7.11: Mutations, Deletions, Duplications & Repair - Biology

7.11: Mutations, Deletions, Duplications & Repair - Biology

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While DNA is used as the universal genetic material of organisms, it is worth remembering that it is a thermodynamically unstable molecule. For example, at a temperature of ~13ºC, half of the phosphodiester bonds in a DNA sample will break after ~520 years214. But, uracil is not normally found in DNA and its presence will be recognized by an enzyme that severs the bond between the uracil moiety and the deoxyribose group215. The absence of a base, due either to spontaneous loss or enzymatic removal, acts as a signal for another enzyme system (the Base Excision Repair complex) that removes a section of the DNA strand with the missing base216. In the human genome there are over 130 genes devoted to repairing damaged DNA217. How frequent are such events? A human body contains ~1014 cells. Each cell contains about ~109 base pairs of DNA. Each cell (whether it is dividing or not) undergoes ~10,000 base loss events per day or ~1018 events per day per person. That's a lot! The basic instability of DNA (and the lack of repair after an organism dies) means that DNA from dinosaurs (the last of which went extinct ~65,000,000 years ago) has disappeared from the earth, makingit impossible to clone (or resurrect) a true dinosaur218. Many of the most potent known mutagens are natural products, often produced by organisms to defend themselves against being eaten or infected by parasites, predators, or pathogens219.

Mutation spectrum of Drosophila CNVs revealed by breakpoint sequencing

The detailed study of breakpoints associated with copy number variants (CNVs) can elucidate the mutational mechanisms that generate them and the comparison of breakpoints across species can highlight differences in genomic architecture that may lead to lineage-specific differences in patterns of CNVs. Here, we provide a detailed analysis of Drosophila CNV breakpoints and contrast it with similar analyses recently carried out for the human genome.


By applying split-read methods to a total of 10x coverage of 454 shotgun sequence across nine lines of D. melanogaster and by re-examining a previously published dataset of CNVs detected using tiling arrays, we identified the precise breakpoints of more than 600 insertions, deletions, and duplications. Contrasting these CNVs with those found in humans showed that in both taxa CNV breakpoints fall into three classes: blunt breakpoints simple breakpoints associated with microhomology and breakpoints with additional nucleotides inserted/deleted and no microhomology. In both taxa CNV breakpoints are enriched with non-B DNA sequence structures, which may impair DNA replication and/or repair. However, in contrast to human genomes, non-allelic homologous-recombination (NAHR) plays a negligible role in CNV formation in Drosophila. In flies, non-homologous repair mechanisms are responsible for simple, recurrent, and complex CNVs, including insertions of de novo sequence as large as 60 bp.


Humans and Drosophila differ considerably in the importance of homology-based mechanisms for the formation of CNVs, likely as a consequence of the differences in the abundance and distribution of both segmental duplications and transposable elements between the two genomes.

Change history

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Bladder cancer is a molecular disease resulted from the multistep accumulation of genetic, epigenetic, and environmental factors 27 . The human tumor suppressor gene p53, which maps to chromosome 17p13.1, consists of 11 exons spanning over 20 kb of DNA and encodes a 393 amino acid protein. TP53 gene is the most common target in human tumors 15 . The ability of TP53 gene to manage apoptosis versus DNA repair is not only important for prevention of malignancy but also could be targeted for therapeutic strategies. Mutations in the TP53 tumor suppressor gene are found at high frequency in a wide range of human cancers 7 . TP53 gene codes for a protein that acts as a transcription factor and serves as a key regulator of the cell cycle. Inactivation of p53 by mutations disrupts the cell cycle and may lead to tumor formation 26 . Tumors with intact TP53 respond more efficiently to chemo and/or radiotherapy 1, 2 . Bladder cancer is one of the most common cancers worldwide. It is the fourth most prevalent cancer in men and the 11th most prevalent cancer in women in the United States 8 . More than 90% of bladder cancers are carcinomas. Some genomic alterations are strongly associated with specific histopathologic tumor features like grade or stage 6 . Very few studies have the molecular genetic abnormalities of bladder carcinoma 9, 16 . According to 6 mutations or deletions in tumor suppressor genes TP53 (70%), RB (37%), and PTEN (35%) are commonly detected in urothelial carcinoma in situ which are frequently accompanied by chromosomal deletion chromosome 6 . Similar studies on molecular genetics pathways of transitional cell carcinoma (TCC) have focused on TP53 gene mutations as a whole without revealing the role of each exon in carcinogenesis of this kind of neoplasm 4, 16, 17, 21 . Mutations of p53 are detected in many cancer types among which urothelial cancer is one of the most prevalent. It is not entirely what is the place and additive value of the current technique as compared to state-of-the-art next-generation sequencing. More background information on the type of p53 aberrations (deletion, mutation, duplication) occurring in Urothelial cell carcinoma is required to facilitate the interpretation of the data presented.

Multiplex ligation-dependent probe amplification (MLPA) has recently been developed as a novel method to detect exonic duplication/deletion mutations in multiple human disease genes 12 . This method utilizes an oligonucleotide ligation assay with embedded universal primer sequences to permit relatively uniform amplification of multiple (up to 40) regions of the genome revealing the accurate copy number of the regions of interest 12 . MLPA technique has been applied in this study for comprehensive analysis of all exons of TP53 to screen deletion/duplication mutations. In addition, role of each exon in the pathogenesis of TCC and the relation between TCC tumor grade and any type of mutation are reported for the first time in this study.


By investigating a large number of diploid isolates subcultured for many generations, we have obtained accurate quantitative and qualitative measurements of the rates of multiple types of mitotic genetic alterations including mutations, deletions and duplications, and LOH events. A summary of the numbers of these alterations is shown in Fig. 6. Our findings are as follows. 1) Rates and spectra of mutations are in general agreement with previous studies. 2) Large (>1 kb) deletions and duplications are primarily the result of homologous recombination between nonallelic, but closely linked, retrotransposons the tandemly repeated rRNA and CUP1 genes have high rates of instability. 3) Translocations and aneuploidy are rare relative to other types of gross chromosome rearrangements. 4) Gene-conversion events and cross-overs have different genomic distributions, suggesting that these events may be initiated and/or resolved by different mechanisms. 5) Finally, mitotic LOH events, both interstitial and terminal, are common, occurring at a rate of about 4.7 × 10 −3 events per genome per cell division. We present a simple method of calculating the probability per cell division of an LOH event for an individual SNP. In S. cerevisiae, these probabilities are largely a function of the distance of the SNP from the centromere.

Numbers of different classes of genomic alterations summed over 93 subcultured WYspo11 isolates. A total of 2,628 events were observed.

Frequency and Spectra of Mutations.

The rate of single-bp mutations in our study was about 2 × 10 −10 per bp per cell division, similar to the rates of 1 to 3 × 10 −10 per bp per cell division observed by others. As in previous studies, the rates of small (<10 bp) in/dels are less than 10% of the rates of single-bp mutations. Most of the in/dels occur within mononucleotide tracts or between short direct repeats, and likely reflect DNA polymerase slippage. These data, based on diploids without genome-destabilizing mutations grown under unstressed conditions, serve as a baseline for examining strains under replication stress or strains with a mutator phenotype.

Large Deletions and Duplications.

In agreement with many previous studies done in S. cerevisiae, most large deletions and duplications are a consequence of homologous recombination between repeated genes. These events usually involve closely linked repeats. Only one translocation was observed, consistent with previous results indicating that recombination occurs more frequently between repeats on the same homolog than between repeats on nonhomologs (56). Translocations were also infrequent in a diverse collection of yeast strains (47) 79 of 100 samples had colinear chromosomes.

As expected from previous mitotic studies of the rRNA and CUP1 gene tandem arrays (57 ⇓ ⇓ –60), we observed frequent size changes of the arrays. These size changes likely reflect a number of mechanisms, including unequal sister-strand recombination, gene conversion, single-strand annealing, and BIR. The relative importance of these mechanisms has not been completely resolved. We found that loss of repeats from one cluster is often associated with gain of repeats from the other cluster, suggesting that there is selection for an optimal number of repeats under the growth conditions of our experiment.

