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    • br Results RNA analysis was

    2018-11-05


    Results RNA analysis was carried out on 361 NF1 affected individuals who fulfilled NIH criteria with at least one non pigmentary criterion. Twenty one familial cases with just pigmentary NIH criteria were excluded (14 NF1 mutations (12 missense mutations); 7 SPRED1 mutations). One hundred and seventy one individuals were from families with multiple affected individuals and 190 were sporadic de novo (no evidence of NF1 family history) affected people. Potentially causative variants in the NF1 gene were identified in 166/171 (97.08%–95% CI 94.56–99.6%) of familial cases compared to 182/190 (95.8%–95% CI 92.93–98.65%) of sporadic samples (p=0.58-Table 2). This dropped to 154/171 (90.06%) and 175/190 (92.10%) respectively if class 3 variants were excluded. Although truncating mutations (nonsense and frameshift) were seen slightly more frequently in sporadic cases and non-truncating mutations in familial affected individuals these differences were not significant (Table 2). Whole gene deletions were present in 6/166 (3.6%) of familial samples with identified mutations and 15/182 (8.2%) of sporadic samples but this difference did not quite reach significance (p=0.08). However, when whole gene deletions found before Nov 2010 were included the 21 found in sporadic de novo cases was significantly higher than the 9 in familial cases (p=0.05). Two sporadic cases had a point mutation detected at low level in their lymphocyte RNA and were assumed to be mosaic for the mutation. One patient with the c.2530C>T (p.Lys844Phe) missense mutation detected at low level in blood lymphocytes (we estimate that the mutation is present in approx. 40% of lymphocytes in this sample, this equates to a ~20% mutant allele fraction) has had an affected child who has the same mutation. On reassessment of her skin although she clearly met NIH criteria with >5 CAL, freckling and neurofibromas her pattern of skin involvement meant various body segments were completely unaffected. A similar proportion of inherited and sporadic samples had mutations identified that were only classifiable by RNA analysis (Table 3). A total of 41/348 (11.8%) of samples with proven mutations met this criteria with ten (2.9%) variants occurring >10 nucleotides from the consensus splice site of which six would have been well outside the boundaries of intron GSK2656157 regions screened in standard DNA analysis. Seven mutations which would have been classified as a variant of uncertain significance (VUS) (6 missense mutations, one synonymous change) and eight patients with six nonsense mutations on DNA analysis alone were also found to affect the normal splicing of NF1 RNA. Table 4 shows the variants which were not truncating and did not affect the splicing at RNA level. These were predominantly missense mutations some of which have previous published evidence for pathogenicity (Griffiths et al., 2007; Upadhyaya et al., 1997; Ahmadian et al., 2003; Valero et al., 2011; Fahsold et al., 2000). However, six samples (4 familial) occurred in an evolutionary conserved region of the 5′ untranslated region (UTR). Although we did confirm bi-allelic NF1 expression in two samples that were heterozygous at the DNA level for transcribed SNPs (c.-272G>A and c.-273A>C), the first variant segregated with disease in the family. Furthermore c.-273A>C was shown to have arisen de novo as it was not carried by the unaffected parents of a sporadic case. Classification of pathogenicity in Table 4 is as per accepted guidelines (Plon et al., 2008; Wallis et al., 2013; Richards et al., 2015). Of the thirteen cases with no identified mutation two had a dysembryoplastic neuroepithelial tumour (DNET) identified on MRI of the brain (Fig. 1a and b) both associated males with epilepsy. One had inherited NF1 from a mother affected with classical features of NF1. The other was sporadic but did appear to have major nerve root nerve sheath tumors on whole body MRI and had tumour resection because of his intractable epilepsy. Histopathology of tissue removed at surgery to relieve epilepsy in the sporadic case showed sections of cortical grey matter revealing indistinct glioneuronal nodules, the familial case histopathology showed oligodendroglia-like cells forming bundles around axonal profiles with occasional floating neurons, both consistent with the radiological diagnosis of DNET. Both patients tested negative for SPRED1 mutations. The only two other NF1 affected individuals with a DNET had identified NF1 nonsense mutations (c.6763G>T p.(Glu2255Ter; c.4084C>T p.(Arg1362Ter). They both had classical NF1 inherited from a clearly affected parent with >6 CAL and neurofibromas, but did not have epilepsy and diagnosis was based on imaging appearances alone. The presence of DNET in 2/13 (15%) negative screens versus 2/348 (0.6%-Relative Risk of 0.004) with mutations is highly significant (p=0.007).