O-287 Preimplantation genetic testing for monogenic disease (PGT-M) where results are only based upon analysis of linked polymorphisms/haplotypes risks serious diagnostic errors
Authors: Haj Ali A , Ampiah M , Abdous H , Babariya D , Raberi A , Wells D 
Published: June 2023

Study question

PGT-M often involves diagnoses based upon the analysis of polymorphisms linked to the mutant gene. Are such methods sufficiently reliable to be used alone?

Summary answer

Several problems can lead to misdiagnosis when linkage analysis is used in isolation. Therefore, PGT-M should also include direct mutation testing (DMT) whenever possible.

What is known already

Many PGT-M strategies involve analysis of DNA sequence polymorphisms in close proximity to the mutant gene, which have specific alleles that are inherited along with the disease. Unlike diagnostics that focus on detection of specific mutations, which can be unique to individual families, PGT-M protocols using linked polymorphisms can usually be reused for multiple families. Indeed, strategies such as karyomapping, which assess thousands of polymorphisms across the genome, provide a single method applicable to numerous diseases. Such methods are attractive since the work-up required for individual cases is minimal, reducing costs and patient waiting times. However, are such methods safe?

Study design, size, duration

Over a period of three years, we carried out 261 PGT-M cases covering 312 different mutations in 116 genes. Each couple requesting PGT-M provided blood samples from which DNA was extracted. The patients underwent IVF and embryos that reached the blastocyst stage were subjected to trophectoderm biopsy. All PGT-M cases involved the use of DMT to interrogate the mutation site(s). The DMT result was supplemented by analysis of multiple informative linked polymorphisms, as described below.

Participants/materials, setting, methods

Embryo biopsy samples were subjected to multiple displacement amplification (MDA). Mutation site(s) were amplified from MDA products using PCR and mutations were revealed using minisequencing, Sanger sequencing, or DNA fragment size analysis. Parental and embryo samples were also analysed using karyomapping, involving the genotyping ∼300,000 polymorphisms scattered across the genome with a microarray. Where possible, samples from additional family members were also tested, allowing determination of which alleles of linked polymorphisms accompanied mutant gene copies.

Main results and the role of chance

Multiple PGT-M cases were identified where DMT prevented potentially serious errors. In five cases, diagnostic reports provided to the PGT laboratory were incorrect. These reports are vital for defining the genetic status of an individual, allowing specific alleles of linked polymorphisms to be correctly associated with mutant or normal gene copies. In three cases, DMT carried out on patient samples during the initial work-up revealed that, contrary to the report, the patient did not carry a mutation. Therefore, PGT-M was not indicated, saving patients from the stress and expense of an unnecessary PGT-M cycle and avoiding discard of healthy embryos. In the other two cases, errors in the reports would have inverted all results based upon linkage analysis, leading to transfer of affected embryos and discard of unaffected embryos, potentially causing a serious misdiagnosis. Thirteen more cases had recombination events extremely close to the mutation site, preventing determination of the status of the embryos based on analysis of nearby polymorphisms. Two further cases displayed consanguinity (undisclosed by the patients), leading to large areas of homozygosity in the genome, precluding use of linked polymorphisms for diagnosis. In all these cases DMT unequivocally confirmed the true status of patients and their embryos.

Limitations, reasons for caution

In ∼8% of PGT-M cases at least one embryo could not be accurately diagnosed without DMT, while in ∼1% of cases our routine use of DMT averted a serious misdiagnosis. Nonetheless, even when DMT and linkage analysis are combined, it must be acknowledged that misdiagnoses remain possible (although extremely rare).

Wider implications of the findings

PGT-M is a valuable reproductive strategy for patients at high-risk of transmitting a single gene disorder. Linkage analysis is a valid strategy for PGT-M, but misdiagnoses are possible when testing relies entirely on such methods. These errors can be virtually eliminated by including direct testing of the mutation site.

Trial registration number

Not applicable