Adrian M. Stütz

Dissecting the genomic complexity underlying medulloblastoma

Medulloblastoma is an aggressively growing tumour, arising in the cerebellum or medulla/brain stem. It is the most common malignant brain tumour in children, and shows tremendous biological and clinical heterogeneity. Despite recent treatment advances, approximately 40% of children experience tumour recurrence, and 30% will die from their disease. Those who survive often have a significantly reduced quality of life. Four tumour subgroups with distinct clinical, biological and genetic profiles are currently identified. WNT tumours, showing activated wingless pathway signalling, carry a favourable prognosis under current treatment regimens. SHH tumours show hedgehog pathway activation, and have an intermediate prognosis. Group 3 and 4 tumours are molecularly less well characterized, and also present the greatest clinical challenges. The full repertoire of genetic events driving this distinction, however, remains unclear. Here we describe an integrative deep-sequencing analysis of 125 tumour–normal pairs, conducted as part of the International Cancer Genome Consortium (ICGC) PedBrain Tumor Project. Tetraploidy was identified as a frequent early event in Group 3 and 4 tumours, and a positive correlation between patient age and mutation rate was observed. Several recurrent mutations were identified, both in known medulloblastoma-related genes (CTNNB1, PTCH1, MLL2, SMARCA4) and in genes not previously linked to this tumour (DDX3X, CTDNEP1, KDM6A, TBR1), often in subgroup-specific patterns. RNA sequencing confirmed these alterations, and revealed the expression of what are, to our knowledge, the first medulloblastoma fusion genes identified. Chromatin modifiers were frequently altered across all subgroups. These findings enhance our understanding of the genomic complexity and heterogeneity underlying medulloblastoma, and provide several potential targets for new therapeutics, especially for Group 3 and 4 patients.

An integrated map of structural variation in 2,504 human genomes

Structural variants are implicated in numerous diseases and make up the majority of varying nucleotides among human genomes. Here we describe an integrated set of eight structural variant classes comprising both balanced and unbalanced variants, which we constructed using short-read DNA sequencing data and statistically phased onto haplotype blocks in 26 human populations. Analysing this set, we identify numerous gene-intersecting structural variants exhibiting population stratification and describe naturally occurring homozygous gene knockouts that suggest the dispensability of a variety of human genes. We demonstrate that structural variants are enriched on haplotypes identified by genome-wide association studies and exhibit enrichment for expression quantitative trait loci. Additionally, we uncover appreciable levels of structural variant complexity at different scales, including genic loci subject to clusters of repeated rearrangement and complex structural variants with multiple breakpoints likely to have formed through individual mutational events. Our catalogue will enhance future studies into structural variant demography, functional impact and disease association.

DELLY: structural variant discovery by integrated paired-end and split-read analysis

Motivation: The discovery of genomic structural variants (SVs) at high sensitivity and specificity is an essential requirement for characterizing naturally occurring variation and for understanding pathological somatic rearrangements in personal genome sequencing data. Of particular interest are integrated methods that accurately identify simple and complex rearrangements in heterogeneous sequencing datasets at single-nucleotide resolution, as an optimal basis for investigating the formation mechanisms and functional consequences of SVs. Results: We have developed an SV discovery method, called DELLY, that integrates short insert paired-ends, long-range mate-pairs and split-read alignments to accurately delineate genomic rearrangements at single-nucleotide resolution. DELLY is suitable for detecting copy-number variable deletion and tandem duplication events as well as balanced rearrangements such as inversions or reciprocal translocations. DELLY, thus, enables to ascertain the full spectrum of genomic rearrangements, including complex events. On simulated data, DELLY compares favorably to other SV prediction methods across a wide range of sequencing parameters. On real data, DELLY reliably uncovers SVs from the 1000 Genomes Project and cancer genomes, and validation experiments of randomly selected deletion loci show a high specificity. Availability: DELLY is available at www.korbel.embl.de/software.html Contact: tobias.rausch@embl.de