Differences in the Distribution of Interstitial and Terminal LOH Events.

The distributions of interstitial and cross-over/BIR breakpoints (Figs. 4 and 5) differ in a variety of ways. In general, the distribution of events appears more uniform for conversion events than the terminal LOH events. As discussed previously, the T-LOH events, unlike the I-LOH events, are enriched near the telomeres. Such patterns are unexpected if both types of events are initiated by the same type of DNA lesion and processed to generate conversions and cross-overs with a fixed probability.

In meiosis in S. cerevisiae, Allers and Lichten (32) showed that intermediates that lead to non–cross-over conversions appear earlier than those that result in cross-overs, and that the two types of exchange are under different genetic regulation. They also showed physical evidence that conversions unassociated with cross-overs are primarily a consequence of the synthesis-dependent strand annealing pathway (SDSA Fig. 7A), whereas cross-overs with associated conversions were produced by formation and resolution of double Holliday junctions (Fig. 7B). In addition, Mancera et al. (61) found that meiotic hotspots for cross-overs and conversions were not always coincident. Our results suggest the same separation may exist in mitosis. Based on the relatively few relevant studies, it is difficult to generalize about the relative frequencies of I- and T-LOH events in human cancers. In retinoblastomas, T-LOH events appear more common (34), whereas, in other types of solid tumors (gastric, glioma, and lung), I-LOH events are the predominant class (62).

Simplified form of the double-strand break-repair model. In this figure, recombination is initiated on the blue chromatid, and the broken ends are resected 5′ to 3′. The broken end invades the homologous chromatid, forming a D-loop. Two possible outcomes of the strand invasion are shown in A and B. It should be emphasized that recent studies of patterns of heteroduplex formation during meiotic and mitotic recombination indicate that additional steps (branch migration, “patchy” mismatch repair, and strand switching) are required to explain some recombination events. (A) Synthesis-dependent strand annealing (SDSA). Following strand invasion, the end of the invading strand is used as a primer for DNA synthesis, resulting in a longer D-loop. The invading strand is then extruded, pairing with the other broken end. The resulting heteroduplex may contain mismatches that can be repaired to produce a conversion event (enclosed in a rectangle) unassociated with a cross-over. (B) Formation of a double Holliday junction. Following strand invasion and DNA synthesis primed from the invading strand, the D-loop pairs with the second broken end. The resulting junctions can be cleaved in a variety of ways as indicated by the numbered arrows. Cleavage at positions 5, 6, 7, and 8 results in a region of conversion without an associated cross-over. Cleavage at positions 2, 4, 5, and 6 results in a conversion tract associated with a cross-over.

The difference in the location of breakpoints for I- and T-LOH events can be explained by a number of mechanisms. It is possible that the DNA lesions responsible for spontaneous I- and T-LOH events are different. For example, spontaneous conversion events could be primarily a consequence of repair of nicked DNA (28) and T-LOH events could reflect repair of DSBs. In addition, recombinogenic DSBs could be generated in two ways: by an interstitial break on the chromosome or by terminal degradation of the end as a consequence of telomere disfunction. One interpretation of the observation that T-LOH events are nonrandomly near the telomeres is that a subset of terminal LOH events result from repair of a terminally degraded chromosome.

Alternatively, the different distribution of I- and T-LOH events could result from different modes of processing the same initiating lesion. In the current models of recombination (28), both the SDSA and DSBR pathways are initiated by a DSB (Fig. 7). In both pathways, the initial step involves 5′-3′ excision of the broken end, followed by invasion of the broken end into the unbroken homolog. In the SDSA model, after limited DNA synthesis, the invading end dissociates and anneals with the other broken end (Fig. 7A). In the DSBR pathway, after more extensive DNA synthesis, the noninvading broken end pairs with the resulting D-loop, forming a double Holliday junction (Fig. 7B). Consistent with these models, the lengths of both meiotic and mitotic conversion tracts are longer for cross-over–associated events than for non–cross-over conversions (53, 61). Based on these observations, one explanation for the different distribution of I-LOH and T-LOH events is that DNA synthesis primed by the invading strand is more processive in some chromosome regions than others, and this processive synthesis is more likely to result in a T-LOH event than an I-LOH event.

Another interesting conclusion from our data is that the numbers of I- and T-LOH events are different by only a factor of 2.4. Assuming that the cross-overs are associated with conversions (26), we conclude that about 30% of the conversions are associated with cross-overs. Although about two thirds of meiotic conversion events are associated with cross-overs (61), in previous mitotic studies, the percentage of conversion events associated with cross-overs varied from 10% to 50% [reviewed by Yim et al., 2014 (29)]. Many of these previous studies, however, were done by selecting recombination between heteroalleles at ectopic chromosome locations, which may bias the results. In studies of allelic gene conversion, Yim et al. (29) found that about 40% of mitotic gene conversions were associated with cross-overs. Our current results are consistent with this estimate.

Calculation of Expected SNP-Specific Rates of LOH.

The expected rates of LOH (RLOH) for individual SNPs are the sum of two rates: the rate expected from I-LOH events (RI-LOH) and the rate expected from T-LOH events (RT-LOH). Since mitotic gene conversions are distributed reasonably uniformly in the genome in our analysis, we will assume that RI-LOH is the same for all SNPs. There are two related methods for calculating RI-LOH: 1) multiply the genomic rate of interstitial LOH times the ratio of the average conversion tract size divided by the total genome size or 2), using Dataset S6, calculate the average number of SNPs that undergo LOH in each isolate and divide by the number of cell divisions per isolate. The details of these calculations are in the SI Appendix. Both methods lead to an RI-LOH value of about 10 −6 per SNP per division.

The second rate (RT-LOH) is a function of the distance of the SNP from the centromere of the chromosome, since a cross-over occurring anywhere in this interval will result in LOH for centromere-distal SNPs. Therefore, we can estimate the rate of LOH caused by cross-overs/BIR events for individual SNPs by multiplying the rate of terminal LOH events in the genome (1.4 × 10 −3 per cell division) by the ratio of the distance of the SNP from the centromere (in kb) divided by the total genome length (12.5 Mb). Thus, RT-LOH will vary from 0 (for SNPs very close to the centromere) to about 2 × 10 −4 per SNP per cell division for a marker located at the end of the right arm of chromosome XII, the longest chromosome arm in the genome.

The RT-LOH values can be used to calculate the expected number of isolates undergoing LOH by multiplying these values by the aggregate number of cell divisions (N = 264,000). The details of these calculations are in the SI Appendix, and a summary of the expected numbers of T-LOH events for SNPs located near the ends of the chromosome arms is in Dataset S7. In general, there is good agreement between the expected and observed numbers of T-LOH events. For example, for an SNP located near the end of chromosome XII, the predicted number of events is about 52 the observed number is 47. One exception to this generalization is the right arm of chromosome IV, where we expected 32 events and observed only 11. As discussed in the SI Appendix, the likely explanation of this discrepancy is that cross-overs on the right arm of IV result in loss of the ade2-1–suppressing SUP4 insertion. Cells homozygous for the unsuppressed ade2-1 allele accumulate a red pigment that results in slow growth and, therefore, a reduced level of such cells in an MA experiment.

In summary, the expected rate of LOH per cell division for SNPs can be estimated by the following equation: rate of LOH per SNP per cell division = 10 −6 + [(CEN–SNP distance in kb/12.5 Mb) × 1.4 × 10 −3 ]. For SNPs located more than 100 kb from the centromere, >90% of the LOH rate will be a consequence of T-LOH events rather than gene conversion. This conclusion assumes that most terminal LOH events are a consequence of repair of random DSBs. The observed numbers of LOH events for each SNP are shown in Fig. 8. A similar pattern of increasing LOH as SNPs get further from the centromere has also been observed in other studies with smaller datasets (14, 22, 31).

