27. CLINICAL APPLICATION OF KARYOMAPPING – EMBRYOS AS REFERENCES?

Abstract

Introduction Karyomapping uses the principles of genetic linkage to detect any monogenic disorder i.e. allowing the diagnosis of the presence or absence of a disease causing allele. The process involves genome-wide single nucleotide polymorphism (SNP) analysis of the parents, a reference (e.g. an affected child) and amplified embryos DNA. ‘Informative loci’ are then identified and compared to the reference DNA in order to establish phase. The genotype of each embryo is established by comparing to the genotype of the reference. Despite its many advantages, Karyomapping has its limitation, for instance, no family members available as reference for karyomapping testing. Therefore, this study is to explore alternatives to counter the limitation. Material & methods A total of 42 blastocysts (8 cases) were biopsied and the cells obtained were amplified using the Repli-g Single Cell Kit (Qiagen). Blood and saliva samples from the couple seeking PGD and family members used as references were obtained and DNA is extracted using QIAmp DNA Midi Kit (Qiagen). DNA samples from the couple, family members and WGA amplified embryo samples were analysed using HumanKaryomap-12 BeadChips protocol (Illumina, USA), and results were evaluated using BlueFuse Multi 4.0, Illumina. DNA samples used as references for phasing of SNP alleles included any of the following: the couple's son/daughter, the parents of the male and/or female patient and WGA amplified embryo samples. The genes involved in each single gene disorder were targeted for assessment via Karyomapping. Direct mutation analysis with PCR and Sanger Sequencing was also performed in parallel with Karyomapping. Two approaches were conducted to assess the feasibility and reliability of the method. Firstly, to assess the feasibility of the method, 15 out of 42 blastocysts (4 cases) were assessed without prior knowledge of phase as couples had neither children nor family members available. These blastocysts were processed using a combination of Karyomapping and direct mutation detection in order to establish the phase. Embryos diagnosed by direct mutation analysis were used as references for karyomapping. Secondly, to assess the reliability of the use of embryos as reference, the remaining 27 blastocysts in which DNA samples from family members were available as references, were processed using Karyomapping in combination with PCR testing. Embryos diagnosed by these methods were used to replace the existing references for Karyomapping and to confirm the results of the existing PGT case detecting monogenic disorder. Results Results obtained were shown to be in complete agreement between karyomapping and PCR testing indicating the feasibility of the method. Diagnoses made were also 100% concordant between family members as references and embryos as references which is indicative of the reliability of the method. Conclusion The results indicated that the use of embryos as references allows informative loci to be determined in cases where reference is not available in some couple. Ideally, more cases should be carried out in the future to reaffirm the reliability of this method. These approaches will be beneficial for couples that have no family members available for karyomapping testing in order to establish phase.