Recurrent somatic alterations of FGFR1 and NTRK2 in pilocytic astrocytoma

Pilocytic astrocytoma, the most common childhood brain tumor, is typically associated with mitogen-activated protein kinase (MAPK) pathway alterations. Surgically inaccessible midline tumors are therapeutically challenging, showing sustained tendency for progression and often becoming a chronic disease with substantial morbidities. Here we describe whole-genome sequencing of 96 pilocytic astrocytomas, with matched RNA sequencing (n = 73), conducted by the International Cancer Genome Consortium (ICGC) PedBrain Tumor Project. We identified recurrent activating mutations in FGFR1 and PTPN11 and new NTRK2 fusion genes in non-cerebellar tumors. New BRAF-activating changes were also observed. MAPK pathway alterations affected all tumors analyzed, with no other significant mutations identified, indicating that pilocytic astrocytoma is predominantly a single-pathway disease. Notably, we identified the same FGFR1 mutations in a subset of H3F3A-mutated pediatric glioblastoma with additional alterations in the NF1 gene. Our findings thus identify new potential therapeutic targets in distinct subsets of pilocytic astrocytoma and childhood glioblastoma.

Assembly and diploid architecture of an individual human genome via single-molecule technologies

We present the first comprehensive analysis of a diploid human genome that combines single-molecule sequencing with single-molecule genome maps. Our hybrid assembly markedly improves upon the contiguity observed from traditional shotgun sequencing approaches, with scaffold N50 values approaching 30 Mb, and we identified complex structural variants (SVs) missed by other high-throughput approaches. Furthermore, by combining Illumina short-read data with long reads, we phased both single-nucleotide variants and SVs, generating haplotypes with over 99% consistency with previous trio-based studies. Our work shows that it is now possible to integrate single-molecule and high-throughput sequence data to generate de novo assembled genomes that approach reference quality.

The Genomic and Transcriptomic Landscape of a HeLa Cell Line

HeLa is the most widely used model cell line for studying human cellular and molecular biology. To date, no genomic reference for this cell line has been released, and experiments have relied on the human reference genome. Effective design and interpretation of molecular genetic studies performed using HeLa cells require accurate genomic information. Here we present a detailed genomic and transcriptomic characterization of a HeLa cell line. We performed DNA and RNA sequencing of a HeLa Kyoto cell line and analyzed its mutational portfolio and gene expression profile. Segmentation of the genome according to copy number revealed a remarkably high level of aneuploidy and numerous large structural variants at unprecedented resolution. Some of the extensive genomic rearrangements are indicative of catastrophic chromosome shattering, known as chromothripsis. Our analysis of the HeLa gene expression profile revealed that several pathways, including cell cycle and DNA repair, exhibit significantly different expression patterns from those in normal human tissues. Our results provide the first detailed account of genomic variants in the HeLa genome, yielding insight into their impact on gene expression and cellular function as well as their origins. This study underscores the importance of accounting for the strikingly aberrant characteristics of HeLa cells when designing and interpreting experiments, and has implications for the use of HeLa as a model of human biology.

A Comprehensive Map of Mobile Element Insertion Polymorphisms in Humans

As a consequence of the accumulation of insertion events over evolutionary time, mobile elements now comprise nearly half of the human genome. The Alu, L1, and SVA mobile element families are still duplicating, generating variation between individual genomes. Mobile element insertions (MEI) have been identified as causes for genetic diseases, including hemophilia, neurofibromatosis, and various cancers. Here we present a comprehensive map of 7,380 MEI polymorphisms from the 1000 Genomes Project whole-genome sequencing data of 185 samples in three major populations detected with two detection methods. This catalog enables us to systematically study mutation rates, population segregation, genomic distribution, and functional properties of MEI polymorphisms and to compare MEI to SNP variation from the same individuals. Population allele frequencies of MEI and SNPs are described, broadly, by the same neutral ancestral processes despite vastly different mutation mechanisms and rates, except in coding regions where MEI are virtually absent, presumably due to strong negative selection. A direct comparison of MEI and SNP diversity levels suggests a differential mobile element insertion rate among populations.

