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

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.     

Evidence for non-homologous end joining and non-allelic homologous recombination in atypical NF1 microdeletions

NF1 microdeletion syndrome is caused by haploinsufficiency of the NF1 gene and of gene(s) located in adjacent flanking regions. Most of the NF1 deletions originate by non-allelic homologous recombination between repeated sequences (REP-P and -M) mapped to 17q11.2, while the remaining deletions show unusual breakpoints. We performed high-resolution FISH analysis of 18 NF1 microdeleted patients with the aims of mapping non-recurrent deletion breakpoints and verifying the presence of additional recombination-prone architectural motifs. This approach allowed us to obtain the sequence of the first junction fragment of an atypical deletion. By conventional FISH, we identified 16 patients with REP-mediated common deletions, and two patients carrying atypical deletions of 1.3 Mb and 3 Mb. Following fibre-FISH, we identified breakpoint regions of 100 kb, which led to the generation of several locus-specific probes restricting the atypical deletion endpoint intervals to a few kilobases. Sequence analysis provided evidence of small blocks of REPs, clustered around the 1.3-Mb deletion breakpoints, probably involved in intrachromatid non-allelic homologous recombination (NAHR), while isolation and sequencing of the 3-Mb deletion junction fragment indicated that a non-homologous end joining (NHEJ) mechanism is implicated.M. Venturin and C. Gervasini contributed equally to the study     

Extensively high load of internal tumors determined by whole body MRI scanning in a patient with neurofibromatosis type 1 and a non-LCR-mediated 2-Mb deletion in 17q11.2

Deletions in 17q11.2 affecting the NF1 gene and surrounding regions occur in 5% of patients with NF1. The two major types of NF1 deletions encompass 1.4-Mb and 1.2-Mb, respectively, and have breakpoints in the NF1 low-copy repeats or in the JJAZ gene and its pseudogene. Deletions larger than 1.4-Mb are rare, and only seven cases have been reported so far. Here, we describe a 26-year-old NF1 patient with an atypical NF1 deletion of 2-Mb. In contrast to the 1.4-Mb deletions, which preferentially occur by interchromosomal recombination during maternal meiosis, the deletion described here occurred intrachromosomally on the paternal chromosome. The centromeric deletion breakpoint lies in an L1-element located 1.3-Mb proximal to the NF1 gene. The telomeric deletion boundary is located in a single copy segment between an AT-rich segment and an AluSx-element in intron 15 of the JJAZ1 gene. Structural analysis implies that non-B DNA conformations at the breakpoints destabilized the duplex DNA and caused double-strand breaks. Although the breakpoints of this 2-Mb deletion are not recurrent, it is conspicuous that one breakpoint is located in the JJAZ1 gene. Paralogous recombination between the JJAZ1 gene and its pseudogene causes the recurrent 1.2 Mb deletions. The genomic architecture of the NF1 gene region, influenced by paralogous sequences such as the JJAZ1 gene and its pseudogene, seems also to stimulate the occurrence of non-recurrent deletions mediated by non-homologous end joining. Patient 442 described here suffers from a very high burden of subdermal neurofibromas. Magnetic resonance imaging of the whole body revealed numerous internal tumors, mainly plexiform neurofibromas and spinal tumors. This demonstrates the value of whole-body MRI scanning in determining the total tumor load, which is an important aspect in genotype/phenotype correlations with regard to large NF1 deletions.     

Uncommon Alu-mediated NF1 microdeletion with a breakpoint inside the NF1 gene

Neurofibromatosis type 1 (NF1) microdeletion syndrome is caused by haploinsufficiency of the NF1 gene and of gene(s) located in adjacent flanking regions. Most of the NF1 deletions originate by nonallelic homologous recombination between repeated sequences (REP-P and -M) mapped to 17q11.2, while a few uncommon deletions show unusual breakpoints. We characterized an uncommon 1.5-Mb deletion of an NF1 patient displaying a mild phenotype. We applied high-resolution FISH analysis allowing us to obtain the sequence of the first junction fragment of an uncommon deletion showing the telomeric breakpoint inside the IVS23a of the NF1 gene. Sequence analysis of the centromeric and telomeric boundaries revealed that the breakpoints were present in the AluJb and AluSx regions, respectively, showing 85% homology. The centromeric breakpoint is localized inside a chi-like element; a few copies of this sequence are also located very close to both breakpoints. The in silico analysis of the breakpoint intervals, aimed at identifying consensus sequences of several motifs usually involved in deletions and translocations, suggests that Alu sequences, probably associated with the chi-like element, might be the only recombinogenic motif directly mediating this large deletion.     

