Alternative splicing of transposable elements in human breast cancer.

Transposable elements (TEs) drive genome evolution and can affect gene expression through diverse mechanisms. In breast cancer, disrupted regulation of TE sequences may facilitate tumor-specific transcriptomic alterations. We examine 142,514 full-length isoforms derived from long-read RNA sequencing (LR-seq) of 30 breast samples to investigate the effects of TEs on the breast cancer transcriptome. Approximately half of these isoforms contain TE sequences, and these contribute to half of the novel annotated splice junctions. We quantify splicing of these LR-seq derived isoforms in 1,135 breast tumors from The Cancer Genome Atlas (TCGA) and 1,329 healthy tissue samples from the Genotype-Tissue Expression (GTEx), and find 300 TE-overlapping tumor-specific splicing events. Some splicing events are enriched in specific breast cancer subtypes - for example, a TE-driven transcription start site upstream of ERBB2 in HER2+ tumors, and several TE-mediated splicing events are associated with patient survival and poor prognosis. The full-length sequences we capture with LR-seq reveal thousands of isoforms with signatures of RNA editing, including a novel isoform belonging to RHOA ; a gene previously implicated in tumor progression. We utilize our full-length isoforms to discover polymorphic TE insertions that alter splicing and validate one of these events in breast cancer cell lines. Together, our results demonstrate the widespread effects of dysregulated TEs on breast cancer transcriptomes and highlight the advantages of long-read isoform sequencing for understanding TE biology. TE-derived isoforms may alter the expression of genes important in cancer and can potentially be used as novel, disease-specific therapeutic targets or biomarkers. One Sentence Summary: Transposable elements generate alternative isoforms and alter post-transcriptional regulation in human breast cancer.

Co-option of endogenous retroviruses through genetic escape from TRIM28 repression.

Endogenous retroviruses (ERVs) have rewired host gene networks. To explore the origins of co-option, we employed an active murine ERV, IAPEz, and an embryonic stem cell (ESC) to neural progenitor cell (NPC) differentiation model. Transcriptional silencing via TRIM28 maps to a 190 bp sequence encoding the intracisternal A-type particle (IAP) signal peptide, which confers retrotransposition activity. A subset of "escapee" IAPs (∼15%) exhibits significant genetic divergence from this sequence. Canonical repressed IAPs succumb to a previously undocumented demarcation by H3K9me3 and H3K27me3 in NPCs. Escapee IAPs, in contrast, evade repression in both cell types, resulting in their transcriptional derepression, particularly in NPCs. We validate the enhancer function of a 47 bp sequence within the U3 region of the long terminal repeat (LTR) and show that escapee IAPs convey an activating effect on nearby neural genes. In sum, co-opted ERVs stem from genetic escapees that have lost vital sequences required for both TRIM28 restriction and autonomous retrotransposition.

Transposable element-mediated rearrangements are prevalent in human genomes.

Transposable elements constitute about half of human genomes, and their role in generating human variation through retrotransposition is broadly studied and appreciated. Structural variants mediated by transposons, which we call transposable element-mediated rearrangements (TEMRs), are less well studied, and the mechanisms leading to their formation as well as their broader impact on human diversity are poorly understood. Here, we identify 493 unique TEMRs across the genomes of three individuals. While homology directed repair is the dominant driver of TEMRs, our sequence-resolved TEMR resource allows us to identify complex inversion breakpoints, triplications or other high copy number polymorphisms, and additional complexities. TEMRs are enriched in genic loci and can create potentially important risk alleles such as a deletion in TRIM65, a known cancer biomarker and therapeutic target. These findings expand our understanding of this important class of structural variation, the mechanisms responsible for their formation, and establish them as an important driver of human diversity.

A comprehensive long-read isoform analysis platform and sequencing resource for breast cancer.

