Targeted deep sequencing of DNA from multiple tissue types improves the diagnostic rate and reveals a highly diverse phenotype of mosaic neurofibromatosis type 2

Abstract

BackgroundAlthough 60% of patients with de novo neurofibromatosis type 2 (NF2) are presumed to have mosaic NF2, the actual diagnostic rate of this condition remains low at around 20% because of the existing difficulties in detecting NF2 variants with low variant allele frequency (VAF). Here, we examined the correlation between the genotype and phenotype of mosaic NF2 after improving the diagnostic rate of mosaic NF2.MethodsWe performed targeted deep sequencing of 36 genes including NF2 using DNA samples from multiple tissues (blood, buccal mucosa, hair follicle and tumour) of 53 patients with de novo NF2 and elucidated their genotype–phenotype correlation.ResultsTwenty-four patients (45.2%) had the NF2 germline variant, and 20 patients with NF2 (37.7%) had mosaic NF2. The mosaic NF2 phenotype was significantly different from that in patients with NF2 germline variant in terms of distribution of NF2-related disease, tumour growth rate and hearing outcome. The behaviour of schwannoma correlated to the extent of VAF with NF2 variant in normal tissues unlike meningioma.ConclusionWe have improved the diagnostic rate of mosaic NF2 compared with that of previous studies by targeted deep sequencing of DNA from multiple tissues. Many atypical patients with NF2 diagnosed with ‘unilateral vestibular schwannoma’ or ‘multiple meningiomas’ presumably have mosaic NF2. Finally, we suggest that the highly diverse phenotype of NF2 could result not only from the type and location of NF2 variant but also the extent of VAF in the NF2 variant within normal tissue DNA.

Analysis of induced pluripotent stem cell clones derived from a patient with mosaic neurofibromatosis type 2

“…For the first sample of meningioma (meningioma 1), targeted amplicon sequencing of NF2 was performed as previously described (Yuzawa et al, 2016), and the loss of NF2 was identified (Figure 2c). Because complete loss of NF2 is frequent in meningiomas but uncommon as a germline mutation of NF2 (Moyhuddin et al, 2003; Teranishi et al, 2021; Yuzawa et al, 2016), NF2 p.Q65X nonsense mutation was considered an initial postzygotic somatic mutation. Based on the peripheral blood genetic analysis, the patient was genetically diagnosed as a mosaic patient.…”

“…The combination of DNA sequencing and multiplex ligation‐dependent probe amplification for blood and separate tumors has revealed mosaicism in 25%–33% of patients with NF2 (Evans et al, 2020; Moyhuddin et al, 2003). Recent studies have reported higher sensitivity and proportions of predicted mosaicism with 37.7%–59.7% using next‐generation sequencing to detect NF2 alteration with low VAF in patients with mosaic NF2 (Evans et al, 2020; Teranishi et al, 2021). Mosaicism in patients with NF2 has been speculated based on the VAF in the blood DNA or by identical mutations in different tumors in previous reports (Evans et al, 2020; Moyhuddin et al, 2003; Teranishi et al, 2021).…”

“…Mosaicism is generally diagnosed based on low variant allele frequency (VAF) in blood DNA or due to the presence of identical mutations in separate tumors (Moyhuddin et al, 2003). However, it is sometimes challenging to diagnose mosaicism in patients with NF2 due to low VAF in the bone marrow‐derived peripheral mononuclear cells (MNCs) (Evans et al, 2020; Teranishi et al, 2021).…”

“…Patients with NF2 develop multiple intracranial and spinal tumors, including schwannomas and meningiomas (Asthagiri et al, 2009). The major forms of NF2 alterations are nonsense, frameshift, splice-site, and missense mutations as well as large exon and in-frame deletions (Asthagiri et al, 2009;Evans et al, 2020Evans et al, , 2007Moyhuddin et al, 2003;Teranishi et al, 2021). These genetic alterations in NF2 are detected in peripheral blood samples of most patients with familial NF2, while many sporadic patients present with mosaicism of an NF2 alteration (Evans et al, 2007;Halliday et al, 2017;Moyhuddin et al, 2003;Teranishi et al, 2021).…”

“…The major forms of NF2 alterations are nonsense, frameshift, splice-site, and missense mutations as well as large exon and in-frame deletions (Asthagiri et al, 2009;Evans et al, 2020Evans et al, , 2007Moyhuddin et al, 2003;Teranishi et al, 2021). These genetic alterations in NF2 are detected in peripheral blood samples of most patients with familial NF2, while many sporadic patients present with mosaicism of an NF2 alteration (Evans et al, 2007;Halliday et al, 2017;Moyhuddin et al, 2003;Teranishi et al, 2021). Mosaicism is generally diagnosed based on low variant allele frequency (VAF) in blood DNA or due to the presence of identical mutations in separate tumors (Moyhuddin et al, 2003).…”