The t(4;8) is mediated by homologous recombination between olfactory receptor gene clusters, but other 4p16 translocations occur at random

The t(4;8)(p16;p23) is the second most common constitutional chromosomal translocation and is caused by an ectopic meiotic recombination between the olfactory receptor gene clusters (ORGC), located on chromosome 4p and 8p. Given that ORGCs are scattered across the genome and make-up about 0.1% of the human genome we reasoned that translocations between 4p16 and other chromosomes might be mediated by ectopic recombination between different ORGC. In 13 patients, we mapped the breakpoints of either a balanced or unbalanced translocation between chromosome 4p16 and different chromosomes. For all four t(4;8) cases, the breakpoints fall within the 4p and 8pter ORGC, confirming that non-allelic homologous recombination (NAHR) between the ORGC is the main mechanism of the t(4;8) formation. For the nine other translocations, the breakpoints on chromosome 4 mapped to different loci, one of them within the ORGC and in two flanking the ORGC. In these three cases, the translocation breakpoint at the reciprocal chromosome did not contain ORGC sequences. We conclude that only the t(4;8) is mediated by NAHR between ORGC.     

Mechanisms for Nonrecurrent Genomic Rearrangements Associated with CMT1A or HNPP: Rare CNVs as a Cause for Missing Heritability

Genomic rearrangements involving the peripheral myelin protein gene (PMP22) in human chromosome 17p12 are associated with neuropathy: duplications cause Charcot-Marie-Tooth disease type 1A (CMT1A), whereas deletions lead to hereditary neuropathy with liability to pressure palsies (HNPP). Our previous studies showed that >99% of these rearrangements are recurrent and mediated by nonallelic homologous recombination (NAHR). Rare copy number variations (CNVs) generated by nonrecurrent rearrangements also exist in 17p12, but their underlying mechanisms are not well understood. We investigated 21 subjects with rare CNVs associated with CMT1A or HNPP by oligonucleotide-based comparative genomic hybridization microarrays and breakpoint sequence analyses, and we identified 17 unique CNVs, including two genomic deletions, ten genomic duplications, two complex rearrangements, and three small exonic deletions. Each of these CNVs includes either the entire PMP22 gene, or exon(s) only, or ultraconserved potential regulatory sequences upstream of PMP22, further supporting the contention that PMP22 is the critical gene mediating the neuropathy phenotypes associated with 17p12 rearrangements. Breakpoint sequence analysis reveals that, different from the predominant NAHR mechanism in recurrent rearrangement, various molecular mechanisms, including nonhomologous end joining, Alu-Alu-mediated recombination, and replication-based mechanisms (e.g., FoSTeS and/or MMBIR), can generate nonrecurrent 17p12 rearrangements associated with neuropathy. We document a multitude of ways in which gene function can be altered by CNVs. Given the characteristics, including small size, structural complexity, and location outside of coding regions, of selected rare CNVs, their identification remains a challenge for genome analysis. Rare CNVs may potentially represent an important portion of “missing heritability” for human diseases.     

Deletions and a translocation interrupt a cloned gene at the neurofibromatosis type 1 locus.

Three new neurofibromatosis type 1 (NF1) mutations have been detected and characterized. Pulsed-field gel and Southern blot analyses reveal the mutations to be deletions of 190, 40, and 11 kb of DNA. The 11 kb deletion does not contain any of the previously characterized genes that lie between two NF1 translocation breakpoints, but it does include a portion of a rodent/human conserved DNA sequence previously shown to span one of the translocation breakpoints. By screening cDNA libraries with the conserved sequence, we identified a number of cDNA clones from the translocation breakpoint region (TBR), one of which hybridizes to an approximately 11 kb mRNA. The TBR gene crosses at least one of the chromosome 17 translocation breakpoints found in NF1 patients. Furthermore, the newly characterized NF1 deletions remove internal exons of the TBR gene. Although these mutations might act by compromising regulatory elements affecting some other gene, these findings strongly suggest that the TBR gene is the NF1 gene.     

