Structural and functional analysis of somatic coding and UTR indels in breast and lung cancer genomes.

Insertions and deletions (Indels) represent one of the major variation types in the human genome and have been implicated in diseases including cancer. To study the features of somatic indels in different cancer genomes, we investigated the indels from two large samples of cancer types: invasive breast carcinoma (BRCA) and lung adenocarcinoma (LUAD). Besides mapping somatic indels in both coding and untranslated regions (UTRs) from the cancer whole exome sequences, we investigated the overlap between these indels and transcription factor binding sites (TFBSs), the key elements for regulation of gene expression that have been found in both coding and non-coding sequences. Compared to the germline indels in healthy genomes, somatic indels contain more coding indels with higher than expected frame-shift (FS) indels in cancer genomes. LUAD has a higher ratio of deletions and higher coding and FS indel rates than BRCA. More importantly, these somatic indels in cancer genomes tend to locate in sequences with important functions, which can affect the core secondary structures of proteins and have a bigger overlap with predicted TFBSs in coding regions than the germline indels. The somatic CDS indels are also enriched in highly conserved nucleotides when compared with germline CDS indels.

RAD51-UTR haplotype genetic polymorphisms and susceptibility to breast cancer in women from Jordanian population.

BACKGROUND: Genetic predisposition to breast cancer (BC) has been extensively explored to achieve an enhanced understanding of the biology of BC. Targeting candidate genes to screen different genetic variants such as RAD51 gene that plays a critical role in DNA repair pathways including the double-strand break repair system is an important task. AIM: To study several single nucleotide polymorphisms (SNPs) within RAD51-UTR gene and to find their relationship with BC risk and prognosis among Jordanian females. MATERIALS AND METHODS: In this case-control study, DNA sequencing technique was used to screen SNPs within the untranslated region (UTR) of RAD51 in 206 cases and 185 controls and the resulting data were statistically analyzed using different types of genetic analyses. Patients' clinical and pathological features were obtained from their medical records to perform genotype-phenotype association analysis. RESULTS: Our findings show a significant association between both SNPs rs528590644, rs1801320 and BC risk (p = 0.016). We estimated the correlation between many of BC prognostic factors and BC risk, and we found an association between rs1801321 and age at first menstruation (p = 0.032) in addition to a strong correlation between age at BC diagnosis and rs1801320 (p = 0.008). CONCLUSION: RAD51-UTR polymorphisms may be involved in BC development and progression.

Targeted sequencing of genes associated with the mismatch repair pathway in patients with endometrial cancer.

Germline variants inactivating the mismatch repair (MMR) genes MLH1, MSH2, MSH6 and PMS2 cause Lynch syndrome that implies an increased cancer risk, where colon and endometrial cancer are the most frequent. Identification of these pathogenic variants is important to identify endometrial cancer patients with inherited increased risk of new cancers, in order to offer them lifesaving surveillance. However, several other genes are also part of the MMR pathway. It is therefore relevant to search for variants in additional genes that may be associated with cancer risk by including all known genes involved in the MMR pathway. Next-generation sequencing was used to screen 22 genes involved in the MMR pathway in constitutional DNA extracted from full blood from 199 unselected endometrial cancer patients. Bioinformatic pipelines were developed for identification and functional annotation of variants, using several different software tools and custom programs. This facilitated identification of 22 exonic, 4 UTR and 9 intronic variants that could be classified according to pathogenicity. This study has identified several germline variants in genes of the MMR pathway that potentially may be associated with an increased risk for cancer, in particular endometrial cancer, and therefore are relevant for further investigation. We have also developed bioinformatics strategies to analyse targeted sequencing data, including low quality data and genomic regions outside of the protein coding exons of the relevant genes.

Pretransplant Genetic Susceptibility: Clinical Relevance in Transplant-Associated Thrombotic Microangiopathy.

Transplant-associated thrombotic microangiopathy (TA-TMA) is a life-threatening complication of allogeneic hematopoietic cell transplantation (HCT). We hypothesized that pretransplant genetic susceptibility is evident in adult TA-TMA and further investigated the association of TMA-associated variants with clinical outcomes. We studied 40 patients with TA-TMA, donors of 18 patients and 40 control non-TMA HCT recipients, without significant differences in transplant characteristics. Genomic DNA from pretransplant peripheral blood was sequenced for TMA-associated genes. Donors presented significantly lower frequency of rare variants and variants in exonic/splicing/untranslated region (UTR) regions, compared with TA-TMA patients. Controls also showed a significantly lower frequency of rare variants in ADAMTS13, CD46, CFH, and CFI. The majority of TA-TMA patients (31/40) presented with pathogenic or likely pathogenic variants. Patients refractory to conventional treatment (62%) and patients that succumbed to transplant-related mortality (65%) were significantly enriched for variants in exonic/splicing/UTR regions. In conclusion, increased incidence of pathogenic, rare and variants in exonic/splicing/UTR regions of TA-TMA patients suggests genetic susceptibility not evident in controls or donors. Notably, variants in exonic/splicing/UTR regions were associated with poor response and survival. Therefore, pretransplant genomic screening may be useful to intensify monitoring and early intervention in patients at high risk for TA-TMA.

