3 mg/kg per day, starting on day –1) until 10 months after HSCT. Infectious prophylaxis included cotrimoxazole, acyclovir and itraconazole, and intravenous immunoglobulin at 400 mg/kg until immune reconstitution at 10 months after HSCT. Posttransplantation course was uneventful with minimal transfusion requirement, low-grade mucositis, and only 1 minor infectious episode during aplasia. The patient engrafted on day +12 for platelets and on day +13 for neutrophils. Full sustained stable donor-derived hematopoietic chimerism was observed (100% XX by FISH) from day +26 onward, and DHR test was 100% from day +34 onward, both up to currently 25 months after HSCT. B-cell immunologic reconstitution was achieved at 10 months after HSCT with sufficient antibody production and peripheral blood mononuclear cell proliferation to mitogen and antigen stimulation after the first test vaccinations with diphtheria and tetanus toxoid and is sustained 25 months after HSCT (Fig 1, Table 2). The child is currently well and off all treatment.


Figure 1
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  		FIGURE 1 Immunologic reconstitution after HSCT. Date of the HSCT is indicated by a black arrow. The percentage of H2O2-producing granulocytes after phorbol myristate acetate (PMA) stimulation by FACS analysis (DHR test) at different time points before and after HSCT is indicated at the top of the graph.

 

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  		TABLE 2 Reconstitution of Immunoglobulin Production and Lymphocyte Proliferation After HSCT

 

DISCUSSION

In the absence of an HLA-genoidentical sibling, in vitro fertilization (IVF) and PGD for sibling HLA matching might be the only realistic chance of cure in patients with X-CGD. Using HLA-genoidentical matched related donor, survival is excellent (86%) for children who do not have infection and are at an early stage of the disease.2,10,11 Results of HSCT by using a matched unrelated donor are less favorable (survival rate: 71%).2,10,12–14 Haploidentical HSCT is considered risky in CGD because of delayed immune reconstitution and graft failure.15–17 Use of unrelated cord blood could be a promising alternative, but published experience is limited to 3 cases.16,18,19 PGD on day 3 embryos is efficient and reliable and does not seem to interfere with viability and implantation or lead to major malformations.20 The combination of PGD and HLA matching performed in 6 centers for reproductive medicine worldwide has resulted in a total of 16 published successful pregnancies and births of healthy HLA-compatible donors for patients with Fanconi anemia,21–24 β-thalassemia,24,25 Blackfan-Diamond anemia,26 Wiskott-Aldrich syndrome,25 hyper-IgM syndrome,24 anhydrotic ectodermal dysplasia with immunodeficiency,24 and leukemia.26 Of these series, only 2 children with Fanconi anemia, 2 with β-thalassemia, 1 with Blackfan-Diamond anemia, and 1 with Wiskott-Aldrich syndrome have been published to be cured by HSCT.21–23,25 The extremely low number of published successful cases may be attributable to 2 major constraints of the method: first, birth rates per cycle are low (15% for the published cases); second, as in our case, the CD34+ cell number obtained from cord blood may be insufficient for HSCT.

PGD combined with HLA typing for providing an HSC donor has been accompanied by considerable controversy. Significant ethical, legal, and policy issues are raised by this procedure, and there is no consensus worldwide as to its authorization.5,27–30 A number of criteria must be met to proceed to this strategy27,31: (1) eligible diseases must be lethal or severe and with known genetic mutations or X-linked and must be potentially benefited by HSCT; (2) only diseases for which HSCT can be performed in an elective manner, implying sufficiently slow disease progression, allow this procedure, because of the minimum of 10 months required for 1 IVF/ICSI and PGD cycle with HLA typing, followed by the period of gestation; (3) maternal age should preferentially be below 38 years for sufficient success rate and number of retrievable oocytes; and (4) careful psychological counseling is mandatory to understand the couple's motivation to have the child to evaluate and to avoid potential instrumentation.6 This risk should be less important if there had been a wish for another child before the illness of the existing child; however, when a child becomes severely ill and knowing that a future child might save the sick sibling, parents may change their mind about the number of children they want. PGD and HLA typing could be less of a burden for parents than other alternatives, such as natural conception followed by prenatal genetic diagnosis and a termination of pregnancy if the fetus is ill. Nevertheless, the low risk for the future child's being valued only as an HSC donor and thus being neglected after birth should be carefully assessed.6

To date, equitable access to this kind of treatment option is not ensured for all patients who could benefit from it, because IVF/ICSI and PGD with HLA typing are allowed only in some countries and because the substantial cost of each clinical cycle of IVF and PGD (approximately $15000), is mostly not reimbursed by insurers. As our patient demonstrates, parents who seek access to this treatment option might be willing to circumvent national legislative and health care systems and consult IVF centers in countries that currently do not ban this approach; therefore, international coordination and harmonization of legislation would be strongly desirable.

ACKNOWLEDGMENTS

This work was supported in part by grants from the FWO Vlaanderen and the UZ Brussels Gepts Foundation for personnel and running costs.

We are grateful to the patient and his family for their trust. We are indebted to the laboratory, nursing, and medical staff of the Centre for Medical Genetics and the Centre for Reproductive Medicine. We are also obliged to Dr A. Keisker and the nursing team of University Children's Hospital Zurich, who cared for the patient during his stay in the bone marrow transplant unit, and to M. Rutishauser and C. Wenk for helpful laboratory support.

FOOTNOTES

Accepted May 21, 2008.

Address correspondence to Janine Reichenbach, MD, University Children's Hospital Zurich, Division of Immunology/Hematology/BMT, Steinwiesstrasse 75, CH-8032 Zürich, Switzerland. E-mail: janine.reichenbach{at}kispi.uzh.ch

The authors have indicated they have no financial relationships relevant to this article to disclose.

Drs Reichenbach, Van de Velde, Seger, and Liebaers contributed equally to this work.

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PEDIATRICS (ISSN 1098-4275). ©2008 by the American Academy of Pediatrics

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H. Van de Velde, M. De Rycke, C. De Man, K. De Hauwere, F. Fiorentino, S. Kahraman, G. Pennings, W. Verpoest, P. Devroey, and I. Liebaers
The experience of two European preimplantation genetic diagnosis centres on human leukocyte antigen typing
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