Tumors display widespread transcriptome alterations, but the full repertoire of isoform-level alternative splicing in cancer is unknown. We developed a long-read (LR) RNA sequencing and analytical platform that identifies and annotates full-length isoforms and infers tumor-specific splicing events. Application of this platform to breast cancer samples identifies thousands of previously unannotated isoforms; ~30% affect protein coding exons and are predicted to alter protein localization and function. We performed extensive cross-validation with -omics datasets to support transcription and translation of novel isoforms. We identified 3059 breast tumor­specific splicing events, including 35 that are significantly associated with patient survival. Of these, 21 are absent from GENCODE and 10 are enriched in specific breast cancer subtypes. Together, our results demonstrate the complexity, cancer subtype specificity, and clinical relevance of previously unidentified isoforms and splicing events in breast cancer that are only annotatable by LR-seq and provide a rich resource of immuno-oncology therapeutic targets.

Extrachromosomal DNA is associated with oncogene amplification and poor outcome across multiple cancers.

Extrachromosomal DNA (ecDNA) amplification promotes intratumoral genetic heterogeneity and accelerated tumor evolution1-3; however, its frequency and clinical impact are unclear. Using computational analysis of whole-genome sequencing data from 3,212 cancer patients, we show that ecDNA amplification frequently occurs in most cancer types but not in blood or normal tissue. Oncogenes were highly enriched on amplified ecDNA, and the most common recurrent oncogene amplifications arose on ecDNA. EcDNA amplifications resulted in higher levels of oncogene transcription compared to copy number-matched linear DNA, coupled with enhanced chromatin accessibility, and more frequently resulted in transcript fusions. Patients whose cancers carried ecDNA had significantly shorter survival, even when controlled for tissue type, than patients whose cancers were not driven by ecDNA-based oncogene amplification. The results presented here demonstrate that ecDNA-based oncogene amplification is common in cancer, is different from chromosomal amplification and drives poor outcome for patients across many cancer types.

Structural variant identification and characterization.

Structural variant (SV) differences between human genomes can cause germline and mosaic disease as well as inter-individual variation. De-regulation of accurate DNA repair and genomic surveillance mechanisms results in a large number of SVs in cancer. Analysis of the DNA sequences at SV breakpoints can help identify pathways of mutagenesis and regions of the genome that are more susceptible to rearrangement. Large-scale SV analyses have been enabled by high-throughput genome-level sequencing on humans in the past decade. These studies have shed light on the mechanisms and prevalence of complex genomic rearrangements. Recent advancements in both sequencing and other mapping technologies as well as calling algorithms for detection of genomic rearrangements have helped propel SV detection into population-scale studies, and have begun to elucidate previously inaccessible regions of the genome. Here, we discuss the genomic organization of simple and complex SVs, the molecular mechanisms of their formation, and various ways to detect them. We also introduce methods for characterizing SVs and their consequences on human genomes.

Comparative Genomic Analyses of the Human NPHP1 Locus Reveal Complex Genomic Architecture and Its Regional Evolution in Primates.

Many loci in the human genome harbor complex genomic structures that can result in susceptibility to genomic rearrangements leading to various genomic disorders. Nephronophthisis 1 (NPHP1, MIM# 256100) is an autosomal recessive disorder that can be caused by defects of NPHP1; the gene maps within the human 2q13 region where low copy repeats (LCRs) are abundant. Loss of function of NPHP1 is responsible for approximately 85% of the NPHP1 cases-about 80% of such individuals carry a large recurrent homozygous NPHP1 deletion that occurs via nonallelic homologous recombination (NAHR) between two flanking directly oriented ~45 kb LCRs. Published data revealed a non-pathogenic inversion polymorphism involving the NPHP1 gene flanked by two inverted ~358 kb LCRs. Using optical mapping and array-comparative genomic hybridization, we identified three potential novel structural variant (SV) haplotypes at the NPHP1 locus that may protect a haploid genome from the NPHP1 deletion. Inter-species comparative genomic analyses among primate genomes revealed massive genomic changes during evolution. The aggregated data suggest that dynamic genomic rearrangements occurred historically within the NPHP1 locus and generated SV haplotypes observed in the human population today, which may confer differential susceptibility to genomic instability and the NPHP1 deletion within a personal genome. Our study documents diverse SV haplotypes at a complex LCR-laden human genomic region. Comparative analyses provide a model for how this complex region arose during primate evolution, and studies among humans suggest that intra-species polymorphism may potentially modulate an individual's susceptibility to acquiring disease-associated alleles.