The role of noncoding mutations in blood cancers.

The search for oncogenic mutations in haematological malignancies has largely focused on coding sequence variants. These variants have been critical in understanding these complex cancers in greater detail, ultimately leading to better disease monitoring, subtyping and prognostication. In contrast, the search for oncogenic variants in the noncoding genome has proven to be challenging given the vastness of the search space, the intrinsic difficulty in assessing the impact of variants that do not code for functional proteins, and our still primitive understanding of the function harboured by large parts of the noncoding genome. Recent studies have broken ground on this quest, identifying somatically acquired and recurrent mutations in the noncoding genome that activate the expression of proto-oncogenes. In this Review, we explore some of the best-characterised examples of noncoding mutations in haematological malignancies, and highlight how a significant majority of these variants impinge on gene regulation through the formation of aberrant enhancers and promoters. We delve into the challenges faced by those that embark on a search for noncoding driver mutations, and provide a framework distilled from studies that have successfully identified such variants to overcome some of the most salient hurdles. Finally, we discuss the current therapeutic strategies being explored to target the oncogenic mechanism supported by recurrent noncoding variants. We postulate that the continued discovery and functional characterisation of somatic variants in the noncoding genome will not only advance our understanding of haematological malignancies, but offer novel therapeutic avenues and provide important insights into transcriptional regulation on a broader scale.

HLA alleles and haplotypes observed in 263 US families.

The 17th International HLA and Immunogenetics Workshop (IHIW) conducted a project entitled "The Study of Haplotypes in Families by NGS HLA". We investigated the HLA haplotypes of 1017 subjects in 263 nuclear families sourced from five US clinical immunogenetics laboratories, primarily as part of the evaluation of related donor candidates for hematopoietic stem cell and solid organ transplantation. The parents in these families belonged to five broad groups - African (72 parents), Asian (115), European (210), Hispanic (118) and "Other" (11). High-resolution HLA genotypes were generated for each subject using next-generation sequencing (NGS) HLA typing systems. We identified the HLA haplotypes in each family using HaplObserve, software that builds haplotypes in families by reviewing HLA allele segregation from parents to children. We calculated haplotype frequencies within each broad group, by treating the parents in each family as unrelated individuals. We also calculated standard measures of global linkage disequilibrium (LD) and conditional asymmetric LD for each ethnic group, and used untruncated and two-field allele names to investigate LD patterns. Finally we demonstrated the utility of consensus DNA sequences in identifying novel variants, confirming them using HLA allele segregation at the DNA sequence level.

Sequences enriched in Alu repeats drive nuclear localization of long RNAs in human cells.

Long noncoding RNAs (lncRNAs) are emerging as key parts of multiple cellular pathways, but their modes of action and how these are dictated by sequence remain unclear. lncRNAs tend to be enriched in the nuclear fraction, whereas most mRNAs are overtly cytoplasmic, although several studies have found that hundreds of mRNAs in various cell types are retained in the nucleus. It is thus conceivable that some mechanisms that promote nuclear enrichment are shared between lncRNAs and mRNAs. Here, to identify elements in lncRNAs and mRNAs that can force nuclear localization, we screened libraries of short fragments tiled across nuclear RNAs, which were cloned into the untranslated regions of an efficiently exported mRNA. The screen identified a short sequence derived from Alu elements and bound by HNRNPK that increased nuclear accumulation. Binding of HNRNPK to C-rich motifs outside Alu elements is also associated with nuclear enrichment in both lncRNAs and mRNAs, and this mechanism is conserved across species. Our results thus identify a pathway for regulation of RNA accumulation and subcellular localization that has been co-opted to regulate the fate of transcripts with integrated Alu elements.

UTR-Dependent Control of Gene Expression in Plants.

