The gene was cloned in and

The gene was cloned in 1990 and initial studies of the large complex gene identified only between 30 and 65% of germline mutations in clearly affected individuals (Shen et al., 1996). Furthermore, phenotype analysis in pedigrees suggested that there would be little significant genotype–phenotype correlation (Easton et al., 1993) and apart from the NF1 microdeletions first reported in 1994 (Kayes et al., 1992), there was little interest in this field until relatively recently. It was not until 10years after the gene was identified that a study using an exhaustive approach including RNA analysis identified 64/67 (95%) of mutations in clearly affected NF1 individuals (Messiaen et al., 2000). Since then mutations in the SPRED1 gene have been identified as the cause of Legius syndrome characterised by multiple (CAL) patches and macrocephaly, but without the tumour features of NF1. Among a cohort of 42 SPRED1 mutated individuals 48% fulfilled NIH NF1 diagnostic criteria based on the presence of >5 CAL with or without freckling or an NF1-compatible family history (Messiaen et al., 2009). Of 94 probands with familial CAL with or without freckling and no other NF1 features, 69 (73%; 95% CI, 63%–80%) had an NF1 orexin antagonist and 18 (19%; 95% CI, 12%–29%) had a pathogenic SPRED1 mutation (Messiaen et al., 2009). Variants have not been identified in any further gene producing resulting in an NF1-like syndrome. However, since the finding that certain NF1 mutations give rise to a CAL and freckling only phenotype (Upadhyaya et al., 2007; Rojnueangnit et al., 2015) and that patients with ‘spinal phenotype’ have an excess of splicing and missense mutations there has been a resurgence of interest in genotype–phenotype correlation. Although post zygotic mutations in embryogenesis leading to mosaicism have been shown to be the cause at least 33% of de novo Neurofibromatosis type 2 (NF2) affected individuals with classical disease fulfilling NIH criteria with bilateral vestibular schwannoma (National Institutes of Health Consensus Development Conference, 1987; Evans et al., 2007), it has rarely been reported as the cause of classical non segmental NF1. As the reference laboratory for the nationally funded highly specialised complex NF1 service in England, from 2009 we have applied comprehensive RNA analysis of the NF1 gene coupled with MLPA based copy number analysis using the approach developed by Messiaen et al. (Messiaen et al., 2000) The current study aimed to determine the sensitivity of this strategy to detect mutations in a large cohort of well characterised individuals with NF1 who met NIH diagnostic with more than just pigmentary criteria. This would potentially identify whether other genes may still cause features compatible with NF1. Furthermore the study aimed to assess the likely contribution of mosaicism to classically affected de novo cases.
Materials and Methods Individuals referred to the Manchester service who fulfilled NIH criteria (Table 1), which was not confined to a body segment and who had at least one non-pigmentary criteria for NF1 in them or their affected relative were included. NF1 affected individuals were divided into familial where there was at least one affected first degree relative who also met NIH criteria and sporadic de novo cases. All the families tested were unrelated and did not contain known multiple branches of the same family. In addition, a separate analysis was carried out on consecutive children with at least 6 CAL with or without freckling, but no other NF1 diagnostic criterion who also had no parent with an NF1 criterion and were aged <20years of age at assessment. This was for a 5-year period from November 2010–November 2015. NF1 mutation analysis was carried out in the Genomic Diagnostic Laboratory at the Manchester Centre for Genomic Medicine in St Mary\'s Hospital, Manchester. This is a clinically accredited medical testing laboratory. RNA and genomic DNA were prepared from peripheral blood samples. RNA was isolated from short term PHA stimulated cultures pre-treated before RNA extraction with Puromycin to inhibit nonsense mediated decay. RNA was reverse transcribed to cDNA using standard procedures, and the cDNA was PCR amplified in 5 overlapping fragments of approx. 2kb in size. Each fragment was then Sanger sequenced with between 8 and 11 primers to give overlapping sequence data of the whole fragment thus highlighting abnormalities in NF1 splicing or mutations within the coding sequence. Short term culture followed by Puromycin treatment was used in preference to RNA stabilising blood collection tubes e.g. PAXgene, due to more consistent and robust RNA quality achieved in preliminary tests. Mutation status was confirmed in genomic DNA. Multiplex ligation dependent probe amplification (MLPA) for dosage analysis was additionally performed in samples without a clearly pathogenic mutation identified on cDNA analysis using the MRC-Holland P081 and P082 probe sets. In samples where no clearly pathogenic NF1 mutation was identified the SPRED1 gene was screened for mutations by bidirectional Sanger sequencing of the whole coding region and flanking splice donor and acceptor sites to ±15bp plus MLPA dosage analysis using the MRC-Holland P295 probe set.