Number of times SNPs underwent LOH at different sites along the yeast chromosomes. The gray and green colors indicate T-LOH and I-LOH, respectively. Red triangles show the positions of centromeres. Numbers on the y axis are the numbers of LOH events at each SNP, and numbers on the x axis are SGD coordinates (in kb). Black dots at the ends of the chromosome indicate the expected level of LOH at the ends of the chromosomes resulting from cross-overs (as calculated in Dataset S7).

Based on Dataset S6, we calculated that the average LOH rate per SNP per division was 2.6 × 10 −5 . As expected, the rates for different SNPs have a very wide range. SNPs near the end of chromosome XII have a rate of LOH of about 1.6 × 10 −4 , and those near the centromere of XII have a rate of 3.8 × 10 −6 . In several other species in the Saccharomycodaceae family, Nguyen et al. (63) calculated average LOH rates per SNP/division varying between 2 and 11 × 10 −6 . In our view, since the rate of LOH per SNP in S. cerevisiae is largely a function of the distance between the SNPs and the centromere, the average rate has limited utility.

Several other points should be mentioned. First, our conclusions are based on S. cerevisiae. Other organisms could have higher or lower rates of recombination or a different ratio of gene conversions to cross-overs. For example, in Daphnia pulex, the rate of LOH per SNP per generation is about 8 × 10 −8 , and most of the LOH events are deletions rather than conversions or cross-overs (64). It is likely that, in organisms (such as S. cerevisiae) in which LOH events per SNP increase as a function of distance from the centromere, cross-overs and BIR events are the drivers of LOH. Second, the LOH events in our experiments were distributed relatively evenly over the isolates (Datasets S4–S6). Thus, our conclusions are not derived from a small subset of cells with unusually high levels of recombination.

In summary, our results provide a global view of spontaneous genomic alterations in unstressed diploid yeast cells. Our analysis and those of others show that mitotic recombination events are frequent enough to be an important source of diversity. In organisms such as S. cerevisiae, these events will produce variants that may have selective advantages in certain environments (65 ⇓ –67). In humans, such events can release cells from normal growth regulation, initiating tumor formation.


A two-step CRISPR/Cas9 strategy generates the first intragenic tandem multi-exonic duplication mouse model

To generate an intragenic duplication mouse model having a duplication of the exons from 18 to 30 in the Dmd gene, we designed four sgRNAs referred to as i17A, i17B, i30A, and i30B, targeting introns 17 and 30 flanking the region to duplicate (Fig 1A). The guides were introduced into mouse embryonic stem cells (mESC) by means of electroporation, followed by clone screening via PCR to identify the duplication junction (Appendix Fig S1). Of the 243 screened clones, three positive clones (1.23%) were expanded, aggregated, and injected into blastocysts which were implanted in pseudo-pregnant mice. Of the three mice positive for the duplication junction, one showed no evidence of germline transmission of the duplication, and another demonstrated a complex rearrangement in lieu of a duplication. Accordingly, these two mice were excluded from further analysis.

Figure 1. A CRISPR/Cas9-based strategy generates a mouse model exhibiting a 137 kb multi-exonic tandem head-to-tail duplication in the Dmd gene

  1. A 137 kb region encompassing exons 18–30 was targeted with 4 sgRNAs in introns 17 and 30 to generate a genomic duplication. Cas9 and the sgRNAs are represented as scissors.
  2. Schematics of the 12,049 bp inversion present in the Dup18-30i mice and of the strategy utilized to correct the inversion. Two sgRNAs targeting the inversion were utilized to rescue the complex rearrangement. Cas9 and the sgRNAs are represented as scissors.
  3. PCR amplification and Sanger sequencing of the duplication junction in the Dup18-30 founder mouse confirming the joining of intron 30 and 17, along with a 96 bp intronic deletion.
  4. The heart, tibialis anterior (TA), and triceps muscle were isolated and analyzed to identify the presence of the duplication from exons 18 to 30 via RT–PCR using primers represented by the arrows. Sanger sequencing confirmed correct splicing of exons 30 and 18 at the duplication junction.
  5. Protein lysates isolated from the TA, triceps, diaphragm, and heart muscles of WT and Dup18-30 mice were probed for dystrophin expression by Western blot. Calnexin serves as a loading control.

As CRISPR/Cas9 editing may generate inadvertent structural variants, the founder mouse was analyzed via whole genome sequencing (WGS), confirming the presence of the predicted 136.8 kb duplication (ChrX: 83,737,872–83,874,709). However, the second copy of the duplication was immediately followed by an unwanted 12,049 bp inversion of the region spanning introns 30 to 34 (ChrX: 83,876,785–83,888,834) (Appendix Table S1). This mouse model, referred as Dup18-30i, presented Dmd splicing abnormalities (Appendix Fig S2A and B), lack of dystrophin expression, and compromised muscle physiology (Appendix Fig S3A and B).

To correct the inversion, we designed two gRNAs flanking the inverted DNA region (Fig 1B), which were electroporated together with the Cas9 protein into Dup18-30i zygotes. We screened newborn mice and detected the predicted re-inversion junctions in 6.25% of them. One founder, the Dup18-30 mouse model, exhibited germline transmission of the re-inverted allele together with the duplication junction. WGS confirmed that the inversion was corrected without any alteration of the tandem duplication (Appendix Table S2).

Molecular analysis via PCR and Sanger sequencing of the duplication junction in the Dup18-30 mouse model revealed joining of intron 30 and 17, along with a 96-bp intronic deletion (Fig 1C). Additionally, RT–PCR analysis of the Dup18-30 Dmd transcript showed the presence of the predicted 2065 bp duplication of the exons from 18 to 30 at the RNA level (Fig 1D, Appendix Fig S2A and C). The correct joining of exons 30 and 18 was confirmed by Sanger sequencing the Dup18-30 cDNA (Fig 1D). Western blotting analysis revealed absent dystrophin expression in skeletal and cardiac muscles of Dup18-30 mice (Fig 1E). These findings confirmed Dup18-30 to be the first Dmd multi-exonic tandem duplication mouse model faithfully recapitulating a patient mutation.

The Dup18-30 mouse model recapitulates DMD disease manifestations

Immunohistochemical analysis of cardiac and skeletal muscles in 15-week-old Dup18-30 mice showed complete absence of dystrophin expression except for a few revertant fibers (RFs) (Figs 2A and EV1A). Dup18-30 muscles showed sporadic clusters of RFs, dystrophin-positive fibers that arise from spontaneous exon skipping events, commonly reported in DMD patients and animal models (Pigozzo et al, 2013 ). The tibialis anterior (TA) and triceps showed spontaneous dystrophin expression in 4.7% and 2.3% of the fibers, respectively (Fig 2C).

Figure 2. The Dup18-30 mouse model recapitulates DMD disease manifestations

  • A. 8-µm cross section of 15-week-old WT and Dup18-30 TA and triceps were analyzed for dystrophin localization by immunofluorescence. Asterisk indicates clusters of revertant fibers. Scale bars, 100 μm.
  • B. The muscle architecture of the same muscles was investigated by H&E staining. A representative image is shown. Scale bars, 100 μm. Asterisk indicates areas with necrotic fibers and fibrosis. Arrows indicate fibers with central nuclei.
  • C. Percentage of dystrophin-positive fibers in the TA and triceps of WT and Dup18-30 mice. WT, n = 4 Dup18-30, n = 6–7.
  • D. Percentage of myofibers with centrally located nuclei in the TA and triceps of WT and Dup18-30 mice. WT, n = 4 Dup18-30, n = 6.
  • E. Forelimb grip strength was measured in 15-week-old WT and Dup18-30 mice. WT, n = 9 Dup18-30, n = 12.
  • F. Specific tetanic force was measured in 15-week-old mice using an in vivo muscle-function analyzer. WT, n = 11 Dup18-30, n = 15 mice.
  • G, H. Mice were tested in an open-field chamber in which total distance traveled (G) and vertical activity (H) were assessed. WT, n = 9 Dup18-30, n = 12.

Data information: Data are represented as means ± SD. Statistical analyses were performed with Student’s t-test. ***P < 0.001, ****P < 0.0001.

