Introduction: Preimplantation genetic diagnosis (PGD) has become an important option for couple at risk of conceiving affected children with single gene disorders. PGD has recently been used in combination with Human Leukocyte Antigen (HLA) Matching, allowing selection and transfer of unaffected embryos also having a close HLA match with those of an existing affected child. In such cases, PGD was used not only to avoid the birth of affected children, but also to conceive healthy children who may be potential HLA-identical donors of haematopoietic stem cells (HSC) for transplantation in siblings with a life-threatening disorder.
This study includes 76 cycles, for 55 couples overall, performed in a 2-years period. In a total of 48 couples preimplantation HLA Matching was performed in combination with PGD for a single gene disorder, from whom 66 cycles were performed, including 65 cycles for 47 couples for -thalassaemia and 1 cycle for Wiscott-Aldrich syndrome. HLA Matching procedure, not involving testing for a causative gene, was performed for 7 couples having a child affected by ALL (5 cycles) and sporadic DBA (5 cycles).
Material and Methods: An indirect single-cell HLA typing protocol based on a multiplex fluorescent polymerase chain reaction (PCR) of short tandem repeat (STR) markers scattered throughout the HLA complex was used. A nested multiplex PCR assay was used to co-amplify all the selected loci. The first round PCR contained the external primers for the amplification of the informative HLA STR markers selected during the preclinical work-up of each PGD case, the gene regions involved by mutations, STR markers linked to these regions for ADO detection and STR markers used for detection of aneuploidies in patients of advanced reproductive age. The first round multiplex PCR was followed by separate second round PCR reactions for each locus. Mutation analysis was performed using the minisequencing method.
Results: A total of 696 embryos were tested, involving analysis of 1178 blastomeres, in 1088 (92.4%) of which a successful amplification was obtained. A reliable HLA haplotype was obtained in 1088 /1088 (100%) of the blastomeres with positive PCR results. Testing for chromosome 6 copy number revealed 53 (7.6%) embryos with aneuploidies, including a total of 5 (0.7%) trisomies, 48 (6.9%) monosomies, which affected the HLA Matching diagnosis for these embryos, leading to a conclusive diagnosis in only 643/696 embryos (92.4%). Recombination was found in 34 (4.9%) embryos, 7 (1.0%) of which were originally unaffected and HLA compatible, but were not considered for transfer because of recombination. In total, 102 (15.9%) embryos revealed an HLA match with the affected siblings, 76 (11.8%) of which resulted unaffected and 64 (10.0%) have been transferred back to patients in 41 cycles. Twelve pregnancies were achieved (29.3% per transfer); 4 pregnancies resulted only biochemical, one spontaneously miscarried and one resulted ectopic, and was then terminated. Five healthy HLA matched children have been already delivered and cord blood stem cells were transplanted to 3 affected siblings, resulting in a successful hematopoietic reconstruction.
Conclusions: These preliminary results are very encouraging and, complemented by other similar experiences in the field, demonstrate that preimplantation HLA Matching represent a valid alternative for the achievement of a successful treatment in children affected by severe congenital or acquired bone marrow disorders, in the absence of a compatible related donor.