Throughout their lives, plants sense many developmental and environmental stimuli, and activation of optimal responses against these stimuli requires extensive transcriptional reprogramming. To facilitate this activation, plant mRNA contains untranslated regions (UTRs) that significantly increase the coding capacity of the genome by producing multiple mRNA variants from the same gene. In this review we compare UTRs of arabidopsis (Arabidopsis thaliana) and rice (Oryza sativum) at the genome scale to highlight their complexity in crop plants. We discuss different modes of UTR-based regulation with emphasis on genes that regulate multiple plant processes, including flowering, stress responses, and nutrient homeostasis. We demonstrate functional specificity in genes with variable UTR length and propose future research directions.

Assessment of the functional impact of germline BRCA1/2 variants located in non-coding regions in families with breast and/or ovarian cancer predisposition.

PURPOSE: The molecular mechanism of breast and/or ovarian cancer susceptibility remains unclear in the majority of patients. While germline mutations in the regulatory non-coding regions of BRCA1 and BRCA2 genes have been described, screening has generally been limited to coding regions. The aim of this study was to evaluate the contribution of BRCA1/2 non-coding variants. METHODS: Four BRCA1/2 non-coding regions were screened using high-resolution melting analysis/Sanger sequencing or next-generation sequencing on DNA extracted from index cases with breast and ovarian cancer predisposition (3926 for BRCA1 and 3910 for BRCA2). The impact of a set of variants on BRCA1/2 gene regulation was evaluated by site-directed mutagenesis, transfection, followed by Luciferase gene reporter assay. RESULTS: We identified a total of 117 variants and tested twelve BRCA1 and 8 BRCA2 variants mapping to promoter and intronic regions. We highlighted two neighboring BRCA1 promoter variants (c.-130del; c.-125C > T) and one BRCA2 promoter variants (c.-296C > T) inhibiting significantly the promoter activity. In the functional assays, a regulating region within the intron 12 was found with the same enhancing impact as within the intron 2. Furthermore, the variants c.81-3980A > G and c.4186-2022C > T suppress the positive effect of the introns 2 and 12, respectively, on the BRCA1 promoter activity. We also found some variants inducing the promoter activities. CONCLUSION: In this study, we highlighted some variants among many, modulating negatively the promoter activity of BRCA1 or 2 and thus having a potential impact on the risk of developing cancer. This selection makes it possible to conduct future validation studies on a limited number of variants.

ADAR1-mediated RNA-editing of 3'UTRs in breast cancer

BACKGROUND: Whole transcriptome RNA variant analyses have shown that adenosine deaminases acting on RNA ( ADAR ) enzymes modify a large proportion of cellular RNAs, contributing to transcriptome diversity and cancer evolution. Despite the advances in the understanding of ADAR function in breast cancer, ADAR RNA editing functional consequences are not fully addressed. RESULTS: We characterized A to G(I) mRNA editing in 81 breast cell lines, showing increased editing at 3'UTR and exonic regions in breast cancer cells compared to immortalized non-malignant cell lines. In addition, tumors from the BRCA TCGA cohort show a 24% increase in editing over normal breast samples when looking at 571 well-characterized UTRs targeted by ADAR1. Basal-like subtype breast cancer patients with high level of ADAR1 mRNA expression shows a worse clinical outcome and increased editing in their 3'UTRs. Interestingly, editing was particularly increased in the 3'UTRs of ATM, GINS4 and POLH transcripts in tumors, which correlated with their mRNA expression. We confirmed the role of ADAR1 in this regulation using a shRNA in a breast cancer cell line (ZR-75-1). CONCLUSIONS: Altogether, these results revealed a significant association between the mRNA editing in genes related to cancer-relevant pathways and clinical outcomes, suggesting an important role of ADAR1 expression and function in breast cancer.

miR-483-5p promotes prostate cancer cell proliferation and invasion by targeting RBM5

ABSTRACT Objective: miR-483-5p has been identified as a miRNA oncogene in certain cancers. However, its role in prostate cancer has not been sufficiently investigated. In this study, we investigated the role of miR-483-5p in prostate cancer and examined RBM5 regulation by miR-483-5p. Material and methods: Expression levels of miR-483-5p were determined by quantitative real-time PCR. The effect of miR-483-5p on proliferation was evaluated by MTT assay, cell invasion was evaluated by trans-well invasion assays, and target protein expression was determined by western blotting in LNCaP, DU-145, and PC-3 cells. Luciferase reporter plasmids were constructed to confirm the action of miR-483-5p on downstream target gene RBM5 in HEK-293T cells. Results: we observed that miR-483-5p was upregulated in prostate cancer cell lines and tissues. A miR-483-5p inhibitor inhibited prostate cancer cell growth and invasion in DU-145 and PC-3 cells. miR-483-5p directly bound to the 3' untranslated region (3'UTR) of RBM5 in HEK-293T cells. RBM5 overexpression inhibited prostate cancer cell growth and invasion in LNCaP cells. Enforced RBM5 expression alleviated miR-483-5p promotion of prostate cancer cell growth and invasion in LNCaP cells. Conclusion: The present study describes a potential mechanism underlying a miR-483-5p/RBM5 link that contributes to prostate cancer development.