Figure EV1. The Dup18-30 mouse model shows generalized dystrophic muscle pathology and locomotor dysfunctions

  • A. Heart, gastrocnemius, and diaphragm cross sections were analyzed for dystrophin localization via immunostaining in 15-week-old WT and Dup18-30 mice. A representative sample is shown. Scale bar, 100 μm.
  • B. The muscle morphology of the gastrocnemious and diaphragm was further investigated via H&E. A representative image is shown. Scale bars, 50 μm.
  • C, D. The mice were tested in an open-field chamber in which total resting time (C) and average speed (D) were assessed. WT, n = 9 Dup18-30, n = 12. Data are represented as means ± SD. Statistical analyses were performed with Student’s t-test. **P < 0.01, ***P < 0.001.

The lack of dystrophin expression resulted in dystrophic muscle architecture, fibrosis, central nuclei, and heterogeneous fiber size which are typical signs of muscular dystrophy (Figs 2B and EV1B). TA and triceps samples of Dup18-30 mice demonstrated 65.4 and 75.9% central nuclei (compared to 0% in WT), respectively (Fig 2D). The muscle strength of the Dup18-30 mice was evaluated with forelimbs grip strength and specific tetanic force measurements in the TA muscle. The grip strength and the tetanic force were decreased by 22.2% (P < 0.001) and 40.5% (P < 0.001) in Dup18-30 mice compared to WT, respectively (Fig 2E–F). The locomotor function of the Dup18-30 was assessed via open-field test. The Dup18-30 mice exhibited decreased performance compared to their WT counterparts with respect to total distance traveled (P < 0.001), vertical activity (P < 0.001), average speed (P < 0.001), and total resting time (P < 0.01) (Figs 2G and H, and EV1C and D).

A single-sgRNA/Cas9 treatment removes the Dmd duplication and restores full-length dystrophin expression in Dup18-30 mice

We employed the single-sgRNA approach, previously used successfully in vitro (Wojtal et al, 2016 ) to remove the duplication in the Dup18-30 mice in vivo. The approach consists of a single-sgRNA guide that together with a Cas9 targets both copies of the Dmd duplication. Upon Cas9 cleavage and re-ligation of the DNA ends, the region encompassed between the two single-sgRNA target sites is removed, restoring the Dmd ORF and dystrophin expression (Fig 3A). To select the single-sgRNA to treat the Dup18-30 mice, we scanned all introns within the duplicated region. We then selected the best sgRNAs based on their off-target scores, while verifying that they would not interfere with any predicted splice sites. The top eight-ranking guides were tested in vitro in N2A cells, and the most active guide, a sgRNA targeting intron 21 of the Dmd gene (i21), was chosen for the in vivo treatment (Appendix Table S3). A Staphylococcus aureus Cas9 (SaCas9) driven by the constitutive CMV promoter and the i21 sgRNA encoding cassettes were packaged into adeno-associated virus 9 (AAV9), which has a known tropism for skeletal and cardiac muscle (Lau & Suh, 2017 ). The AAV9-Cas9-i21sgRNA viral particles were delivered to neonatal mice in the first 2 days of life via temporal vein injection at a dosage of 3 × 10 12 VG (viral genomes) per mouse (Fig 3B). Outcomes were analyzed 7 weeks post-injection.

Figure 3. A single-sgRNA/Cas9 treatment removes the Dmd duplication and restores full-length dystrophin expression in the Dup18-30 mouse model

  • A. Schematic of the single-sgRNA CRISPR/Cas9 strategy to remove the duplication of the exons from 18 to 30 in the Dup18-30 mouse model. A single-sgRNA targeting intron 21 (i21) was utilized to cut both copies of the duplicated region. Cas9 and the sgRNAs are represented as scissors.
  • B. Two-day-old neonatal Dup18-30 pups were injected with AAV9 carrying Cas9 and i21 gRNA (n = 11 3 × 10 12 viral genomes) via temporal vein and sacrificed 7 weeks later.
  • C. The efficiency of the removal of the duplicated region at the DNA level was assessed via qPCR analysis by normalizing the signal obtained from the duplication junction to that of the total Dmd signal. Dup18-30 untreated, n = 3–4 Dup18-30 treated, n = 5–9.
  • D. The RNA editing efficiency was quantified in the same tissues analyzed in panel B via qPCR utilizing the expression ratio between the duplication junction and the WT Dmd transcript. Dup18-30 untreated, n = 4 Dup18-30 treated, n = 4–5.
  • E. RT–PCR analyzing the removal of the duplication from the heart, TA and triceps muscle of WT, Dup18-30-untreated, and Dup18-30-treated mice. Arrows correspond to primers in exon 17 and exon 33.
  • F, G. Western blotting detected restoration of dystrophin expression in (F) heart and (G) TA in the Dup18-30 mice. 25% and 50% of the WT proteins compared to Dup18-30 mice have been loaded on the gel. Calnexin was used as a loading control.
  • H. Immunostaining showed restoration of dystrophin expression in the heart and TA. A representative sample is shown. Scale bars, 100 μm.
  • I. Quantification of dystrophin Western blot in panels F and G. Dup18-30 untreated, n = 2 Dup18-30 treated, n = 4.
  • J. Percentage of dystrophin-positive fibers from the immunostaining in the heart and TA of Dup18-30-untreated and Dup18-30-treated mice. Dup18-30 untreated, n = 5 Dup18-30 treated, n = 10–11.

Data information: All data are represented as the mean ± SD. Statistical analyses were performed with Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

We analyzed the efficacy of duplication removal at the DNA level by qPCR, comparing the region encompassing the duplication junction, and a normal intron-exon junction (outside the duplicated region). The treatment group showed an average 26.92% reduction in the presence of the duplication junction in the heart, translating into editing of 9.5–58.4% (P < 0.01) in individual mice (Fig 3C). A similar trend was noted for TA (12.22% P = 0.15) and triceps (20.2% P = 0.085), with editing efficiencies of up to 30% and 42% in individual mice, respectively (Fig 3C). Additionally, targeted deep amplicon sequencing of the sgRNA target site in the heart and TA muscles revealed on average 5.7% and 3.1% indels formation, respectively (Appendix Fig S4A–D). Deep sequencing of the top 11 potential off-target sites did not show any non-specific activity of the i21 sgRNA in Dup18-30 Cas9 + i21 treated compared to untreated mice (Appendix Table S4).

The efficiency of editing at the RNA level was further assessed via qRT–PCR. A 60.9% reduction in the presence of the duplication was noted in the hearts of treated compared to untreated mice (P < 0.001) (Fig 3D). A similar trend was noted for TA (19.39% P = 0.28) and triceps (27.3% P = 0.24) (Fig 3D). Editing efficiency in individual mice was found to be up to 46% in TA and up to 62% in triceps at the RNA level. Furthermore, RT–PCR analysis of the Dmd transcript showed removal of the duplicated region and restoration of the wild-type Dmd transcript in heart and skeletal muscles (Fig 3E).

Seven weeks following treatment, dystrophin level in hearts of treated mice, as measured by Western blotting, was found to be on average 16.42% of WT [range: 5–25% 0% in untreated mice (P < 0.05)] (Fig 3F and I). Dystrophin expression improved in other tissues as well, ranging from 4 to 18% of WT dystrophin protein in the diaphragm, TA, and triceps of treated mice (P < 0.05 compared to untreated mice) (Figs 3G and I, and EV2A–C). The percentage of dystrophin-positive fibers was further assessed by immunofluorescence, ranging from 22 to 36% for heart, and 12–68% for skeletal muscle (P < 0.0001 compared to untreated mice) (Figs 3H and J, and EV2D and E).

