Tissue-specific differences in the proportion of mosaic large NF1 deletions are suggestive of a selective growth advantage of hematopoietic del(+/-) stem cells.

Type-2 NF1 deletions spanning 1.2 Mb are frequently of postzygotic origin and hence tend to be associated with mosaicism for normal cells and those harboring the deletion (del(+/-) cells). Eleven patients with mosaic type-2 deletions were investigated by FISH and high proportions (94-99%) of del(+/-) cells were detected both in whole blood and in isolated CD3+, CD14+, CD15+, and CD19+ leukocytes. Significantly lower proportions of del(+/-) cells (24-82%) were however noted in urine-derived epithelial cells. A patient harboring an atypical large NF1 deletion with nonrecurrent breakpoints was also found to have a much higher proportion of del(+/-) cells in blood (96%) than in urine (51%). The tissue-specific differences in the proportions of del(+/-) cells as well as the X chromosome inactivation (XCI) patterns observed in these mosaic patients suggest that the majority of the deletions had occurred before or during the preimplantation blastocyst stage before the onset of XCI. We postulate that hematopoietic del(+/-) stem cells present at an early developmental stage are characterized by a selective growth advantage over normal cells lacking the deletion, leading to a high proportion of del(+/-) cells in peripheral blood from the affected patients.

Characterization of the nonallelic homologous recombination hotspot PRS3 associated with type-3 NF1 deletions.

Nonallelic homologous recombination (NAHR) is the major mechanism underlying recurrent genomic rearrangements, including the large deletions at 17q11.2 that cause neurofibromatosis type 1 (NF1). Here, we identify a novel NAHR hotspot, responsible for type-3 NF1 deletions that span 1.0 Mb. Breakpoint clustering within this 1-kb hotspot, termed PRS3, was noted in 10 of 11 known type-3 NF1 deletions. PRS3 is located within the LRRC37B pseudogene of the NF1-REPb and NF1-REPc low-copy repeats. In contrast to other previously characterized NAHR hotspots, PRS3 has not developed on a preexisting allelic homologous recombination hotspot. Furthermore, the variation pattern of PRS3 and its flanking regions is unusual since only NF1-REPc (and not NF1-REPb) is characterized by a high single nucleotide polymorphism (SNP) frequency, suggestive of unidirectional sequence transfer via nonallelic homologous gene conversion (NAHGC). By contrast, the previously described intense NAHR hotspots within the CMT1A-REPs, and the PRS1 and PRS2 hotspots underlying type-1 NF1 deletions, experience frequent bidirectional sequence transfer. PRS3 within NF1-REPc was also found to be involved in NAHGC with the LRRC37B gene, the progenitor locus of the LRRC37B-P duplicons, as indicated by the presence of shared SNPs between these loci. PRS3 therefore represents a weak (and probably evolutionarily rather young) NAHR hotspot with unique properties.

Extended runs of homozygosity at 17q11.2: an association with type-2 NF1 deletions?

Large deletions in the NF1 gene region at 17q11.2 are caused by nonallelic homologous recombination (NAHR). The recurrent type-2 NF1 deletions span 1.2 Mb, with breakpoints in the SUZ12 gene and SUZ12P. Type-2 NF1 deletions occur preferentially during mitosis and are associated with somatic mosaicism. A panel of 16 type-2 NF1 deletions was used as a model system in which to investigate whether extended homozygosity across 17q11.2 might be associated with somatic deletion. Using SNP arrays, a 3.2 Mb interval encompassing the NF1 deletion region was found to harbor runs of homozygosity (ROHs) in different human populations. However, ROHs >or=500 kb directly flanking the NF1 deletion region on both sides were not found to occur disproportionately in NF1 patients harboring type-2 deletions compared to controls. Although low allelic diversity in 17q11.2 is unlikely to be a key factor in promoting NAHR-mediated somatic type-2 deletions, a specific ROH of 588 kb (roh1), located some 525 kb proximal to the deletion interval, was found to occur more frequently (P=0.012) in the type-2 deletion patients compared with controls. We postulate that roh1 may act remotely, via an as yet unknown mechanism, to increase the frequency of somatic recombination between the distally duplicated SUZ12 sequences.

Intrachromosomal mitotic nonallelic homologous recombination is the major molecular mechanism underlying type-2 NF1 deletions.

Nonallelic homologous recombination (NAHR) is responsible for the recurrent rearrangements that give rise to genomic disorders. Although meiotic NAHR has been investigated in multiple contexts, much less is known about mitotic NAHR despite its importance for tumorigenesis. Because type-2 NF1 microdeletions frequently result from mitotic NAHR, they represent a good model in which to investigate the features of mitotic NAHR. We have used microsatellite analysis and SNP arrays to distinguish between the various alternative recombinational possibilities, thereby ascertaining that 17 of 18 type-2 NF1 deletions, with breakpoints in the SUZ12 gene and its highly homologous pseudogene, originated via intrachromosomal recombination. This high proportion of intrachromosomal NAHR causing somatic type-2 NF1 deletions contrasts with the interchromosomal origin of germline type-1 NF1 microdeletions, whose breakpoints are located within the NF1-REPs (low-copy repeats located adjacent to the SUZ12 sequences). Further, meiotic NAHR causing type-1 NF1 deletions occurs within recombination hotspots characterized by high GC-content and DNA duplex stability, whereas the type-2 breakpoints associated with the mitotic NAHR events investigated here do not cluster within hotspots and are located within regions of significantly lower GC-content and DNA stability. Our findings therefore point to fundamental mechanistic differences between the determinants of mitotic and meiotic NAHR.

Delineation of the clinical phenotype associated with non-mosaic type-2 NF1 deletions: two case reports.

INTRODUCTION: Large deletions of the NF1 gene and its flanking regions are frequently associated with a severe clinical manifestation. Different types of gross NF1 deletion have been identified that are distinguishable both by their size and the number of genes included within the deleted regions. Type-1 NF1 deletions encompass 1.4 Mb and include 14 genes, whereas the much less common type-2 NF1 deletions span 1.2 Mb and contain 13 genes. Genotype-phenotype correlations in patients with large NF1 deletions are likely to be influenced by the nature and number of the genes deleted in addition to the NF1 gene. Whereas the clinical phenotype associated with type-1 NF1 deletions has been well documented, the detailed clinical characterization of patients with non-mosaic type-2 NF1 deletions has not so far been reported. CASE PRESENTATION: In the present report we characterized two Caucasian European patients with non-mosaic (germline) type-2 NF1 deletions. Our first patient was a 13-year-old girl with dysmorphic facial features, mild developmental delay, large hands and feet, hyperflexibility of the joints, macrocephaly and T2 hyperintensities in the brain. A whole-body magnetic resonance imaging scan indicated two internal plexiform neurofibromas. Our second patient was an 18-year-old man who exhibited dysmorphic facial features, developmental delay, learning disability, large hands and feet, hyperflexibility of the joints, macrocephaly and a very high subcutaneous and internal tumor load as measured volumetrically on whole-body magnetic resonance imaging scans. At the age of 18 years, he developed a malignant peripheral nerve sheath tumor and died from secondary complications. Both our patients exhibited cardiovascular malformations. CONCLUSIONS: Our two patients with non-mosaic type-2 NF1 deletions exhibited clinical features that have been reported in individuals with germline type-1 NF1 deletions. Therefore, a severe disease manifestation is not confined to only patients with type-1 NF1 deletions but may also occur in individuals with type-2 NF1 deletions. Our findings support the concept of an NF1 microdeletion syndrome with severe clinical manifestation that is caused by type-1 as well as type-2 NF1 deletions.