Preimplantation genetic testing of pathogenic variants in genes SMN1, PKD1, RHD with high homologous copies

Introduction Preimplantation genetic testing for the monogenic disease (PGT-M) compared with conventional genetic diagnosis is complicated by extremely small amounts of biomaterial. This feature leads to increased allele dropout (ADO) rates, contamination significance, failed amplification events. Therefore, the development of PGT-M systems for pathogenic variants in the genes with homologous copies is more complicated challenge. PGT-M test-systems for the SMN1, RHD and PKD1 genes were developed in our laboratory and successfully applied in PGT-M cases. Material & methods The test-systems combine direct diagnosis of the pathogenic variants by PCR- RFLP or fragment analysis and linkage analysis of highly heterozygous STRs. The nested PCR was used for DNA amplification from different sources: single cells or whole genome amplification products (WGA) of embryo biopsy, total DNA. Results The test-system for PGT-M in the presence of homologous genes or pseudogene copies of the target gene has to exactly discriminate the allele with and without pathogenic variant and detect miss target amplification to evaluate the probability of misdiagnosis. PCR-RFLP was used to discriminate the single nucleotide difference between exon 7 of the SMN1 (NC_000005.9:g.70247773C) and SMN2 (NC_000005.9:g.69372353T) genes. This method cannot detect the number of the SMN1 copies, but confirms the presence of at least one copy. PGT-M for the SMN1 was performed in 7 cycles for 36 embryos. RHD detection was based on the difference of PCR products length from the RHD and RHCE intron 7 (NC_000001.10: 25636476-25636480GGGGT, 25639738-25639741GGCA in the RHD gene) and 8 (NC_000001.10: 25698440-25698442TAA in the RHCE gene). These nucleotide variations were performed for PGT-M previously, but we optimized this approach for WGA products. PGT for Rh- status was performed in 2 cycles for 11 embryos. Similarity of genes SMN1/SMN2 and RHD/RHCE sequences requires careful usage of STRs for indirect diagnostics. Among the STRs, selected for the test-system, we found some duplicate repeats, flanked by similar sequences and located in a similar way in each target/nontarget locus. D1S2561/ D1S2572 and D1S2564/D1S2569 for RHD/RHCE and D5S1556/ D5S6934 forSMN1/SMN2. The segregation analysis of such markers is not univocal and can lead to misdiagnosis. Exons 1–33 of all 46 exons of the PKD1have high similarity with six pseudogenes located on chromosome 16. Therefore, it is important to distinguish PCR product from the gene and pseudogenes as well as amplification of miss targets. Amplification of several miss targets is conjugated with high load of PCR reaction and can lead to increased ADO rate. The previously performed PGT test-system based on long-range PCR is not applicable with WGA products ( Zeevi et al. 2013 ). So, we developed new test-system and specified amplification by using “touchdown” amplification program and high- precision polymerase in combination with miss target PCR products detection by PCR-RFLP. PGT-M for PKD1 was performed in 2 cycles for 3 embryos. Conclusions The PGT-M test-system in the presence of homologous copies of target gene has to be able to distinguish target and nontarget pathogenic variant location. The reported test-systems for the SMN1, PKD1, RHD genes satisfy this condition, detect pathogenic variant with high efficacy and can be used with biomaterials of different types.