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Multiplex PCR of polymorphic markers flanking the CYP21 gene as a general approach for preimplantation genetic diagnosis of 21-hydroxylase deficiency

Biricik A., Ercelen N., Magli M.C., Podini D., Nuccitelli A, Vitale N., Brardinoni L., Baldi M., Fiorentino F. and Gianaroli L.

EmbryoGen – Preimplantation Genetic Diagnosis Centre, Rome – Italy

Reproductive Genetics Centre, Istanbul American Hospital, Istanbul, Turkey

Introduction: Congenital adrenal hyperplasia (CAH) is one of the most common forms of inborn metabolism error. Deficiency of steroid 21-hydroxylase accounts for 90-95% of all cases. The most common mutation causing a severe form of CAH is a splicing mutation in the second intron of the 21-hydroxylase gene (CYP21) in which an A or C, at nucleotide (nt) 656, is replaced by G.

A consanguineous couple who had a child affected by 21-hydroxylase deficiency was referred to our centre for preimplantation genetic diagnosis (PGD). After mutation analysis of CYP21 gene, both father and mother resulted homozygote for nt656 mutation, yet showing no clinical signs of the disease. Non-amplification of the normal allele at nt656 is a well known phenomenon described in asymptomatic carriers. These putative nt656 G/G individuals are incorrectly typed due to normal haplotype drop-out during PCR amplification.

We have developed and clinically applied a multiplex microsatellite polymerase chain reaction (PCR) protocol for PGD of 21-hydroxylase deficiency. This strategy was optimized in order to resolve ambiguities at nt656 using an indirect genotyping through linkage analysis of polymorphic linked extragenic CYP21 gene markers, in alternative of a mutation-based PGD protocol.

Material and methods:

Seven highly polymorphic short tandem repeat (STR) markers (MIB, MIC-A, 62, TNFa, LH-1, D3A, DRA-CA), flanking the CYP21 gene, were selected. In order to evaluate the reliability of the protocol before proceeding to PGD, primers used were first tested on single lymphocytes collected from both parents and the affected child. The PGD strategy involved a first round multiplex PCR for simultaneous amplification of the selected linked polymorphisms, followed by separate second round fluorescent PCR reactions for each locus.

Results:

In 100% (60/60) of the single lymphocytes tested, multiplex PCR results were obtained. Amplification rates were generally high for all loci tested, ranging from 96.7% to 100%. The ADO rates varied among the different loci investigated, ranging from 0% to 5.0%. Eight blastomeres were retrieved from 4 embryos; results were obtained for all the markers investigated in 8/8 (100%) cells enabling diagnosis for all embryos. Three embryos resulted affected due to the presence of both affected haplotypes; one embryo was diagnosed as carrier and transferred back to patient. Unfortunately no pregnancy was achieved. Later reanalysis of untrasferred embryos confirmed the cycle results.

Conclusions:

We have optimized a 7-plex assay allowing the amplification of 7 highly polymorphic STR markers linked to CYP21 gene from single cells. This method has revealed to be a robust and flexible approach applicable to PGD of a wide spectrum of different genotype combinations causing 21-hydroxylase deficiency. Such strategy can also obviate to the problem of non-amplification of the normal allele affecting nt656 mutation.