LAUSR

Introduction Thalassaemia affects 4.5% of the population in Malaysia. Complications of β- thalassaemia major in children pose a heavy load on transfusion and paediatric services. Preimplantation Genetic Diagnosis (PGD) in conjunction with IVF to avoid transferring affected blastocyst is one strategy to eradicate this disease. Allele drop out in PGD however may lead to misdiagnosis. Single nucleotide polymorphism (SNP) is a useful marker to confirm the inheritance of the parental wild-type or mutant allele thus increasing the accuracy of the diagnosis. The objective of this study is to evaluate the efficiency of an in-house developed SNP assay for PGD used in conjunction with aneuploidy screening. Methodology Beta-globin gene common mutation at CD41/42 and IVS2-654 were studied. Haplotyping was carried out to determine the SNPs that present in the wild-type and mutant allele of the parent. Five pairs of primers were designed specifically to amplify the region containing potential SNPs (3 pairs) and the mutations stated earlier (2 pairs). A total of 27 blastocysts were biopsied from 3 couples. Biopsies were amplified using Sureplex whole genome amplification kit. Aliquots of amplified DNA were subjected to second round of amplification to determine the presence of SNP and the mutation. Gel electrophoresis and sequencing were carried out and the resulting sequences were aligned. The genotype of the blastocysts was compared and confirmed with the SNP that present either in the wild-type or the mutant allele. The unaffected and heterozygous carrier blastocysts will subsequently be subjected to aneuploidy screening using Veriseq PGS Protocol (Illumina). Only blastocysts that were not affected or aneuploidy were suitable for blastocyst transfer. Results Of the 27 blastocysts tested for beta globin gene mutation at CD41/42 and IVS2-654, 7 was shown to be unaffected, 11 heterozygous carriers for thalassaemia and 9 were affected. For the first couple, the SNP is present in the paternal wild-type allele while for second couple, the SNP is present in the maternal mutant allele. For third couple the SNP is present in the paternal mutant allele and maternal wild-type allele. The results from SNP analysis were then compared with the genotype results obtained from direct sequencing on the targeted mutation, and the comparison showed 100% concordance. Aneuploidy screening on unaffected and heterozygous carrier blastocyst revealed 6 euploid, 7 mosaic and 5 aneuploid blastocysts. Five blastocysts (3 unaffected euploid, 1 heterozygous carrier euploid and 1 heterozygous carrier mosaic) were transferred with three successful implantations (2 unaffected euploid and 1 heterozygous carrier mosaic). Conclusion The presence of wild-type allele means that the blastocyst is either unaffected or is a heterozygous carrier and vice-versa. On the other hand, the presence of mutant allele means that the blastocyst is either affected or is a heterozygous carrier and vice-versa. This in-house developed SNP assay shows concordance results with the established mutation screening assay. The combination of SNP and mutation screening assay improves the accuracy of PGD by minimizing the risk of misdiagnosis caused by allele drop out.