Figure EV2. The single-sgRNA treatment restores dystrophin expression in the triceps and diaphragm of Dup18-30 mice

  • A, B. Western blotting detected restoration of dystrophin expression in (A) triceps and (B) diaphragm in the Dup18-30 mice. 25% and 50% of the WT proteins compared to Dup18-30 mice have been loaded on the gel. Calnexin was used as a loading control.
  • C. Quantification of dystrophin Western blot (A and B). Dup18-30 untreated, n = 2 Dup18-30 treated, n = 4.
  • D. Immunostaining showed restoration of dystrophin expression in the triceps and diaphragm. A representative image is shown. Scale bars, 100 μm.
  • E. Percentage of dystrophin-positive fibers from the immunostaining in the triceps and diaphragm of Dup18-30-untreated and Dup18-30-treated mice. Dup18-30 untreated, n = 4–5 Dup18-30 treated, n = 6–10.

Data information: All data are represented as the mean ± SD. Statistical analyses were performed with Student’s t-test. *P < 0.05, ***P < 0.001.

CRISPR/Cas9-mediated duplication correction improves dystrophic phenotypes in the Dup18-30 mice

To further investigate treatment efficacy, we performed immunostaining for components of the dystrophin-associated glycoprotein complex (DGC). In DMD, lack of dystrophin destabilizes the DGC, resulting in loss of normal muscle architecture (Campbell & Kahl, 1989 Ervasti et al, 1990 ). As expected, untreated Dup18-30 mice revealed absent localization of DGC components at the sarcolemma. Treated mice showed partially restored expression of the DGC components, including alpha-syntrophin, beta-sarcoglycan, and neuronal nitric oxide synthase (nNOS) across all muscle samples analyzed (Figs 4A and EV3A and B). H&E staining performed on TA, triceps, and diaphragm of Dup18-30-treated mice showed overall improved muscle pathology with a reduction in the typical hallmarks of dystrophic muscles, including infiltration, fibrosis, and central nuclei (Fig 4B). In regard to central nuclei, a 68.5% reduction compared to the untreated group was noted in the TA and diaphragm (P < 0.0001), with a 60.3% reduction noted in triceps (P < 0.001) (Fig 4C). Further analysis of the treatment showed a beneficial bystander effect exerted by dystrophin restoration on dystrophin-negative unedited fibers. In treated mice, only 14.7% of dystrophin-negative fibers presented central nuclei, marking a 75.4% reduction in central nuclei in dystrophin-negative fibers compared with untreated mice (Fig EV4A and B).

Figure 4. The single-sgRNA/Cas9 treatment restores DGC expression and improves muscle pathology in Dup18-30 mice

  1. Immunofluorescence staining for alpha-syntrophin and beta-sarcoglycan in the TA and triceps muscles of the Dup18-30 mice. A representative image is shown. Scale bars, 100 μm.
  2. TA, triceps, and diaphragm muscle architecture were analyzed by H&E staining. Scale bars, 50 μm.
  3. Central nuclei were quantified in the TA, triceps, and diaphragm muscles. Dup18-30 untreated, n = 4–5 Dup18-30 treated, n = 6–9. All data are represented as means ± SD. Statistical analyses were performed with Student’s t-test. ***P < 0.001, ****P < 0.0001.

Figure EV3. The Cas9 treatment restores expression of DGC components

  1. Immunofluorescence staining for alpha-syntrophin and beta-sarcoglycan in the diaphragm and heart of WT, Dup18-30-untreated, and Dup18-30-treated mice. A representative image is shown. Scale bars, 100 μm.
  2. Immunofluorescence staining for nNOS in TA, triceps, and diaphragm of WT, Dup18-30-untreated, and Dup18-30-treated mice. A representative image is shown. Scale bars, 100 μm.

Figure EV4. The Cas9 treatment reduces central nuclei in dystrophin-negative muscle fibers

  1. Immunofluorescence staining for laminin alpha-2 and dystrophin in the TA of Dup18-30-untreated, and Dup18-30-treated mice. A representative image is shown. Scale bars, 100 μm.
  2. Quantification of central nuclei in dystrophin-negative fibers in Dup18-30-untreated and Dup18-30-treated mice. Dup18-30 untreated, n = 5 Dup18-30 Cas9 treated, n = 6. All data are represented as the mean ± SD. Statistical analyses were performed with Student’s t-test. ****P < 0.0001.

We subsequently assessed the diaphragm, which is severely affected in DMD patients representing a substantial cause of morbidity and mortality. Masson’s Trichrome staining was performed to analyze diaphragmatic fibrosis, showing a 61.2% reduction of fibrosis in Dup18-30-treated mice compared with untreated controls (Fig 5A and B). In addition, we evaluated forelimb grip strength and contractile force measurements in the TA. In the treated group, grip strength was increased by 75.3% (P < 0.01) (Fig 5C), while specific tetanic force was 48.4% higher compared to untreated mice (P < 0.01) (Fig 5D). Notably, specific tetanic force was not significantly different between the WT and treated group (P = 0.98). We evaluated our treated mice functionally via open-field testing, and they demonstrated WT-like performance across all parameters including total distance traveled (P = 0.11), vertical activity (P = 0.072), total resting time (P = 0.64), and average speed (P = 0.30). This represents a significant improvement in all open-field parameters compared to untreated mice [total distance traveled (P < 0.0001), vertical activity (P < 0.0001), total resting time (P < 0.001), and average speed (P < 0.05)] (Figs 5E and F, and EV5A and B).

Figure 5. The single-sgRNA/Cas9 treatment improves DMD phenotypes in Dup18-30 mice

  • A. Fibrosis was analyzed in the diaphragm via Masson Trichrome staining. Scale bars, 100 μm.
  • B. Quantification of diaphragm fibrosis in WT, Dup18-30-untreated, and Dup18-30-treated mice. WT, n = 5 Dup18-30 untreated, n = 5 Dup18-30 treated, n = 8.
  • C–F. WT, Dup18-30-untreated, and Dup18-30-treated mice were tested 7 weeks after treatment by measuring (C) forelimbs grip strength, (D) specific tetanic force, and (E) total distance traveled and (F) vertical activity in an open-field chamber. WT, n = 8–9 Dup18-30 untreated, n = 6–7 Dup18-30 treated, n = 6–7.

Data information: All data are represented as means ± SD. Statistical analyses were performed with Student’s t-test. n.s. not significant, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure EV5. The Cas9 treatment improves DMD disease phenotypes

  • A, B. WT, Dup18-30-untreated, and Dup18-30-treated mice were tested 7 weeks post-treatment in an open-field chamber measuring (A) total resting time in the arena and (B) average speed. WT, n = 9 Dup18-30 untreated, n = 7 Dup18-30 Cas9 treated, n = 7. All data are represented as the mean ± SD. Statistical analyses were performed with Student’s t-test. n.s. not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.


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Identification of optimal guide RNAs to target 12 different exons associated with hotspot regions of DMD mutations

A list of the top 12 exons that, when skipped, can potentially restore the dystrophin open reading frame in most of the hotspot regions of DMD mutations is shown in Table 1. As an initial step toward correcting a majority of human DMD mutations by exon skipping, we screened pools of guide RNAs to target the top 12 exons of the human DMD gene (Fig. 1, A and B). We selected three to six PAM sequences (NAG or NGG) to target the 3′ or 5′ splice sites, respectively, of each exon (Fig. 1A and Table 1). These guide RNAs were cloned in plasmid SpCas9-2A-GFP, as described previously (9). Indels that remove essential splice donor or acceptor sequences allow for skipping of the corresponding target exon. On the basis of the frequency of known DMD mutations, these guide RNAs would be predicted to be capable of rescuing dystrophin function in up to 60% of DMD patients (30).


Speciation inherently involves interactions. All levels of biological function are involved in such interactions, behavioural, physiological, genetic and molecular, including that between respective parental chromosomes in the offspring during meiosis. Failure to crossover leads to chromosomal anomalies in germ cells and reduced fertility in many organisms including S. cerevisiae. Previously, it was shown that the mismatch repair system has a significant effect on chromosome segregation between species having low hybrid fertility (∼1%) ( Hunter et al., 1996 ). The results provided herein show that the mismatch repair system has substantial effects on fertility within species, between populations that may be at early stages of speciation.