DNA REPAIR. Mus81 and converging forks limit the mutagenicity of replication fork breakage.

Most spontaneous DNA double-strand breaks (DSBs) result from replication-fork breakage. Break-induced replication (BIR), a genome rearrangement-prone repair mechanism that requires the Pol32/POLD3 subunit of eukaryotic DNA Polδ, was proposed to repair broken forks, but how genome destabilization is avoided was unknown. We show that broken fork repair initially uses error-prone Pol32-dependent synthesis, but that mutagenic synthesis is limited to within a few kilobases from the break by Mus81 endonuclease and a converging fork. Mus81 suppresses template switches between both homologous sequences and diverged human Alu repetitive elements, highlighting its importance for stability of highly repetitive genomes. We propose that lack of a timely converging fork or Mus81 may propel genome instability observed in cancer.

Global transcriptional disturbances underlie Cornelia de Lange syndrome and related phenotypes.

Cornelia de Lange syndrome (CdLS) is a genetically heterogeneous disorder that presents with extensive phenotypic variability, including facial dysmorphism, developmental delay/intellectual disability (DD/ID), abnormal extremities, and hirsutism. About 65% of patients harbor mutations in genes that encode subunits or regulators of the cohesin complex, including NIPBL, SMC1A, SMC3, RAD21, and HDAC8. Wiedemann-Steiner syndrome (WDSTS), which shares CdLS phenotypic features, is caused by mutations in lysine-specific methyltransferase 2A (KMT2A). Here, we performed whole-exome sequencing (WES) of 2 male siblings clinically diagnosed with WDSTS; this revealed a hemizygous, missense mutation in SMC1A that was predicted to be deleterious. Extensive clinical evaluation and WES of 32 Turkish patients clinically diagnosed with CdLS revealed the presence of a de novo heterozygous nonsense KMT2A mutation in 1 patient without characteristic WDSTS features. We also identified de novo heterozygous mutations in SMC3 or SMC1A that affected RNA splicing in 2 independent patients with combined CdLS and WDSTS features. Furthermore, in families from 2 separate world populations segregating an autosomal-recessive disorder with CdLS-like features, we identified homozygous mutations in TAF6, which encodes a core transcriptional regulatory pathway component. Together, our data, along with recent transcriptome studies, suggest that CdLS and related phenotypes may be "transcriptomopathies" rather than cohesinopathies.

The Alu-rich genomic architecture of SPAST predisposes to diverse and functionally distinct disease-associated CNV alleles.

Intragenic copy-number variants (CNVs) contribute to the allelic spectrum of both Mendelian and complex disorders. Although pathogenic deletions and duplications in SPAST (mutations in which cause autosomal-dominant spastic paraplegia 4 [SPG4]) have been described, their origins and molecular consequences remain obscure. We mapped breakpoint junctions of 54 SPAST CNVs at nucleotide resolution. Diverse combinations of exons are deleted or duplicated, highlighting the importance of particular exons for spastin function. Of the 54 CNVs, 38 (70%) appear to be mediated by an Alu-based mechanism, suggesting that the Alu-rich genomic architecture of SPAST renders this locus susceptible to various genome rearrangements. Analysis of breakpoint Alus further informs a model of Alu-mediated CNV formation characterized by small CNV size and potential involvement of mechanisms other than homologous recombination. Twelve deletions (22%) overlap part of SPAST and a portion of a nearby, directly oriented gene, predicting novel chimeric genes in these subjects' genomes. cDNA from a subject with a SPAST final exon deletion contained multiple SPAST:SLC30A6 fusion transcripts, indicating that SPAST CNVs can have transcriptional effects beyond the gene itself. SLC30A6 has been implicated in Alzheimer disease, so these fusion gene data could explain a report of spastic paraplegia and dementia cosegregating in a family with deletion of the final exon of SPAST. Our findings provide evidence that the Alu genomic architecture of SPAST predisposes to diverse CNV alleles with distinct transcriptional--and possibly phenotypic--consequences. Moreover, we provide further mechanistic insights into Alu-mediated copy-number change that are extendable to other loci.

