Classification of NF1 microdeletions and its importance for establishing genotype/phenotype correlations in patients with NF1 microdeletions

Abstract

An estimated 5-11% of patients with neurofibromatosis type-1 (NF1) harbour large deletions encompassing the NF1 gene and flanking regions. These NF1 microdeletions are subclassified into type 1, 2, 3 and atypical deletions which are distinguishable from each other by their extent and by the number of genes included within the deletion regions as well as the frequency of mosaicism with normal cells. Most common are type-1 NF1 deletions which encompass 1.4-Mb and 14 protein-coding genes. Type-1 deletions are frequently associated with overgrowth, global developmental delay, cognitive disability and dysmorphic facial features which are uncommon in patients with intragenic pathogenic NF1 gene variants. Further, patients with type-1 NF1 deletions frequently exhibit high numbers of neurofibromas and have an increased risk of malignant peripheral nerve sheath tumours. Genes located within the type-1 NF1 microdeletion interval and co-deleted with NF1 are likely to act as modifiers responsible for the severe disease phenotype in patients with NF1 microdeletions, thereby causing the NF1 microdeletion syndrome. Genotype/phenotype correlations in patients with NF1 microdeletions of different lengths are important to identify such modifier genes. However, these correlations are critically dependent upon the accurate characterization of the deletions in terms of their extent. In this review, we outline the utility as well as the shortcomings of multiplex ligation-dependent probe amplification (MLPA) to classify the different types of NF1 microdeletion and indicate the importance of high-resolution microarray analysis for correct classification, a necessary precondition to identify those genes responsible for the NF1 microdeletion syndrome.

Fig. 1

Schema of the 1.4-Mb spanning type-1 NF1 microdeletion region and its flanking regions indicating the relative positions of the 14 protein-coding genes, the SUZ12P pseudogene and the 5 microRNA genes located there. The relative extent of type-1, type-2, atypical deletions of group #2A and type-3 NF1 deletions are indicated by grey horizontal bars. The vertical red and green arrows represent the binding sites of the MLPA-probes included in the SALSA^® MLPA^® Probemix P122-D2 NF1-area (MRC-Holland). Red arrows represent MLPA-probes targeting genomic regions encompassed by the respective deletions whereas green arrows represent MLPA-probes targeted to regions which are not deleted and present in two copies. For example, in case of type-1 NF1 deletions encompassing 1.4-Mb, the target sequences for the probes shown in red are present in only one copy because they are located within the deletion region. By contrast, the target sequences for the MLPA-probes shown in green are present in two copies since they are not located within the deletion region. The MLPA-probe SUZ12P intron 4 is shaded in green and red because the region targeted by this probe is deleted in some but not all type-2 and atypical group #2A NF1 deletions. Type-2 deletions and atypical group #2A deletions cannot be distinguished by means of MLPA. cen centromeric; tel telomeric

Fig. 2

Schema of the type-1 NF1 microdeletion region, which includes 14 protein-coding genes as well as the SUZ12P pseudogene and 5 microRNA genes. The relative locations of these genes are indicated by black rectangles. Indicated below is the extent of the 21 known atypical group #2 NF1 deletions represented by vertical black bars. The patient IDs are indicated on the left. The low-copy repeats, NF1-REPa and NF1-REPc, are located at the boundaries of the type-1 NF1 microdeletion region. The atypical NF1 deletions of group #2, which exhibit breakpoints located within the boundaries of the type-1 NF1 microdeletion region, are smaller than type-1 NF1 deletions and do not encompass all of the genes located within the type-1 NF1 microdeletion region. As yet, 30 atypical group #2 NF1 deletions have been reported; indicated are 21 of these atypical group #2 deletions which can be distinguished from type-1 NF1 deletions by MLPA. Patients with IDs indicated in red were analysed by Vogt et al. (2012, 2014), those indicated in blue were analysed by Pasmant et al. (2010), in pink by Bianchessi et al. (2015) and in green by Zhang et al. (2015). Patient 171 was analysed by Ferrari et al. (2017), patients #1 and #2 by Serra et al. (2019), patients 134/260 by Büki et al. (2021) and patient 310221 by Kehrer-Sawatzki et al. submitted for publication). The deletions of the 5 patients whose IDs are marked by an asterisk do not encompass 4 of the 5 microRNA genes located within the type-1 NF1 microdeletion region. cen centromeric; tel telomeric

Fig. 3

Schema of the type-1 NF1 microdeletion region, which includes 14 protein-coding genes as well as the SUZ12P pseudogene and 5 microRNA genes. The relative locations of these genes are indicated by black rectangles. Indicated below is the extent of the 9 known atypical group #2A NF1 deletions which are represented by vertical black bars. Patient IDs are indicated on the left. The low-copy repeats, NF1-REPa and NF1-REPc, are located at the boundaries of the type-1 NF1 microdeletion region. The atypical NF1 deletions of group #2A, which exhibit breakpoints located within the boundaries of the type-1 NF1 microdeletion region, cannot be distinguished from type-2 deletions using MLPA. The vertical red and green arrows represent the binding sites of the MLPA-probes included in the SALSA^® MLPA^® Probemix P122-D2 NF1-area (MRC-Holland). Red arrows represent MLPA-probes targeting genomic regions encompassed by the respective heterozygous deletions whereas green arrows represent MLPA-probes targeted to regions which are not deleted and present in two copies. The MLPA-probe SUZ12P intron 4 is shaded in green/red because the region targeted by this probe is deleted only in the patients marked by an asterisk, and not in the other 5 atypical group #2A deletions which are depicted here. cen: centromeric; tel: telomeric