Novel <i>CIC</i> Point Mutations and an Exon-Spanning, Homozygous Deletion Identified in Oligodendroglial Tumors by a Comprehensive Genomic Approach Including Transcriptome Sequencing

<div><p>Oligodendroglial tumors form a distinct subgroup of gliomas, characterized by a better response to treatment and prolonged overall survival. Most oligodendrogliomas and also some oligoastrocytomas are characterized by a unique and typical unbalanced translocation, der(1,19), resulting in a 1p/19q co-deletion. Candidate tumor suppressor genes targeted by these losses, <i>CIC</i> on 19q13.2 and <i>FUBP1</i> on 1p31.1, were only recently discovered. We analyzed 17 oligodendrogliomas and oligoastrocytomas by applying a comprehensive approach consisting of RNA expression analysis, DNA sequencing of <i>CIC</i>, <i>FUBP1</i>, <i>IDH1/2</i>, and array CGH. We confirmed three different genetic subtypes in our samples: i) the “oligodendroglial” subtype with 1p/19q co-deletion in twelve out of 17 tumors; ii) the “astrocytic” subtype in three tumors; iii) the “other” subtype in two tumors. All twelve tumors with the 1p/19q co-deletion carried the most common <i>IDH1</i> R132H mutation. In seven of these tumors, we found protein-disrupting point mutations in the remaining allele of <i>CIC</i>, four of which are novel. One of these tumors also had a deleterious mutation in <i>FUBP1</i>. Only by integrating RNA expression and array CGH data, were we able to discover an exon-spanning homozygous microdeletion within the remaining allele of <i>CIC</i> in an additional tumor with 1p/19q co-deletion. Therefore we propose that the mutation rate might be underestimated when looking at sequence variants alone. In conclusion, the high frequency and the spectrum of <i>CIC</i> mutations in our 1p/19q-codeleted tumor cohort support the hypothesis that <i>CIC</i> acts as a tumor suppressor in these tumors, whereas <i>FUBP1</i> might play only a minor role. </p> </div

Exon-spanning deletion in BT1 leads to change in expression of <i>CIC</i>, <i>PAFAH1B3</i> and <i>PR19</i>.

<p><b>3a</b> Genomic region chr19:42,788,249-42,815,094 that includes the <i>CIC, PAFAH1B3</i> and <i>PR19</i> gene. The red bar represents the deletion in BT1. The minimum extent (thick) and the maximum extend (thin) of the deletion according to array CGH and qPCR results are shown. <b>3b</b>: Expression data (RNA-seq) for tumor samples (BT1-15) with both alleles of 1p and 19q (middle gray), 1p/19q co-deletion (light gray) and normal brain (nb1-3, dark gray). BT1 harboring the exon-spanning deletion shows a strong down regulation of expression in <i>CIC</i> and <i>PAFAH1B3</i> and an upregulation in <i>PR19</i> (red arrows). RPKM = Reads Per Kilobase of exon model per Million mapped reads (gene counts/total counts of each sample).</p

Distribution of <i>CIC</i> mutations in this study.

<p>The dark blue boxes represent exons. HMG-box denotes the highly conserved DNA-interacting high-mobility group domain. Exon 19 and exon 20 harbor a globular domain (GlobDom). An annotated protein-protein interacting domain is located within exon 20 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076623#B22" target="_blank">22</a>]. Mutations marked by a frame indicate novel mutations not yet described in the literature. While somatic missense mutations were only found in the HMG-box and GlobDom of CIC (BT14,5,2,16), stop-, frameshift- and splice site mutations were found across the CIC protein (BT4,15,7). The two missense mutations that were not located in exons 5 or 19 (BT17,11) were already present in the normal DNAs and had a very low PolyPhen-2 Score, indicating that these SNVs are probably polymorphisms. The red bar represents the exon-spanning deletion in BT1 which extends up to the adjacent <i>PAFAH1B3</i> and <i>PR19</i> gene (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076623#pone-0076623-g002" target="_blank">Figure 2</a>).</p

<i>CIC</i> Expression analysis and homozygous deletion of <i>CIC</i> in array CGH in tumor sample BT1.