Pediatric Gliomas: Current Concepts on Diagnosis, Biology, and Clinical Management

Gliomas are the most common CNS tumors in children and adolescents, and they show an extremely broad range of clinical behavior. The majority of pediatric gliomas present as benign, slow-growing lesions classified as grade I or II by the WHO classification of CNS tumors. These pediatric low-grade gliomas (LGGs) are fundamentally different from IDH-mutant LGGs occurring in adults, because they rarely undergo malignant transformation and show excellent overall survival under current treatment strategies. However, a significant fraction of gliomas develop over a short period of time and progress rapidly and are therefore classified as WHO grade III or IV high-grade gliomas (HGGs). Despite all therapeutic efforts, they remain largely incurable, with the most aggressive forms being lethal within months. Thus, the intentions of neurosurgeons, pediatric oncologists, and radiotherapists to improve care for pediatric patients with glioma range from increasing quality of life and preventing long-term sequelae in what is often a chronic, but rarely life-threatening disease (LGG), to uncovering effective treatment options to prolong patient survival in an almost universally fatal setting (HGG). The last decade has seen unprecedented progress in understanding the molecular biology underlying pediatric gliomas, fueling hopes to achieve both goals. Large-scale collaborative studies around the globe have cataloged genomic and epigenomic alterations in gliomas across ages, grades, and histologies. These studies have revealed biologic subgroups characterized by distinct molecular, pathologic, and clinical features, with clear relevance for patient management. In this review, we summarize hallmark discoveries that have expanded our knowledge in pediatric LGGs and HGGs, explain their role in tumor biology, and convey our current concepts on how these findings may be translated into novel therapeutic approaches.

Pediatric low-grade gliomas (LGGs) or glioneuronal tumors (WHO grade I or II) are a highly heterogeneous collection of entities accounting for 25% to 30% of all childhood CNS tumors. They are roughly as common as malignant gliomas and embryonal tumors combined. 1,2 The most common single entity is pilocytic astrocytoma (PA > 15% of tumors in patients age 0 to 19 years 1 ), with ganglioglioma, dysembryoplastic neuroepithelial tumor (DNET), and diffuse glioma, each composing a notable minority. Some additional subsets are so rare that they are only now starting to be described. 3 Overlapping morphology (eg, variants of DNET, entrapped v neoplastic ganglion cells, and microvascular tumors resembling higher-grade tumors) can pose a diagnostic challenge. Furthermore, the natural proliferative potential of the developing CNS may complicate assessments of malignancy, meaning that slightly increased mitotic indices or Ki-67 immunostaining do not automatically preclude a benign course.

In stark contrast to adult lower-grade gliomas, IDH mutations are almost absent in children, and malignant progression is extremely rare in pediatric LGGs. Outcomes are typically good, with 5-year overall survival of approximately 95% (see Stokland et al 4 ). Thus, particularly for tumors not amenable to gross resection, LGGs often become a chronic disease, and affected children may experience a protracted reduction in quality of life. 5 Although there are some reported prognostic indicators (eg, Stokland et al 4 ), we currently know little about the mechanisms by which these tumors relapse or progress. One exception to this may be BRAF V600E mutant and 9p21 (CDKN2A/B)–deleted tumors (hallmark lesions of pleomorphic xanthoastrocytoma 2 ), which likely display an increased propensity for progression and a worse outcome. 6

Also in contrast to most adult gliomas, a notable fraction of pediatric LGGs can be linked to a hereditary component. For example, subependymal giant cell astrocytoma is closely associated with germline mutations in TSC1 or TSC2 and occurs in up to 20% of patients with tuberous sclerosis complex. 7 A similar proportion of patients with neurofibromatosis type I (NF1) develop a pilocytic astrocytoma during the first decade of life, typically in the optic pathway. 8 There are also links between a second RASopathy, namely Noonan syndrome, and pilocytic astrocytoma. 9-11 This syndrome is often a result of germline mutations in PTPN11, which was recently found to be somatically mutated together with FGFR1 in a subset of PAs. 12

Now seen as a canonical single-pathway disease, essentially 100% of PAs harbor an alteration in the MAPK axis, 12,13 most commonly KIAA1549:BRAF fusion. 14 A variety of additional alterations in this pathway have been identified in other LGG histologies, including additional BRAF fusions and mutations RAF1 fusions mutations, fusions, or kinase domain duplications of FGFR1 and fusions of the NTRK gene family. 12,13,15 The link between individual alterations and particular histologies is not 100% clear, as summarized in Figure 1 . Although BRAF fusions are almost exclusive to PA, BRAF V600E mutation, for example, is seen in some PAs as well as a substantial fraction of ganglioglioma and pleomorphic xanthoastrocytoma. 16 One notable exception with BRAF fusions is the recently described diffuse leptomeningeal glioneuronal tumor (also known as disseminated oligodendroglioma-like leptomeningeal neoplasm), which often shows a KIAA1549:BRAF fusion together with isolated 1p or combined 1p/19q deletion. 17 FGFR1 alterations also occur across histologies but with an apparent enrichment in DNETs. 12,13,18,22

Fig 1. Distribution of pediatric low-grade glioma histologies and molecular genetic alterations by anatomic tumor location. Inner pie charts represent relative frequencies of the most common pediatric low-grade glioma histologic entities represented by colors as indicated. Outer rings represent the most common molecular genetic alterations associated with each histologic entity in a given location. Original data from the German Cancer Research Center in Heidelberg aligned with published data from other studies. 1,2,6,8,12,13,16-21 DA, diffuse astrocytoma DNET, dysembryoplastic neuroepithelial tumor GG, ganglioglioma PA, pilocytic astrocytoma PXA, pleomorphic xanthoastrocytoma SEGA, subependymal giant cell astrocytoma.

Thus, MAPK alterations underlie many low-grade glial/glioneuronal entities. MAPK-related oncogene-induced senescence is also likely one reason for their relatively benign behavior. 19,23 However, additional signaling programs are altered in some subsets. A role for amplification and/or rearrangement of MYB/MYBL1, for example, has been identified in a proportion of LGGs, particularly with a diffuse astrocytic or angiocentric morphology. 13,20,21 Although the ultimate downstream consequences are not yet fully clear, it is thought that the most common fusion event (MYB:QKI) acts through a triple mechanism of MYB truncation, increased expression through enhancer hijacking, and loss of QKI tumor suppressor function. 24

The mainstay of current LGG therapy is surgical excision, which may be curative where total resection is possible. In areas where subtotal (or no) resection is possible, however, the chances of progression or relapse are substantial. Here, chemotherapy with either a vincristine plus carboplatin or vinblastine monotherapy regimen is usually given. 25-27 Of note is that temozolomide, the treatment of choice for adult diffuse gliomas, is no better than standard therapy for pediatric LGG. 28 Although current chemotherapies are associated with good overall survival, long-term treatment (especially over several rounds) is often associated with significant morbidity. 5 A more tailored approach is therefore needed to improve quality of life.

To address issues such as translation of biologic knowledge into planning of future LGG trials, a group of scientists and clinicians recently established a consensus-finding group. 29 Their recommendations noted that functional outcomes, not just survival, should be considered as key end points that molecular analysis through resection or biopsy should be performed before adjuvant therapy, and that a combined histologic and molecular stratification should be routinely implemented to facilitate assignment to novel therapeutic studies. It is hoped that a targeted approach may deliver improvements in tumor control and in functional measures with fewer adverse effects, focusing on quality of survival rather than absolute rates. A success story in LGG is the use of mTOR inhibitors for treating subependymal giant cell astrocytoma, a safe and effective treatment which is now approved. 30 On the basis of growing knowledge of activated signaling pathways in other LGGs, several early-phase clinical trials looking at MAPK-targeted therapy have recently been completed or are currently in progress.