PGM3 mutations cause a congenital disorder of glycosylation with severe immunodeficiency and skeletal dysplasia.

Human phosphoglucomutase 3 (PGM3) catalyzes the conversion of N-acetyl-glucosamine (GlcNAc)-6-phosphate into GlcNAc-1-phosphate during the synthesis of uridine diphosphate (UDP)-GlcNAc, a sugar nucleotide critical to multiple glycosylation pathways. We identified three unrelated children with recurrent infections, congenital leukopenia including neutropenia, B and T cell lymphopenia, and progression to bone marrow failure. Whole-exome sequencing demonstrated deleterious mutations in PGM3 in all three subjects, delineating their disease to be due to an unsuspected congenital disorder of glycosylation (CDG). Functional studies of the disease-associated PGM3 variants in E. coli cells demonstrated reduced PGM3 activity for all mutants tested. Two of the three children had skeletal anomalies resembling Desbuquois dysplasia: short stature, brachydactyly, dysmorphic facial features, and intellectual disability. However, these additional features were absent in the third child, showing the clinical variability of the disease. Two children received hematopoietic stem cell transplantation of cord blood and bone marrow from matched related donors; both had successful engraftment and correction of neutropenia and lymphopenia. We define PGM3-CDG as a treatable immunodeficiency, document the power of whole-exome sequencing in gene discoveries for rare disorders, and illustrate the utility of genomic analyses in studying combined and variable phenotypes.

Whole-exome sequencing links TMCO1 defect syndrome with cerebro-facio-thoracic dysplasia.

Whole-exome sequencing (WES) is a type of disruptive technology that has tremendous influence on human and clinical genetics research. An efficient and cost-effective method, WES is now widely used as a diagnostic tool for identifying the molecular basis of genetic syndromes that are often challenging to diagnose. Here we report a patient with a clinical diagnosis of cerebro-facio-thoracic dysplasia (CFTD; MIM#213980) in whom we identified a homozygous splice-site mutation in the transmembrane and coiled-coil domains 1 (TMCO1) gene using WES. TMCO1 mutations cause craniofacial dysmorphism, skeletal anomalies characterized by multiple malformations of the vertebrae and ribs, and intellectual disability (MIM#614132). A retrospective review revealed that clinical manifestations of both syndromes are very similar and overlap remarkably. We propose that mutations of TMCO1 are not only responsible for craniofacial dysmorphism, skeletal anomalies and mental retardation syndrome but also for CFTD.

Optimization of feline immunodeficiency virus vectors for RNA interference.

RNA interference (RNAi) occurs naturally in plant and animal cells as a means for modulating gene expression. This process has been experimentally manipulated to achieve targeted gene silencing in cells, tissues, and animals, using a variety of vector systems. Here, we tested the hypothesis that vectors based on feline immunodeficiency virus (FIV) could be used for coexpression of reporter constructs and RNAi expression cassettes. We found, unexpectedly, in our initial constructs that placement of RNAi expression cassettes downstream from a polymerase II (pol II)-expressed reporter gene inhibited reporter expression but not vector titer. Through a series of intermediate vector constructs, we found that placement of the RNAi expression cassette relative to the Rev response element and the pol II expression cassette was critical for efficient RNAi and reporter gene expression. These results suggested that steric factors, including RNA structure and recruitment of competing transcriptional machinery, may affect gene expression from FIV vectors. In a second series of studies, we show that target sequence silencing can be achieved in cells transduced by FIV vectors coexpressing reporter genes and 3' untranslated region resident microRNAs. The optimized FIV-based RNAi expression vectors will find broad use given the extensive tropism of pseudotyped FIV vectors for many cell types in vitro and in vivo.