DIAGNOSIS OF 21-HYDROXYLASE DEFICIENCY BY HLA TYPING IN PRE-IMPLANTATION EMBRYOS
S. Israel, J. Seligman, D. Manor, J. Itskovitz-Eldor, C. Brautbar Tissue Typing Unit - Hadassah Hospital, Jerusalem, Developmental Biology Lab., Rambam Hospital, Rappaport Faculty of Medicine, Technion, Haifa.

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The 21-hydroxylase gene coding the adrenal cytochrome P450c21 is located on the short arm of chromosome 6 between HLA class I and class II. The close linkage between CYP21B and HLA complex has previously permitted 21-hydroxylase deficiency prenatal diagnosis via HLA typing of cultured amniotic fluid cells derived from a fetus at risk. Here, we have set up a procedure for pre-implantation genetic diagnosis (PGD) of 21-hydroxylase deficiency in embryos by using HLA typing of DRB1 and DQB1 loci simultaneously with a Y-chromosomal marker. We tested our procedure to diagnose embryos of a couple in which the 21-hydroxylase deficient father carries three different mutations: Q318X, V281L, I172N and the heterozygous mother who carries the Q318X mutation. Linkage of DRB1*1305 and DQB1*0301 haplotype with the normal allele of CYP21B has been identified. For PGD, we have also added an Y chromosomal marker to define the sex of the embryos. In these cases, males would be a feasible choice since they do not manifest ambiguous genitalia as mutant females do. Twelve frozen embryos were thawed for PGD. Out of the twelve embryos, six have survived the stage of 8 cells. These six embryos were examined by our PGD assay. Following embryo biopsy, single blastomers were analyzed by PCR. Amplification of DRB1 and DQB1 loci as well as Y-chromosomal marker was performed on single bastomers. The PCR products of the HLA genes were further analyzed using the PCR SSOP method in order to define the different HLA DRB1 and DQB1 alleles. Out of six embryos examined for PGD, two were diagnosed as mutants since they carried the mutation linked allele DQB1*02; three were diagnosed as normal (carriers only) and one failed to amplify. Our results show the possibility of HLA typing in single blastomers. Moreover, we implemented this method in 21-hydroxylase deficiency diagnosis.