The chromosome 9p21.3 region harbours a cluster of important growth regulatory genes (CDKN2A/ARF and CDKN2B) that are deleted or transcriptionally silenced in a wide range of tumours such as plexiform neurofibromas (PNF) [12, 22]. The proteins encoded by the CDKN2A/CDKN2B genes act as inhibitors of the CDK4/6 cyclin-dependent kinases, thereby regulating the growth suppressive activity of the RB family of proteins. By contrast, the ARF protein binds to and inhibits the oncoprotein MDM2 which activates p53 . The expression of CDKN2A, ARF and CDKN2B is very low in both young and non-neoplastic cells but increases during cell aging and oncogene-induced hyperproliferation, suggesting that the coordinated expression of these genes is a means to regulate senescence and prevent oncogene-driven hyperproliferation . The polycomb repressive complexes PRC1 and PRC2 have been shown to initiate and maintain the silenced state of the CDKN2A/ARF, CDKN2B gene cluster [23, 24]. PRC1 and PRC2 are recruited to these loci by the 3.8-kb non-coding RNA ANRIL in order to regulate their expression [8, 10, 25, 26].
In a family-based association study, Pasmant et al.  investigated a total of five tag SNPs located at 9p21.3 in 1105 individuals (740 NF1 patients and 365 unaffected individuals from 306 NF1 families) and observed a significant association between the number of PNF and one of these five SNPs, rs2151280. This SNP, located within intron 3 of the ANRIL gene, was found to be associated with the number of PNF under a dominant model, with preferential transmission of the derived T-allele to those NF1 patients possessing a higher number of PNF. By contrast, the number of dermal neurofibromas (DNF) was not found to be associated with rs2151280. Importantly, the T-allele of rs2151280 is associated with a reduced ANRIL expression level suggesting either a functional role for SNP rs2151280 or that this SNP is in linkage disequilibrium with an additional as yet unknown functional variant which influences ANRIL expression . Taken together, these findings suggested that modulation of ANRIL expression mediates PNF susceptibility in patients with NF1. It is unclear how many patients with NF1 microdeletions were included in the study of Pasmant et al. . However, only 5% of patients with NF1 exhibit NF1 microdeletions and familial cases are very rare.
In this study, we investigated a putative association between the number or volume of PNF and rs2151280 in 29 patients with non-mosaic NF1 microdeletions. These patients were extremely well characterized by whole-body MRI. We did not observe an association between the T-allele of rs2151280 and either PNF number or PNF volume in these patients, suggesting that this SNP does not exert a strong effect on PNF susceptibility in this group of NF1 microdeletion patients. However, we cannot rule out the possibility of a weak association that might have remained undetected owing to the small number of patients investigated. Under the assumption of an ordered categorical distribution, we estimated that it would have been necessary to analyze approximately 300 NF1 patients to detect a significant association between tumour volume and the T-allele with a power of 80% using the Mann–Whitney–Wilcoxon test (α=5%). This estimation is however based on the observations we made in the 29 patients and implies that the distribution of tumour volumes observed is representative for the whole population of NF1 microdeletion patients. Since NF1 microdeletions are rare (occurring with an estimated prevalence of 1:70,000), the whole-body MRI investigation of 300 patients with NF1 microdeletions is scarcely feasible. As deduced from the data obtained from the analysis of the 29 NF1 microdeletion patients, a strong association between the T-allele of SNP rs2151280 and the PNF load is not obvious.
Patients with NF1 microdeletions have been reported to exhibit a more severe clinical phenotype than patients with intragenic NF1 mutations, as evidenced by an increased risk of MPNSTs, severe learning disability, cognitive impairment, developmental delay and dysmorphic facial features [16, 27–30]. However, the number of PNF, as determined by whole-body MRI, was not found to differ significantly between patients with NF1 microdeletions as a group and NF1 patients lacking large NF1 deletions . Nevertheless, differences in PNF development and biology may well exist between both patient groups i.e. those with NF1 microdeletions and those with intragenic NF1 mutations. The most common type of NF1 microdeletion encompasses 1.4-Mb (termed type-1 NF1 deletion) and is associated with the loss of 14 protein-coding genes inclusive of the NF1 gene [31–33]. Potentially, the loss of one or several of the genes located within the NF1 microdeletion region in addition to the deletion of the NF1 gene, may influence tumour biology or progression. A good candidate for such a modifier gene influencing tumour development is SUZ12 which is located within the 1.4-Mb NF1 microdeletion region. One allele of SUZ12 is deleted in all patients investigated in our study. The SUZ12 protein is an essential component of the polycomb repressive complex 2 (PRC2) and somatic mutations as well as deletions of SUZ12 have recently been identified in various haematological malignancies indicating an important role for chromatin modifiers (such as the polycomb repressive complexes) in tumorigenesis [34–39]. Remarkably, the polycomb repressive complexes 1 and 2 (PRC1 and PRC2) have also been shown to regulate the expression of the CDKN2A/ARF and CDKN2B genes. ANRIL directly binds to SUZ12, an essential component of PRC2 and is required for SUZ12 occupancy of the CDKN2B locus as well as for the epigenetic silencing of CDKN2B.
The loss of one SUZ12 allele in patients with germline NF1 microdeletions may well influence ANRIL-mediated expression regulation of the CDKN2A/CDKN2B tumour suppressor genes. Although somatic inactivation of the NF1 wild-type allele is considered to be the PNF-initiating event in NF1 patients with intragenic mutations and patients with NF1 microdeletions , both patient groups may differ with regard to tumour progression because of the heterozygous constitutional deletion of SUZ12 present only in patients with NF1 microdeletions. Consistent with this hypothesis, an extremely high total PNF volume (>3,000 ml) was noted significantly more frequently in patients with NF1 microdeletions than in NF1 patients without large deletions .