Unequal meiotic crossover: a frequent cause of NF1 microdeletions

Neurofibromatosis type 1 is a common autosomal dominant disorder caused by mutations of the NF1 gene on chromosome 17. In only 5%-10% of cases, a microdeletion including the NF1 gene is found. We analyzed a set of polymorphic dinucleotide-repeat markers flanking the microdeletion on chromosome 17 in a group of seven unrelated families with a de novo NF1 microdeletion. Six of seven microdeletions were of maternal origin. The breakpoints of the microdeletions of maternal origin were localized in flanking paralogous sequences, called "NF1-REPs." The single deletion of paternal origin was shorter, and no crossover occurred on the paternal chromosome 17 during transmission. Five of the six cases of maternal origin were informative, and all five showed a crossover, between the flanking markers, after maternal transmission. The observed crossovers flanking the NF1 region suggest that these NF1 microdeletions result from an unequal crossover in maternal meiosis I, mediated by a misalignment of the flanking NF1-REPs.     

Detection and characterisation of large SERPINC1 deletions in type I inherited antithrombin deficiency

Methods routinely used for investigating the molecular basis of antithrombin (AT) deficiency do not detect large SERPINC1 rearrangements. Between 2000 and 2008, 86 probands suspected of having AT-inherited type I deficiency were screened for SERPINC1 mutations in our laboratory. Mutations causally linked to the deficiency were identified by sequencing analysis in 63 probands. We present here results of multiplex ligation-dependent probe amplification (MLPA) analysis performed in 22 of the 23 remaining probands, in whom sequencing had revealed no mutation. Large deletions, present at the heterozygous state, were detected in 10 patients: whole gene deletions in 5 and partial deletions removing either exon 6 (n = 2), exons 1–2 (n = 1) or exons 5–7 (n = 2) in 5 others. Exon 6 partial deletions are a 2,769-bp deletion and a 1,892-bp deletion associated with a 10-bp insertion, both having 5′ and/or 3′ breakpoints located within Alu repeat elements. In addition, we identified the 5′ breakpoint of a previously reported deletion of exons 1–2 within an extragenic Alu repeat. Distinct mutational mechanisms explaining these Alu sequence-related deletions are proposed. Overall, in this series, large deletions detected by MLPA explain almost half of otherwise unexplained type I AT-inherited deficiency cases.     

Tandem Repeats and G-Rich Sequences Are Enriched at Human CNV Breakpoints

Chromosome breakage in germline and somatic genomes gives rise to copy number variation (CNV) responsible for genomic disorders and tumorigenesis. DNA sequence is known to play an important role in breakage at chromosome fragile sites; however, the sequences susceptible to double-strand breaks (DSBs) underlying CNV formation are largely unknown. Here we analyze 140 germline CNV breakpoints from 116 individuals to identify DNA sequences enriched at breakpoint loci compared to 2800 simulated control regions. We find that, overall, CNV breakpoints are enriched in tandem repeats and sequences predicted to form G-quadruplexes. G-rich repeats are overrepresented at terminal deletion breakpoints, which may be important for the addition of a new telomere. Interstitial deletions and duplication breakpoints are enriched in Alu repeats that in some cases mediate non-allelic homologous recombination (NAHR) between the two sides of the rearrangement. CNV breakpoints are enriched in certain classes of repeats that may play a role in DNA secondary structure, DSB susceptibility and/or DNA replication errors.     

Molecular characterization of germline NF2 gene rearrangements

Neurofibromatosis type 2 (NF2) is an autosomal dominant disease that causes a predisposition to nervous system tumors. Deleterious point mutations have been found in about 55% of NF2 patients, and large genomic deletions account for approximately 33% of NF2 gene alterations. The majority of these deletions are larger than 50 kb, with a breakpoint usually lying outside the NF2 gene. We identified two cases of intragenic deletion with loss of 1.5 and 40 kb, respectively. In both cases, one boundary of the deletion was located in or at the proximity of an SVA sequence in NF2 intron 4. No sequence identity longer than 5 bases and no signal of specific recombination have been evidenced on either side of the deletion breakpoints. These observations are compatible with a nonhomologous recombination being responsible for the genomic deletions. In a third case, a paracentric inversion of chromosome 22 was found. This chromosomal rearrangement breaks the NF2 gene in two parts and carries the first NF2 exon in a juxta-centromeric position. The variability in position of the deletions and the observation of a new chromosomal rearrangement in the NF2 gene underscore the importance of FISH analysis in the molecular diagnosis of NF2.     