Enhancer hijacking activates GFI1 family oncogenes in medulloblastoma.

Medulloblastoma is a highly malignant paediatric brain tumour currently treated with a combination of surgery, radiation and chemotherapy, posing a considerable burden of toxicity to the developing child. Genomics has illuminated the extensive intertumoral heterogeneity of medulloblastoma, identifying four distinct molecular subgroups. Group 3 and group 4 subgroup medulloblastomas account for most paediatric cases; yet, oncogenic drivers for these subtypes remain largely unidentified. Here we describe a series of prevalent, highly disparate genomic structural variants, restricted to groups 3 and 4, resulting in specific and mutually exclusive activation of the growth factor independent 1 family proto-oncogenes, GFI1 and GFI1B. Somatic structural variants juxtapose GFI1 or GFI1B coding sequences proximal to active enhancer elements, including super-enhancers, instigating oncogenic activity. Our results, supported by evidence from mouse models, identify GFI1 and GFI1B as prominent medulloblastoma oncogenes and implicate ‘enhancer hijacking’ as an efficient mechanism driving oncogene activation in a childhood cancer.

Nucleotide-resolution analysis of structural variants using BreakSeq and a breakpoint library

Structural variants (SVs) are a major source of human genomic variation; however, characterizing them at nucleotide resolution remains challenging. Here we assemble a library of breakpoints at nucleotide resolution from collating and standardizing ~2,000 published SVs. For each breakpoint, we infer its ancestral state (through comparison to primate genomes) and its mechanism of formation (e.g., nonallelic homologous recombination, NAHR). We characterize breakpoint sequences with respect to genomic landmarks, chromosomal location, sequence motifs and physical properties, finding that the occurrence of insertions and deletions is more balanced than previously reported and that NAHR-formed breakpoints are associated with relatively rigid, stable DNA helices. Finally, we demonstrate an approach, BreakSeq, for scanning the reads from short-read sequenced genomes against our breakpoint library to accurately identify previously overlooked SVs, which we then validate by PCR. As new data become available, we expect our BreakSeq approach will become more sensitive and facilitate rapid SV genotyping of personal genomes.

Whole-exome sequencing links caspase recruitment domain 11 (CARD11) inactivation to severe combined immunodeficiency

BACKGROUND Primary immunodeficiencies represent model diseases for the mechanistic understanding of the human innate and adaptive immune response. They are clinically highly relevant per se because in patients with severe combined immunodeficiency (SCID), infections caused by opportunistic pathogens are typically life-threatening early in life. OBJECTIVES We aimed at defining and functionally characterizing a novel form of SCID in an infant of consanguineous parents who presented with life-threatening Pneumocystis jirovecii pneumonia using a comprehensive immunologic and whole-exome genetic diagnostic strategy. METHODS Analysis of leukocyte subpopulations was performed by using multicolor flow cytometry and was combined with stimulation tests for T-cell function. The search for a disease-causing mutation was performed with diagnostic whole-exome sequencing and systematic variant categorization. Reconstitution assays were used for validating the loss-of-function mutation. RESULTS The novel entity of SCID was characterized by agammaglobulinemia and profoundly deficient T-cell function despite quantitatively normal T and B lymphocytes. Genetic analysis revealed a single pathogenic homozygous nonsense mutation of the caspase recruitment domain 11 (CARD11) gene. In reconstitution assays we demonstrated that the patient-derived truncated CARD11 protein is defective in antigen receptor signaling and nuclear factor κB activation. CONCLUSION We show that an inactivating CARD11 mutation links defective nuclear factor κB signaling to a novel cause of autosomal recessive SCID.