<p><b>2a</b> Column plot of expression of the <i>CIC</i> gene in tumor samples (BT1-15) and normal brain (nb1-3) analyzed using RNA-seq. Tumor samples harboring point mutations are marked as green columns, samples without point mutations as gray columns. Compared to other tumor samples, BT1 (gray arrow) and BT7 (green arrow) show a strong down regulation of <i>CIC</i>. This can be explained by a homozygous deletion of <i>CIC</i> in BT1 (compare <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076623#pone-0076623-g002" target="_blank">Figure 2b</a>) and nonsense-mediated mRNA decay due to a frameshift mutation in BT7. RPKM = Reads Per Kilobase of exon model per Million mapped reads (gene counts/total counts of each sample). <b>2b</b>: Array CGH results of BT1 harboring a partial homozygous deletion of <i>CIC</i> (red circle). One oligonucleotide-probe situated within <i>CIC</i> and another in <i>PAFAH1B3</i> (A_16_P21013642: [hg19] 42,795,949-42,796,008 and A_14_P124269: [hg19] 42804518-42804577) showed a log₂-ratio of -2, while the whole long arm of chromosome 19 displayed a log₂-ratio of -0.9 (corresponding to one allele in the tumor and two alleles in the control probe in array CGH). The log₂-ratio of -2 indicates a homozygous deletion in about 75% of the cell population, corresponding to a background due to normal cells in the tumor tissue. Array CGH narrows the exon-spanning deletion of the second allele of <i>CIC</i> to a minimum size of 8.6 kb and a maximum size of 17.3 kb.</p

Photographs of patient #15 (<i>MECP2</i> mutation).

<p>The patient at 3 years and 6 months. The patient was diagnosed with a de novo mutation p.(Arg133Cys) in <i>MECP2</i>. Note arched eyebrows with slight synophrys, short anteverted nose, thin upper lip and smooth long philtrum.</p

Photographs of patients #7 and #29 (<i>MED13L</i> mutation).

<p>The patient (A-G) at 3 months of age (A,B,D) and at 2 years and 2 months of age (C) as well as patient 47961 (H-J) at 4 years and 2 months of age. Both patients were diagnosed with MED13L syndrome. Note long eyelashes, broad nasal tip and open mouth appearance as well as preauricular tags in both patients.</p

Sequence coverage and occurrence of <i>ADAMTSL2</i> variants.

<p>The data is shown in the UCSC genome browser “multi region view” (<a href="http://genome.ucsc.edu/" target="_blank">http://genome.ucsc.edu</a>), which displays exons in full length (dark blue boxes), flanked by 50 bp of intronic sequence (dark blue vertical line). The scale on top refers to the condensed sequence shown here. The full <i>ADAMTSL2</i> gene comprises 40.6 kb of genomic DNA (chr9:136399975–136440641, hg19). Green: read coverage, target position and variants identified in this cohort; black: corresponding data in gnomAD.</p

Summary of clinical information and mutation detection rates for subgroups of the cohort.

<p>Summary of clinical information and mutation detection rates for subgroups of the cohort.</p

<i>ERCC2</i> frameshift mutation c.1703_1704delTT (p.Phe568fs) in familial breast and ovarian cancer pedigrees.

<p>Individuals with breast cancer (BC), ovarian cancer (OC) or both (BC, OC) are shown as circles filled in black. Individuals tested positive for the familial mutation are indicated in detail; those with WT (wild-type) have been tested negative. All affected individuals with BC or OC not tested for germline mutations in ERCC2 were either deceased or refused testing. (A) German, (B) Lithuanian and (C-E) Czech pedigrees.</p

<i>ERCC2</i> allele frequencies (%) in BC/OC patients and corresponding control cohorts.

<p>The allele frequency is counted on the basis of sample size (in brackets) and number of observed cases (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006248#pgen.1006248.t001" target="_blank">Table 1</a>) with hetero- and homozygosity.</p