Initial results with MEK inhibitors (MEKi), which should block pathway activity regardless of the precise upstream alteration, seem to be promising. Both selumetinib and trametinib have completed phase II trials, and plans for phase III trials are in advanced stages. Initial evidence suggests a particularly strong signal in NF1-associated tumors, which would be in keeping with recent results in NF1-associated plexiform neurofibroma. 31

Studies with drugs targeting the V600E-mutant form of BRAF have also shown positive results, with at least disease stabilization seen in almost all patients in a dabrafenib study. 32 Care must be taken, however, when considering treatment with these type I BRAF V600E–specific inhibitors, because some (such as sorafenib 33 ) can show paradoxical stimulation of tumor growth in the context of the more common KIAA1549:BRAF fusion. 34 The next round of early-phase trials includes both type II RAF inhibitors, which should overcome this activation, 35 and BRAF inhibitor/MEKi combinations ( identifier: NCT02124772), whereas both FGFR1 and NTRK kinases represent additional possible targets. Thus, although they have a ways to go before they become standard of care, there is reason for optimism about the impact that personalized medicine may have on the survival and especially the quality of life of children with LGG.

Pediatric high-grade glioma (HGG) essentially includes anaplastic astrocytoma (WHO grade III) and glioblastoma multiforme (GBM WHO grade IV), both malignant, diffuse, infiltrating astrocytic tumors. 2 Gliomatosis cerebri, a highly infiltrative HGG manifestation affecting multiple brain regions, is thought to represent a phenotypic extreme rather than a distinct entity. 36 Diffuse intrinsic pontine glioma (DIPG), a diagnosis frequently established by a combination of clinical symptoms (rapidly developing brain stem dysfunction and/or cerebrospinal fluid obstruction) and radiologic criteria (large, expansile brain stem mass occupying more than two thirds of the pons), shows a uniformly aggressive behavior, even when displaying lower-grade histology. 37 This is partly reflected in the updated WHO 2016 criteria, whereby diffuse midline gliomas with K27M histone mutations (including most DIPGs) are classed as WHO grade IV, regardless of histology. 2 The morphology and neuropathologic characteristics of anaplastic astrocytoma (ie, foci of increased cell density, nuclear atypia, and mitotic activity) and glioblastoma (additional microvascular proliferation and/or necrosis) usually correspond with a poorly defined tumor mass on magnetic resonance imaging. Analysis of adult glioma has shown that most IDH-wild-type grade III astrocytomas have a dismal prognosis, which mimics that of GBM, 38 and the prognostic/biologic relevance of histologically distinguishing between grade III and grade IV in children is also not clear.

Pediatric HGGs may manifest across all ages and anatomic CNS compartments and are among the most common malignant CNS tumors in children. The reported age-adjusted incidence of 0.26 per 100,000 population 1 is likely an underestimate, because DIPGs with low-grade histology or without histologic assessment are not assigned as HGG in epidemiologic registries, and poorly differentiated HGG variants previously may have been diagnosed as primitive neuroectodermal tumors 39 or tumors with mixed ependymal, glial, or glioneuronal features. Improved profiling through methods such as DNA methylation analysis may help with the latter issue.

Phenotypically indistinguishable from the adult disease, early molecular profiling studies suggested a different biology underlying childhood HGG. 40-45 International next-generation sequencing efforts shortly thereafter discovered somatic histone mutations as a hallmark of HGG in children and young adults, namely K27M and G34R/V mutations in H3.3- and H3.1-coding genes. 46,47 Subsequently, numerous reports have investigated the impact of these mutations on the epigenome, 48-51 and associations with other molecular, 52-56 pathologic, 56-59 or clinical 49,60-63 features, highlighting a pivotal role in gliomagenesis. The resulting insights have formed our current concept of molecular HGG subgroups: that distinct cell-of-origin populations of the developing CNS, susceptible to specific oncogenic hits, give rise to biologically and clinically distinct groups of tumors that are likely to respond to different therapies. An overview of key alterations by location is summarized in Figure 2 , and an example visualization of distinct subclasses of both LGGs and HGGs is provided in Figure 3 .

Fig 2. Molecular patterns and clinical features of pediatric high-grade glioma subgroups. (A) Oncoprints schematically depicting selected common molecular alterations in pediatric high-grade gliomas by anatomic location. Colored boxes indicate alterations being present. (B) Pie charts representing relative frequencies of common pediatric high-grade glioma molecular alterations represented by colors corresponding to (A) and (C). (C) Age distribution of common pediatric high-grade glioma molecular alterations in children and young adults (age younger than 30 years). Original data from the German Cancer Research Center in Heidelberg aligned with published data from other studies. 2,39-46,48,49,52-55,63-65 amp., amplification del., deletion.

Fig 3. Molecular subgroups of pediatric low-grade gliomas (LGGs) and high-grade gliomas (HGGs) by DNA methylation patterns. T-distributed stochastic neighbor embedding (TSNE) analysis of selected groups of pediatric gliomas by genome-wide DNA methylation patterns (Illumina Infinium BeadChip Array 5,000 most variable CpG probes by standard deviation). Patients (n = 10 per class) were selected to emphasize group differences. Each circle represents one sample. Subgroup associations are represented by colors as indicated. Original data from the German Cancer Research Center in Heidelberg were partly published in Sturm et al. 39 DMG K27, diffuse midline glioma with H3 K27 mutations DNT, dysembryoplastic neuroepithelial tumor GG, ganglioglioma HGG pedRTK1, HGG enriched for PDGFRA amplifications 69 HGG pedRTK2, HGG enriched for EGFR amplifications 69 HGG G34, HGG with H3.3 G34 mutations HGG MYCN, HGG enriched for MYCN/MYC amplifications LGG MYB, LGG enriched for MYB/MYBL1 alterations. PA, pilocytic astrocytoma PXA, pleomorphic xanthoastrocytoma SEGA, subependymal giant cell astrocytoma. (*) These groups in particular can contain patients with either typical HGG or more primitive neuroectodermal tumor–like morphology.

The majority of pediatric diffuse midline gliomas arising in the brain stem (ie, DIPG > 90%), 61 thalamus (approximately 50%), 61 and spinal cord (approximately 60%) 63 harbor mutations at position 27 (K27M) in genes coding for histone 3 variants (H3F3A, approximately three fourths HIST1H3B/C, approximately one fourth, and other rare variations). 61 The K27M-mutant histone 3 protein inhibits polycomb repressive complex 2 (PRC2) activity via sequestration of its catalytic subunit EZH2, 48 resulting in globally decreased H3 K27 trimethylation (H3 K27me3). 50 Emerging patterns suggest further biologic diversity within K27M-mutated tumors: H3.3 mutations are found across midline structures (co-occurring with FGFR1 and/or NF1 mutations in some thalamic gliomas 53 ), typically affect children age 7 to 10 years and are associated with very poor outcome. 61 In contrast, H3.1 mutations are largely restricted to DIPG with earlier onset (age 4 to 6 years), have been associated with distinct clinicopathologic and radiologic features and a slightly better prognosis, and frequently co-occur with ACVR1 mutations. 52-55,61 Initially thought to be pathognomonic for high-grade astrocytic tumors, 57,66 the spectrum of CNS tumors with H3 K27M mutations has recently been expanded to include rare examples of lower-grade midline gliomas and posterior fossa ependymomas, 12,13,67,68 in which their prognostic impact is yet to be defined.

