Somatic structural variation targets neurodevelopmental genes and identifies SHANK2 as a tumor suppressor in neuroblastoma

Abstract

Neuroblastoma, like many childhood cancers, exhibits a relative paucity of somatic single nucleotide variants (SNVs). Here, we assess the contribution of structural variation (SV) in neuroblastoma using a combination of whole genome sequencing (WGS; n=135) and single nucleotide polymorphism (SNP) genotyping (n=914) of matched tumor-normal pairs. Our study design provided means for orthogonal validation of SVs as well as validation across genomic platforms. SV frequency, type, and localization varied significantly among high-risk tumors, with MYCN non-amplified tumors harboring an increased SV burden overall (P=1.12x10 -5 ). Genes disrupted by SV breakpoints were enriched in neuronal lineages and autism spectrum disorder. The postsynaptic adapter protein-coding gene SHANK2, located on chromosome 11q13, was disrupted by SVs in 14% and 10% of MYCN non-amplified highrisk tumors based on WGS and SNP array cohorts, respectively. Forced expression of SHANK2 in neuroblastoma cell models resulted in significant growth inhibition (P=2.62x10 -2 to 3.4x10 -5 ) and accelerated neuronal differentiation following treatment with all-trans retinoic acid (P=3.08x10 -13 to 2.38x10 -30 ). These data further define the complex landscape of structural variation in neuroblastoma and suggest that events leading to deregulation of neurodevelopmental processes, such as inactivation of SHANK2, are key mediators of tumorigenesis.Neuroblastoma is a cancer of the developing sympathetic nervous system that most commonly affects children under 5 years of age, with a median age at diagnosis of 17 months 1 . Approximately 50% of cases present with disseminated disease at the time of diagnosis, and despite intense multi-modal therapy, the survival rate for this high-risk subset remains less than 50% 1 . Recent whole genome and exome sequencing studies of neuroblastoma have revealed relatively few recurrent protein-coding somatic mutations including single nucleotide variations (SNVs) and small (<50b) insertion/deletions (indels) 2-5 .Large-scale structural variations (SVs) such as deletions, insertions, inversions, tandem duplications and translocations can arise from mutational processes that alter chromosome structure and evade innate mechanisms of maintaining genomic stability. These diverse SVs are commonly acquired somatically and act as driver mutations 6 .A plethora of approaches have been applied to detect SVs across large cancer datasets 6-9 .First, methods that identify copy number variations (CNVs) can be applied to intensity log R ratios from genotyping and comparative genomic hybridization (CGH) arrays as well as read-depth measures from next generation sequencing. Different segmentation algorithms have been applied to either platform in order to obtain copy number gain and loss calls, which range from a few hundred base-pair size to whole chromosomal alterations. These methods are dosage sensitive, allowing numerical quantification of amplifications and homozygous deletions. Analysis of CNVs in neuroblastoma primary tumor and matched...

Integrative genomics identifies lncRNA regulatory networks across 1,044 pediatric leukemias and extra-cranial solid tumors

“…"somaticCnvDetailsDiploidBeta" containing ploidy estimates and tumor/blood coverage along 2kb bins across the genome. To create segmentation files, we used custom scripts to reformat CGI coverage data to meet requirements of the "copynumber" R bioconductor package as previously described [44]. We used the winsorize function in this package, which performs data smoothing and segmentation via a piecewise constant segmentation (pcf) algorithm (kmin =2 and gamma= 1000).…”

“…Many pediatric cancers are marked by a lower single nucleotide variant (SNV) and insertion-deletion (indel) burden than observed in adult cancers [36]. Instead, large chromosomal events, such as somatic copy number aberrations (SCNAs) and other structural variants (SVs) have been shown to dysregulate protein coding driver genes [36,44]. However, the extent to which large chromosomal alterations impact lncRNAs in pediatric cancers remains unknown.…”

“…While SCNAs can cause the dysregulation of lncRNA expression based on gene dosage, structural variant (SV) breakpoints within a lncRNA could cause loss or gain of function [36,44]. We utilized WGS data to identify lncRNAs disrupted by SV breakpoints using a previously described combination approach involving copy number read-depth and discordant junction approach [44] (Online Methods). There were 650 unique expressed lncRNA genes disrupted by SVs, 89% of which were found in only one sample ( Supplementary Fig.…”

“…are very likely to be functional given that low or rare expression can be an indicator of transcriptional noise [80]. In addition to having a limited number of RNA matched WGS samples, the Complete Genomics short read technology limits the detection of structural variants based on size as previously described [36,44]. The use of long-read sequencing and greater sequencing depth in future studies will enable more accurate copy number and structure variant detection in pediatric cancers.…”

Intratumoral immunosuppression profiles in 11q‐deleted neuroblastomas provide new potential therapeutic targets

“…To validate the results, we used the data from the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) project (GSE131189). Of the total cohort, 215 patients belonged to the high-risk NB subgroup and had both expression and CNV data [23]. The validation cohort was composed of 95 patients with 11q-deletion, 56 patients with MNA, 60 patients with neither of the two alterations, and 4 with both alterations that were excluded from this study.…”