High frequency of mosaicism among patients with neurofibromatosis type 1 (NF1) with microdeletions caused by somatic recombination of the JJAZ1 gene

Abstract

Detailed analyses of 20 patients with sporadic neurofibromatosis type 1 (NF1) microdeletions revealed an unexpected high frequency of somatic mosaicism (8/20 [40%]). This proportion of mosaic deletions is much higher than previously anticipated. Of these deletions, 16 were identified by a screen of unselected patients with NF1. None of the eight patients with mosaic deletions exhibited the mental retardation and facial dysmorphism usually associated with NF1 microdeletions. Our study demonstrates the importance of a general screening for NF1 deletions, regardless of a special phenotype, because of a high estimated number of otherwise undetected mosaic NF1 microdeletions. In patients with mosaicism, the proportion of cells with the deletion was 91%-100% in peripheral leukocytes but was much lower (51%-80%) in buccal smears or peripheral skin fibroblasts. Therefore, the analysis of other tissues than blood is recommended, to exclude mosaicism with normal cells in patients with NF1 microdeletions. Furthermore, our study reveals breakpoint heterogeneity. The classic 1.4-Mb deletion was found in 13 patients. These type I deletions encompass 14 genes and have breakpoints in the NF1 low-copy repeats. However, we identified a second major type of NF1 microdeletion, which spans 1.2 Mb and affects 13 genes. This type II deletion was found in 8 (38%) of 21 patients and is mediated by recombination between the JJAZ1 gene and its pseudogene. The JJAZ1 gene, which is completely deleted in patients with type I NF1 microdeletions and is disrupted in deletions of type II, is highly expressed in brain structures associated with learning and memory. Thus, its haploinsufficiency might contribute to mental impairment in patients with constitutional NF1 microdeletions. Conspicuously, seven of the eight mosaic deletions are of type II, whereas only one was a classic type I deletion. Therefore, the JJAZ1 gene is a preferred target of strand exchange during mitotic nonallelic homologous recombination. Although type I NF1 microdeletions occur by interchromosomal recombination during meiosis, our findings imply that type II deletions are mediated by intrachromosomal recombination during mitosis. Thus, NF1 microdeletions acquired during mitotic cell divisions differ from those occurring in meiosis and are caused by different mechanisms.

Figure 1

Map of the NF1 gene region indicating the duplicated sequences, the genes included in type I and type II deletions, and the probes used for FISH analyses. The black horizontal arrows represent the 14 genes between and within the proximal and distal NF1 LCRs (highlighted by gray rectangles). The functional genes are underlined; pseudogenes are indicated with “P.” The green rectangles represent the regions with homology to segments on chromosome 19. The positions of FISH probes DJ1576/1578, DJHK10/11, and DJ1686/1863 are indicated by black vertical lines. BACs and PACs that build up the contig spanning the NF1 gene region are shown by horizontal bars. Those BACs/PACs marked by asterisks were used as FISH probes. The corresponding accession numbers are RP11-271K11 (GenBank accession number AC005562); CTD-2349P21 (GenBank accession number AC127024); RP11-785C15 (GenBank accession number AC109516); RP11-142O6 (GenBank accession number AC079915); RP11-805L22 (GenBank accession number AC007923); HCIT-307A16 (GenBank accession number AC003041); and RP11-55J8 (GenBank accession number AC015941).

Figure 2

Position of the deletion breakpoints in patients SB and KCD (investigated in this study) and in patient IL (described elsewhere) (Petek et al. 2003). The precise breakpoints in the JJAZ1 gene and its pseudogene (JJAZ-P) were identified by use of somatic cell hybrids that contain only the deleted chromosome 17 (cell lines KCD-3, IL-39, and SB-B9). Black bars indicate the nondeleted regions; white bars represent the deletion intervals. Exon numbers are given above, and the regions covered by PCR products P1–P4 are marked by gray rectangles.

Figure 3

Areas of strand exchange in the recombinant JJAZ1 sequences of patients KCD (A), SB (B), and IL (C), as determined by use of somatic cell hybrids that contain only the chromosome 17 with the deletion found in the respective patient. The regions of strand exchange, marked by black rectangles, were identified by the analysis of nucleotide differences between the JJAZ1 gene located distal and its pseudogene located proximal to the NF1 gene. The nucleotides typical for the JJAZ1 gene are given above the respective lines, and those characteristic for the pseudogene are given below. White ovals indicate that the sequence amplified from the hybrid cell line corresponds to the JJAZ1 pseudogene sequence; black ovals denote matches to the functional JJAZ1 gene. Noninformative polymorphisms (sequence differences among individuals) are represented as gray ovals. The numbers indicate the position of the respective PSV according to AC090616 (BAC 640N20).

Figure 4

Genotype analysis to determine that the type II deletion in patients KCD (A) and WB (B) occurred by intrachromosomal recombination. Eight polymorphic markers that flank the NF1 gene region and are located outside of the deletion interval were investigated. In addition, two markers within the deletion interval (RsaI and SNP2) were analyzed. A, The haplotypes were determined by the analysis of hybrid cell line KCD-3 with the microdeleted chromosome 17, hybrid cell line KCD-25 with chromosome 17 prior to the deletion, and hybrid cell line KCD-101, which retains the unaffected chromosome 17. B, Patient WB shows mosaicism for the type II deletion. The phase of markers was determined in her daughter, SB, by use of hybrid cell line SB-10 with the normal chromosome 17 and cell line SB-B9 with the microdeleted chromosome 17. The gray rectangles highlight the haplotype of the chromosome with the microdeletion.

Figure 5

Expression pattern of the mouse Jjaz1 mRNA in coronal sections of the adult mouse brain (A–D), horizontal sections of ED9,5 (E and F), and sagittal sections of ED14,5 embryos (G and H). Bright-field images are stained with Giemsa (A, C, E, and G). The corresponding dark-field images (B, D, F, and H) indicate as white areas the regions of highest Jjaz1 expression. Amy = amygdala; CA1–3 = regions of the hippocampus; db = decidua basalis; DG = dentate gyrus; e = embryo; fv = primordium of follicle of vibrissae; ge = ganglionic eminence; gtr = trophoblast giant cell; GrCb = granular cell layer of cerebellum; k = kidney; l = labyrinth; li = liver; lu = lung; MHb = medial habenular nucleus; nc = neocortex; Hy = hypothalamus; sb = submandibular gland; to = upper and lower molar tooth; Pur = Purkinje cells; Pir = piriform cortex; ys = yolk sac; and Zi = zona incerta.