Decoding NF1 Intragenic Copy-Number Variations

Genomic rearrangements can cause both Mendelian and complex disorders. Currently, several major mechanisms causing genomic rearrangements, such as non-allelic homologous recombination (NAHR), non-homologous end joining (NHEJ), fork stalling and template switching (FoSTeS), and microhomology-mediated break-induced replication (MMBIR), have been proposed. However, to what extent these mechanisms contribute to gene-specific pathogenic copy-number variations (CNVs) remains understudied. Furthermore, few studies have resolved these pathogenic alterations at the nucleotide-level. Accordingly, our aim was to explore which mechanisms contribute to a large, unique set of locus-specific non-recurrent genomic rearrangements causing the genetic neurocutaneous disorder neurofibromatosis type 1 (NF1). Through breakpoint-spanning PCR as well as array comparative genomic hybridization, we have identified the breakpoints in 85 unrelated individuals carrying an NF1 intragenic CNV. Furthermore, we characterized the likely rearrangement mechanisms of these 85 CNVs, along with those of two additional previously published NF1 intragenic CNVs. Unlike the most typical recurrent rearrangements mediated by flanking low-copy repeats (LCRs), NF1 intragenic rearrangements vary in size, location, and rearrangement mechanisms. We propose the DNA-replication-based mechanisms comprising both FoSTeS and/or MMBIR and serial replication stalling to be the predominant mechanisms leading to NF1 intragenic CNVs. In addition to the loop within a 197-bp palindrome located in intron 40, four Alu elements located in introns 1, 2, 3, and 50 were also identified as intragenic-rearrangement hotspots within NF1.     

Breakpoint determination of 15 large deletions in Peutz–Jeghers subjects

The Peutz–Jeghers Syndrome (PJS) is an autosomal dominant polyposis disorder with increased risk of multiple cancers. STK11/LKB1 (hereafter named STK11) germline mutations account for the large majority of PJS cases whereas large deletions account for about 30% of the cases. We report here the first thorough molecular characterization of 15 large deletions identified in a cohort of 51 clinically well-characterized PJS patients. The deletions were identified by MLPA analysis and characterized by custom CGH-array and quantitative PCR to define their boundaries. The deletions, ranging from 2.9 to 180 kb, removed one or more loci contiguous to the STK11 gene in six patients, while partial STK11 gene deletions were present in the remaining nine cases. By means of DNA sequencing, we were able to precisely characterize the breakpoints in each case. Of the 30 breakpoints, 16 were located in Alu elements, revealing non-allelic homologous recombination (NAHR) as the putative mechanism for the deletions of the STK11 gene, which lays in a region with high Alu density. In the remaining cases, other mechanisms could be hypothesized, such as microhomology-mediated end-joining (MMEJ) or non-homologous end-joining (NHEJ). In conclusion we here demonstrated the non-random occurrence of large deletions associated with PJS. All our patients had a classical PJS phenotype, which shows that haploinsufficiency for SBNO2, C19orf26, ATP5D, MIDN, C19orf23, CIRBP, C19orf24,and EFNA2, does not apparently affect their clinical phenotype.     

Analysis of chromosome 22 deletions in neurofibromatosis type 2-related tumors.

The neurofibromatosis type 2 (NF2) gene has been hypothesized to be a recessive tumor suppressor, with mutations at the same locus on chromosome 22 that lead to NF2 also leading to sporadic tumors of the types seen in NF2. Flanking markers for this gene have previously been defined as D22S1 centromeric and D22S28 telomeric. Identification of subregions of this interval that are consistently rearranged in the NF2-related tumors would aid in better defining the disease locus. To this end, we have compared tumor and constitutional DNAs, isolated from 39 unrelated patients with sporadic and NF2-associated acoustic neuromas, meningiomas, schwannomas, and ependymomas, at eight polymorphic loci on chromosome 22. Two of the tumors studied revealed loss-of-heterozygosity patterns, which is consistent with the presence of chromosome 22 terminal deletions. By using additional polymorphic markers, the terminal deletion breakpoint found in one of the tumors, an acoustic neuroma from an NF2 patient, was mapped within the previously defined NF2 region. The breakpoint occurred between the haplotyped markers D22S41/D22S46 and D22S56. This finding redefines the proximal flanking marker and localizes the NF2 gene between markers D22S41/D22S46 and D22S28. In addition, we identified a sporadic acoustic neuroma that reveals a loss-of-heterozygosity pattern consistent with mitotic recombination or deletion and reduplication, which are mechanisms not previously seen in studies of these tumors. This finding, while inconsistent with models of tumorigenesis that invoke single deletions and their gene-dosage effects, lends further support to the recessive tumor-suppressor model.     

