Written by Ms Florentia Romanou, Cyprus Institute of Neurology and Genetics, Cyprus
Preimplantation Genetic Testing for Monogenic Disorders (PGT-M) involves examining nuclear or mitochondrial DNA pathogenic variants that cause monogenic disorders with autosomal dominant, autosomal recessive, or X-linked transmission patterns. It also includes exclusion testing and Human Leukocyte Antigen (HLA)-typing with or without simultaneous testing for monogenic disorders. PGT-M is a highly established preventive tool, capable of identifying nearly every monogenic disorder, or combination thereof, provided the pathogenic variant is unequivocally diagnosed.
Cyprus ranks among the countries with the highest prevalence of β-thalassaemia carriers (12%) and pioneered by establishing the National Prevention Programme, launched in 1978, based on premarital screening and prenatal testing. PGT-M for β-thalassemia started in 2004 as an additional prevention strategy, today assessing around 40 couples at risk yearly.
One significant limitation that is commonly encountered in PGT-M for β-thalassaemia is the absence of additional family members making it difficult to determine the disease-related haplotype. Similar challenges arise for couples with De-Novo Mutations (DNMs) detected in one partner or an affected child but are not inherited from either parent.
Therefore, there is a pressing need for a new approach which will allow the detection of polymorphic markers co-inherited with the pathogenic variation without requiring other family members or prior knowledge of the inheritance pattern. This approach would help determine the inheritance pattern of the variation and address challenges associated with limited family members. Long-read amplicon sequencing is a very promising and effective approach that has been successfully implemented for PGT-M diagnosis, especially in cases where additional family members are unavailable or where DNMs occur.
The purpose of my mobility to the Constitutional Cytogenetics unit at the Department of Human Genetics, in Leuven, Belgium, was to receive training from the expert Dr Eftychia Dimitriadou, on Long-Read Sequencing using the ONT MinION platform. Oxford Nanopore Technology (ONT) long-read sequencing allows for direct- real-time sequencing of single molecules able to reduce the bias.
During my stay, I gained not only theoretical knowledge but also practical experience in designing and implementing ONT sequencing for PGT-M. Specifically, we successfully designed and validated an ONT panel capable of simultaneously performing direct and indirect detection of every β-thalassaemia related variation. As a follow-up, we plan to adopt the ONT technology and use this panel to examine, more families at risk of β-thalassaemia in-house. Our long-term goal is to implement these techniques and develop customized PGT-M panels for families without additional members or those with DNMs.
Oxford nanopore long-read sequencing is a cutting-edge method, recently applied in the field of PGT-M. We are particularly interested in implementing this technology for the first time in Cyprus, made possible by the support of the HELIOS action. Thanks to the opportunity that the STSM program gave me, we are now able not only to develop & implement such tools but also to disseminate all the knowledge and experience to other members of the CING or the HELIOS networks.
Funded by HELIOS COST Action CA22119