Preimplantation genetic testing for neurofibromatosis type 1: molecular genetic aspects and impact on reproductive counseling

STUDY QUESTION How do the genetic complexities of neurofibromatosis type 1 (NF1) impact reproductive counseling, preimplantation genetic testing (PGT) design, and PGT treatment? SUMMARY ANSWER We established association between both incidence and tissue mosaicism with multiple exon deletions and specific single-nucleotide variants (SNVs) in neurofibromin 1 (NF1), a clinical actionable finding that we structured as a flowchart outlining challenges in and an approach for reproductive counseling, PGT design, and PGT treatment for NF1. WHAT IS KNOWN ALREADY NF1 has a prevalence of 1 in 2500–3000 and is one of the most frequently requested autosomal dominant indications for PGT. NF1 is a large gene with a high mutation rate, resulting in a 50% de novo occurrence, many different reported variants scattered across the gene and relatively frequent mosaicism. STUDY DESIGN, SIZE, DURATION We conducted a retrospective, observational cohort study on PGT molecular design for NF1 in three large PGT centers (n=281 couples), starting from the first assay for NF1 developed in 2004 until 2022. PARTICIPANTS/MATERIALS, SETTING, METHODS A PGT assay was developed for 281 couples with 218 different variants in NF1. Newly described variants (n=76) were scored using the American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP) classification system and submitted prior to publication in the Leiden Open Variation Database (LOVD). The employed techniques were PCR-based PGT using short tandem repeat markers (n=230), SNP-array-based PGT (n=39), and next-generation sequencing (NGS)-based PGT (n=12). Minisequencing (SNAPshot) or double amplification refractory mutation system (D-ARMS) was used to incorporate SNVs. Small deletions and insertions were incorporated using fragment length analysis. All PGT assays were designed and validated according to local protocols and ESHRE guidelines. MAIN RESULTS AND THE ROLE OF CHANCE Mosaicism was present in 8% of the sporadic cases (n=13/168), of which about half were unknown prior to PGT (n=6/13). Mosaicism was significantly higher in patients with multiple exon deletions (n=4/6) as compared to patients with SNVs (n=9/162) (P<0.001, Fisher’s exact test). Additionally, two recurrent SNVs were significantly associated with mosaicism (P <0.0167, Fisher’s exact test). Importantly, three unrelated families with different NF1 variants in close relatives were identified. LIMITATIONS, REASONS FOR CAUTION Due to its retrospective design, not all details on the genetic test results and clinical phenotype could be retrieved for some cases (n=6). The extent to which our findings are applicable to centers worldwide depends on their local procedures and legislation. WIDER IMPLICATIONS OF THE FINDINGS Our findings substantially impact reproductive counseling for couples with NF1, enabling informed reproductive decision-making. For couples affected with NF1 proceeding with PGT, our findings alert colleagues worldwide on NF1-specific pitfalls in PGT molecular design and treatment.