PGD for Fanconi Anemia with Preimplantation HLA Matching

One of the severe congenital disorders requiring stem cell transplantation from a family member is FA, which was the first disease for which cord blood transplantation was introduced [3]. FA is an autosomal recessive disorder, characterised by inherited bone marrow failure, congenital malformations, and an increased predisposition to the development of leukemia. It is genetically heterogeneous, involving different complementation groups (FANCA, FANCB, FANCC, FANCD, and FANCE), one of the most severe being FANCC mutation leading to aberrantly spliced transcripts (IVS4+4A-T), which result in inactivating FANCC protein [4-6]. Bone marrow transplantation is the only treatment, which restores definitively hematopoiesis in FA patients. However, because any modification of the conditioning is too toxic for these patients leading to a high rate of transplant-related mortality, the HLA-identical cord blood transplantation from a sibling is particularly valuable for FA, to avoid late complications due to severe GVH [7, 8].

A couple presented for PGD with both parents being unaffected carriers of IVS 4+4 A-T mutation in FANCC gene. Their affected 6-year-old daughter had two copies of this mutation, requiring an HLA-compatible donor for bone marrow transplantation. The couple requested PGD for FANCC, together with HLA testing of embryos, so to have an unaffected child who may also be a compatible cord blood donor for their affected daughter.

PGD was performed using a standard IVF protocol combined with micromanipulation procedure to biopsy single blastomeres from the day 3 cleaving embryos, as described in Chapter 2. Blas-tomeres were tested for IVS 4+4 A-T mutation in FANCC gene using polyacrylamide gel analysis of PCR product digested with Sca I restriction enzyme, according to the method of single-cell PCR analysis, also described in Chapter 2.

The outer primers IVS4-1 (5'-GTCATAAAAG-GCACTTGCAT-3') and IVS4-2 (5'-GGCACATT-CAGCATTAAACA-3') were designed for performing the first round of amplification, while using the previously described inner primers [9] for the second round of PCR. The second-round PCR produces a 131-bp product, undigested by Sca I restriction enzyme, corresponding to the mutant allele, and two restriction fragments of 108 bp and 23 bp, corresponding to the normal

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