NF1-related locus on chromosome 15

A neurofibromatosis type I (NF1)-related locus has been identified on chromosome 15. It contains a partial copy of the NF1 GAP-related domain, which is known to interact with the ras protooncogenes. However, the chromosome 15 sequence contains multiple deletions resulting in frameshift mutations and stop codons in several highly conserved sequence blocks. The locus on chromosome 15 therefore represents an NF1 pseudogene. This nonprocessed NF1 pseudogene may produce additional fragments in Southern blotting, pulsed-field gel, and PCR experiments with some NF1 cDNA probes or oligonucleotides. In addition, certain regions of the NF1 gene also cross-hybridize with a locus on chromosome 14. These loci must be considered in mutation analysis of patients with NF1 since aberrant findings may not always reflect changes in the NF1 gene.     

A chromosomal rearrangement hotspot can be identified from population genetic variation and is coincident with a hotspot for allelic recombination

Insights into the origins of structural variation and the mutational mechanisms underlying genomic disorders would be greatly improved by a genomewide map of hotspots of nonallelic homologous recombination (NAHR). Moreover, our understanding of sequence variation within the duplicated sequences that are substrates for NAHR lags far behind that of sequence variation within the single-copy portion of the genome. Perhaps the best-characterized NAHR hotspot lies within the 24-kb-long Charcot-Marie-Tooth disease type 1A (CMT1A)-repeats (REPs) that sponsor deletions and duplications that cause peripheral neuropathies. We investigated structural and sequence diversity within the CMT1A-REPs, both within and between species. We discovered a high frequency of retroelement insertions, accelerated sequence evolution after duplication, extensive paralogous gene conversion, and a greater than twofold enrichment of SNPs in humans relative to the genome average. We identified an allelic recombination hotspot underlying the known NAHR hotspot, which suggests that the two processes are intimately related. Finally, we used our data to develop a novel method for inferring the location of an NAHR hotspot from sequence variation within segmental duplications and applied it to identify a putative NAHR hotspot within the LCR22 repeats that sponsor velocardiofacial syndrome deletions. We propose that a large-scale project to map sequence variation within segmental duplications would reveal a wealth of novel chromosomal-rearrangement hotspots.     

Fine structure DNA mapping studies of the chromosomal region harboring the genetic defect in neurofibromatosis type 1

To better map the location of the von Recklinghausen neurofibromatosis (NF1) gene, we have characterized a somatic cell hybrid designated 7AE-11. This microcell-mediated, chromosome-transfer construct harbors a centromeric segment and a neo-marked segment from the distal long arm of human chromosome 17. We have identified 269 cosmid clones with human sequences from a 7AE-11 library and, using a panel of somatic cell hybrids with a total of six chromosome 17q breakpoints, have mapped 240 of these clones on chromosome 17q. The panel included a hybrid (NF13) carrying a der(22) chromosome that was isolated from an NF1 patient with a balanced translocation, t(17;22) (q11.2;q11.2). Fifty-three of the cosmids map into a region spanning the NF13 breakpoint, as defined by the two closest flanking breakpoints (17q11.2 and 17q11.2-q12). RFLP clones from a subset of these cosmids have been mapped by linkage analysis in normal reference families, to localize the NF1 gene more precisely and to enhance the potential for genetic diagnosis of this disorder. The cosmids in the NF1 region will be an important resource for testing DNA blots of large-fragment restriction-enzyme digests from NF1 patient cell lines, to detect rearrangements in patients' DNA and to identify the 17;22 NF1 translocation breakpoint.