Pre-implantation genetic testing for Marfan syndrome using mini-sequencing

Marfan syndrome (MFS1) is an autosomal dominant condition causing aortopathy including fatal aortic dissection. This study aimed to perform clinical PGT-M in a family with a history of MFS1 for two generations. A family with two members affected by MFS1 approached the hospital for PGT-M. The couple decided to join the project following extensive counselling and informed consent was obtained. The mutation contributory to MFS1 was identified using whole-exome sequencing (WES). A novel PGT-M protocol using multiplex fluorescent PCR and mini-sequencing was developed and tested. Ten blastocysts were subjected to PGT-M in two clinical PGT cycles. Mini-sequencing revealed four normal and six affected embryos. Microsatellite-based linkage analysis confirmed mutation analysis results in all samples. The embryos diagnosed as normal (non-MFS1) were chosen for transfer. A pregnancy was obtained in the third embryo transfer. Invasive prenatal diagnosis confirmed the normal genotype of the baby. This study demonstrated comprehensive management using the application of clinical-based diagnosis, WES for mutation identification within the MFS1 gene, mini-sequencing for embryo selection and microsatellite-based linkage analysis for backup of PGT-M results and contamination detection to assist couples in having a healthy child when there was a family history of Marfan syndrome.Impact StatementWhat is already known on this subject? Marfan syndrome (MFS1, OMIM#154700) is an autosomal dominant condition causing aortopathy including fatal aortic dissection. Pre-implantation genetic testing (PGT) is an alternative to traditional invasive prenatal diagnosis (PND) giving the couples the chance of starting pregnancy with the confidence that the baby will be unaffected. Most of the previous PGT reports employed microsatellite-based linkage analysis. A few PGT studies used sequencing, mini-sequencing and mutation analysis; however, the details of the techniques were not described.What do the results of this study add? Single-cell PCR protocol using multiplex fluorescent PCR and mini-sequencing was developed and validated. Two clinical PGTs cycles for Marfan syndrome were performed. A healthy baby was resulted. The details of multiplex fluorescent PCR and mini-sequencing protocols are described in this study so that the procedures can be reproduced.What are the implications of these findings for clinical practice and/or further research? Embryo selection can help the family suffering from Marfan syndrome for two generations to start a pregnancy with confidence that their child will be unaffected. This study also shows the use of a mini-sequencing protocol for PGT, which can be a universal protocol for other mutations by changing the PCR primers and mini-sequencing primers.