Development and clinical application of a strategy for preimplantation genetic diagnosis of single gene disorders combined with HLA matching

Abstract

Preimplantation HLA matching has recently emerged as a tool for couples desiring to conceive a potential donor progeny for transplantation in a sibling with a life-threatening disorder. In this paper we describe a strategy optimized for preimplantation genetic diagnosis (PGD) of haemoglobinopathies combined with HLA matching. This procedure involves a minisequencing-based genotyping of HLA regions A, B, C and DRB combined with mutation analysis of the gene regions involved by mutation. Analysis of at least eight polymorphic short tandem repeat (STR) markers scattered through the HLA complex has also been included to detect potential contamination and crossing-over occurrences between HLA genes. The above assay can also be used for preimplantation HLA matching as a primary indication. The strategy was clinically applied for HLA matching in 17 cycles (14 for beta-thalassaemia, one for Wiscott-Aldrich syndrome and two for leukaemia). A reliable HLA genotype was achieved in 255/266 (95.9%) of the blastomeres. In total, 22 (14.8%) embryos were obtained that were HLA-matched with the affected siblings, 14 (9.4%) of which were unaffected and transferred back to the patients. Four clinical pregnancies were obtained, three of which (one twin, two singletons) are ongoing and were confirmed as healthy and HLA-identical with the affected children. Minisequencing-based HLA typing combined with HLA STR haplotyping has been shown to be a reliable strategy for preimplantation HLA matching. The major advantage of this approach is that the validation of a single assay can be done once and then used for the majority of the patients, reducing notably time needed for preclinical set-up of each case.

Hematopoietic cell transplantation for thalassemia and sickle cell disease: past, present and future

“…Reports have confirmed the feasibility of this approach. [45][46][47][48] The financial cost is considerable and serious ethical concerns have been raised by the practice of selecting an embryo solely for the purpose of HSC donation.…”

Short tandem repeats haplotyping of the HLA region in preimplantation HLA matching

“…The primer sequence of 29 of these STRs and their clinical application, included in a protocol involving a minisequencing-based HLA typing procedure, have been described previously. 4 An additional set of 21 STRs was also included in the present study to achieve a fine mapping of the extended HLA complex. The primer sequence of these markers is shown in Table 1.…”

“…4 A panel of 50 different STR markers was studied to ensure a sufficient informativity in all families. Informativity was also evaluated for STR markers linked to the gene regions, which were involved by mutation, used to avoid a possible misdiagnosis due to the well-known allele drop-out (ADO) phenomena, and for STR markers used to determine the copy number of chromosomes 21, 18, 13, X and Y, applied for patients of advanced reproductive age.…”

“…PCR conditions used were reported previously. 4 Fluorescent PCR products were analysed by 20 min of capillary electrophoresis on an automatic DNA sequencer ABI Prism 3100t (Applied Biosystems). Mutation analysis was performed using the minisequencing method, as described elsewhere.…”

Preimplantation genetic diagnosis of P450 oxidoreductase deficiency and Huntington Disease using three different molecular approaches simultaneously

“…The development of different technologies allows accurate diagnosis and the extension of the application of PGD, not only to the most common single gene disorders but also to more complicated cases such HLA typing. In fact, HLA typing has been combined with PGD for monogenic blood diseases or aneuploidy screening [3][4][5]. Also, some cases involving use of PGD for monogenic disorders plus agerelated aneuploidies screening have been reported [6].…”