Preimplantation Genetic Diagnosis for Benign Hematological Disorders Combined with HLA Matching and Stem Cells Development.

Abstract 3167

Preimplantation genetic diagnosis (PGD) for molecular disorders involves development of disease- and family-specific protocols that allow simultaneous amplification of the mutation with multiple polymorphic markers in single cells. We present the development and results of PGD for 6 non-lethal hematological disorders: dyskeratosis congenita, Gaucher type 1, Fanconi C and A, hemophilia A, and beta thalassemia. For beta thalassemia and Fanconi A we performed combined PGD for the disease and HLA matching since each family had an affected child requiring bone marrow transplant. For each of the 6 diseases we developed a fluorescent PCR protocol that included the familial mutation and at least 6 informative markers surrounding the mutation. Mechanical biopsy technique was performed and a single cell of a 6-cell embryo was analyzed. For 17 families, 43 PGD cycles were performed by blastomere analysis. The efficiency of single cell amplification was 96%. 12 healthy children were born (and 3 on-going pregnancies). PGD for dyskeratosis congenita was performed in 2 families: one carrying the Arg 631Gln mutation and the other the Arg901Trp in the TERT gene. A total of 3 cycles were performed and 1 healthy child was born. Analysis for Gaucher type 1 disease was performed in 4 families (1 couple was carriers of both Tay-Sachs and Gaucher mutations) in which the offspring were predicted to have severe type 1 Gaucher involvement based on genotypes of the following mutations in the beta-glucocerebrosidase gene: IVS2+1 and IVS12+1, 84GG and R359Q, 84GG and R496H, N370S homozygous patient and 84GG carrier with 1 affected child requiring treatment. 13 PGD cycles were performed by blastomere biopsy and 6 healthy children were born (3 children to the same couple from different cycles). Mutant embryos were used for development of stem cell lines and 1 line (N370S/84GG) was achieved. PGD for Fanconi C and A was performed in 4 couples. For Fanconi A combined PGD for the disease and HLA matching was performed in 9 cycles; 2 healthy children were born and 1 on-going pregnancy. For Hemophilia A, 3 PGD cycles were performed for 2 families resulting in birth of 1 healthy boy. PGD for thalassemia beta was performed in 5 families with a total of 10 cycles, 5 of them combined with HLA matching; 1 healthy child was born. The overall pregnancy rate/embryo transfer was 34% (highest for Gaucher, 53%, lowest for beta thalassemia, 10%). We suggest that PGD is a safe and effective method for hematological disorders to prevent birth of an affected child. Combining analysis for the specific disease with HLA matching is useful for hematological diseases requiring bone marrow transplant. Mutant embryos can be developed in stem cell lines that are a valuable source for research.