Up to one third of hemispheric pediatric HGGs carry mutations at position 34 (G34R/V) in H3F3A. 46,47,49,52 Although the exact consequences of H3.3 G34 mutations are not yet understood, associations with mutations in ATRX and subtelomeric hypomethylation may indicate a role for telomerase-independent telomere maintenance mechanisms (ie, alternative lengthening of telomeres) in this subset of tumors. 46,49 Other molecular features include a high percentage of TP53 mutations (> 85%) and MGMT promoter methylation, which is absent from other pediatric HGG subgroups. 56 G34-mutated tumors also have a divergent histopathologic appearance, with some displaying a more primitive morphology. 39,56,58 However, their hemispheric-restricted location, typical manifestation during adolescence or young adulthood (age 10 to 25 years), and association with slightly prolonged survival compared with other HGGs strongly argue for a biologically uniform entity. 49,56

Only a small number of HGGs in older adolescents display hotspot mutations in IDH1/2 genes, thereby representing the lower age spectrum of adult gliomas (reviewed in Sturm et al 64 ). From the remaining heterogeneous fraction of H3/IDH-wild-type pediatric HGGs (approximately 50%), more subgroups are beginning to emerge. For example, amplifications of MYCN, often co-amplified with ID2, may drive a subset of DIPGs and supratentorial tumors with variable glioma or primitive neuroectodermal tumor–like morphology. 39,54 Other subgroups are enriched for amplifications or mutations in receptor tyrosine kinase genes such as PDGFRA or EGFR. 49,65,69 Initial evidence points toward possible prognostic differences in these subsets. 69 Other recently detected alterations include fusions involving MET 70 and NTRK1-3 genes, the latter being enriched in infant HGGs and pointing to some overlap with LGG biology in this age group. 52

An estimated 5% to 10% of pediatric HGGs harbor BRAF V600E mutations. These tumors are predominantly cortical, share histologic and epigenetic characteristics with pleomorphic xanthoastrocytoma (PXA), and frequently harbor homozygous CDKN2A/B deletions. 6 The slightly better clinical outcome of patients with these tumors may explain some of the long-term survivors seen in HGG clinical trials. 71 More importantly, targeted therapy for this molecularly defined group of patients 72-74 is currently being tested in clinical trials ( identifiers: NCT01677741 and NCT01748149). Of note, BRAF V600E mutations are also commonly encountered in epithelioid GBM, which can display histologic features similar to those of PXA but typically with a worse prognosis. 75,76 The association between these two entities both clinically and biologically (eg, whether epithelioid GBM may represent a malignant transformation of PXA) is worthy of additional investigation.

A small number of pediatric HGGs are thought to result from cancer predisposition syndromes. Some GBMs arise in patients with constitutional mismatch repair deficiency (caused by homozygous mutations in mismatch repair genes PMS2, MLH1, MSH2, and MSH6), and exhibit a greatly increased mutational burden. Recent reports of responses of such tumors to immune checkpoint inhibition, likely through presenting a high load of T-cell activating neoantigens, have implications for constitutional mismatch repair deficiency–associated GBM and other HGGs with an acquired hypermutator phenotype. 77

Such translational progress is urgently needed, because current treatment strategies generally bring minimal benefit. The standard therapy in diffuse midline (and therefore unresectable) gliomas is radiotherapy (and best supportive care), temporarily improving quality of life but barely increasing survival. 78,79 Most patients die within 1 year after diagnosis. For supratentorial/hemispheric HGG, maximal surgical resection is followed by radiotherapy (for patients older than age 4 years) and concomitant/adjuvant chemotherapy. On the basis of positive adult data with temozolomide 80 and its decreased toxicity compared with other regimens, 81 radiochemotherapy with temozolomide is widely considered as the therapeutic backbone. However, evidence for efficacy of the latter is currently unclear.

Future clinical trials will need to recognize the diversity of these tumors as opposed to an all-comers approach. This will require upfront molecular characterization of tumor tissue, including for DIPGs. When performed in a safe, standardized setting, stereotactic biopsy of DIPGs allows identification of actionable alterations as part of molecularly informed studies (eg, Fontebasso et al 53 and Worst et al 82 ). Furthermore, increased efforts are required to ascertain tumor material at relapse (or at autopsy), which would give important information about disease progression. Although core drivers may be both spatially and temporally stable, additional modifying alterations in subpopulations can also play important roles (see Nikbakht et al 83 ).

In contrast to MEKi for LGG, the heterogeneity of HGG means that any single drug is unlikely to work for a large proportion of patients. Molecularly informed trials will therefore require global collaboration to conduct adequately powered studies. Initiatives such as international DIPG registries will help improve characterization of these tumors and facilitate trial planning. 84,85 Individual examples of bench-to-bedside translation also indicate that studying acquired resistance mechanisms will be another challenge. 70 Expanding the repertoire of patient-derived preclinical models will help when testing epigenetic modifier therapies for HGG, 86,87 some of which are now entering clinical trials ( identifier: NCT02717455).

Although hurdles such as drug delivery across the blood-brain barrier (especially in DIPGs) remain to be overcome, recent progress in understanding these tumors means that enthusiasm within the research community is greater than ever.

Here we have provided an overview of current concepts on diagnosis, biology, and clinical management for the extremely heterogeneous group of pediatric gliomas. For more detail on some of these aspects, we direct the reader to additional recent reviews, a selection of which is provided in Table 1. Although our knowledge of the biology of pediatric gliomas has expanded enormously in recent years, significant challenges remain in translating these insights into clinical practice. For example, the true intertumoral heterogeneity of this group is far wider than anticipated and also broader than what is captured by current diagnostic practice. Definition of combined histo-molecular subgroups of glioma for prognostication and stratification onto (targeted) treatment trials will therefore be of key importance—something which hopefully will be addressed through initiatives such as the Consortium to Inform Molecular and Practical Approaches to CNS Tumor Taxonomy (cIMPACT) group. 88 In particular, precise prognostic markers and subgroups for BRAF V600E–mutated tumors would be of substantial value, because tumors with this readily druggable target show a spectrum of low- and high-grade histologies and a varied clinical course.

Table 1. Selected Recent Review Articles Summarizing Our Current Understanding of Pediatric Gliomas (see also references therein)

Thus, informative clinical trials in pediatric glioma will require comprehensive molecular characterization at diagnosis to enable precision therapy. The optimal time point for targeted therapies in patients with LGGs needs to be determined, and ranges from adjuvant therapy, even for completely resected tumors, to approaches restricted to relapsed or refractory disease. In HGGs, the delineation of distinct risk groups based on DNA methylation or gene sequencing data could be a next step toward an improved interpretation of clinical trial data. International multicenter trials will be the only way to address these issues rapidly, given the rarity of distinct subgroups.

More detailed characterization of the oncogenic effects of alterations such as histone and ATRX mutations in HGG or MYB in LGG will also be essential for providing additional insight and possible therapeutic vulnerabilities. The importance of understanding signaling networks and feedback loops within the MAPK pathway, for example, is seen from the paradoxical activation by first-generation RAF inhibitors. Such data may also help identify mechanisms of treatment resistance and suggest rational combinations to overcome them.

The availability of good preclinical in vitro and in vivo models, particularly for LGG, is another translational bottleneck. The development of such models will enable more functional studies such as high-throughput genetic or compound screening for novel drug targets. In addition, these models need to be used in a more sophisticated way when planning preclinical studies to improve their predictive value (eg, comparison with standard-of-care therapy and use of multiple models to better mimic clinical trials).

There is much work still to be done, but recent advances in LGGs and HGGs provide a framework for the road ahead. For some entities, that road is relatively clear (eg, second-generation RAF inhibitors with or without MEKi for V600E-mutant tumors), whereas for others, the path will likely have more twists and turns (eg, K27-mutant DIPGs). Overall, however, our improved understanding provides grounds for optimism that meaningful clinical benefit can be achieved in the not-too-distant future.

Conception and design: All authors

Financial support: Stefan M. Pfister

Collection and assembly of data: Dominik Sturm, David T.W. Jones

Data analysis and interpretation: Dominik Sturm, David T.W. Jones

Manuscript writing: All authors

Final approval of manuscript: All authors

Accountable for all aspects of the work: All authors

The following represents disclosure information provided by authors of this manuscript. All relationships are considered compensated. Relationships are self-held unless noted. I = Immediate Family Member, Inst = My Institution. Relationships may not relate to the subject matter of this manuscript. For more information about ASCO's conflict of interest policy, please refer to or

Patents, Royalties, Other Intellectual Property: PCT/CA2012/050834 Mutations of histone proteins associated with proliferative disorders

Patents, Royalties, Other Intellectual Property: PCT/CA2012/050834 Mutations of histone proteins associated with proliferative disorders

Patents, Royalties, Other Intellectual Property: PCT/CA2012/050834 Mutations of histone proteins associated with proliferative disorders

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