Dual function of a cis-acting RNA element that acts as a replication enhancer and a translation repressor in a plant positive-stranded RNA virus.

The genome of red clover necrotic mosaic virus is divided into two positive-stranded RNA molecules of RNA1 and RNA2, which have no 5' cap structure and no 3' poly(A) tail. Previously, we showed that any mutations in the cis-acting RNA replication elements of RNA2 abolished its cap-independent translational activity, suggesting a strong link between RNA replication and translation. Here, we investigated the functions of the 5' untranslated region (UTR) of RNA2 and revealed that the basal stem-structure (5'BS) predicted in the 5' UTR is essential for robust RNA replication. Interestingly, RNA2 mutants with substitution or deletion in the right side of the 5'BS showed strong translational activity, despite their impaired replication competency. Furthermore, nucleotide sequences other than the 5'BS of the 5' UTR were essential to facilitate the replication-associated translation. Overall, these cis-acting RNA elements seem to coordinately regulate the balance between RNA replication and replication-associated translation.

Rare Inherited and De Novo CNVs Reveal Complex Contributions to ASD Risk in Multiplex Families.

Rare mutations, including copy-number variants (CNVs), contribute significantly to autism spectrum disorder (ASD) risk. Although their importance has been established in families with only one affected child (simplex families), the contribution of both de novo and inherited CNVs to ASD in families with multiple affected individuals (multiplex families) is less well understood. We analyzed 1,532 families from the Autism Genetic Resource Exchange (AGRE) to assess the impact of de novo and rare CNVs on ASD risk in multiplex families. We observed a higher burden of large, rare CNVs, including inherited events, in individuals with ASD than in their unaffected siblings (odds ratio [OR] = 1.7), but the rate of de novo events was significantly lower than in simplex families. In previously characterized ASD risk loci, we identified 49 CNVs, comprising 24 inherited events, 19 de novo events, and 6 events of unknown inheritance, a significant enrichment in affected versus control individuals (OR = 3.3). In 21 of the 30 families (71%) in whom at least one affected sibling harbored an established ASD major risk CNV, including five families harboring inherited CNVs, the CNV was not shared by all affected siblings, indicating that other risk factors are contributing. We also identified a rare risk locus for ASD and language delay at chromosomal region 2q24 (implicating NR4A2) and another lower-penetrance locus involving inherited deletions and duplications of WWOX. The genetic architecture in multiplex families differs from that in simplex families and is complex, warranting more complete genetic characterization of larger multiplex ASD cohorts.

A unified analytic framework for prioritization of non-coding variants of uncertain significance in heritable breast and ovarian cancer.

BACKGROUND: Sequencing of both healthy and disease singletons yields many novel and low frequency variants of uncertain significance (VUS). Complete gene and genome sequencing by next generation sequencing (NGS) significantly increases the number of VUS detected. While prior studies have emphasized protein coding variants, non-coding sequence variants have also been proven to significantly contribute to high penetrance disorders, such as hereditary breast and ovarian cancer (HBOC). We present a strategy for analyzing different functional classes of non-coding variants based on information theory (IT) and prioritizing patients with large intragenic deletions. METHODS: We captured and enriched for coding and non-coding variants in genes known to harbor mutations that increase HBOC risk. Custom oligonucleotide baits spanning the complete coding, non-coding, and intergenic regions 10 kb up- and downstream of ATM, BRCA1, BRCA2, CDH1, CHEK2, PALB2, and TP53 were synthesized for solution hybridization enrichment. Unique and divergent repetitive sequences were sequenced in 102 high-risk, anonymized patients without identified mutations in BRCA1/2. Aside from protein coding and copy number changes, IT-based sequence analysis was used to identify and prioritize pathogenic non-coding variants that occurred within sequence elements predicted to be recognized by proteins or protein complexes involved in mRNA splicing, transcription, and untranslated region (UTR) binding and structure. This approach was supplemented by in silico and laboratory analysis of UTR structure. RESULTS: 15,311 unique variants were identified, of which 245 occurred in coding regions. With the unified IT-framework, 132 variants were identified and 87 functionally significant VUS were further prioritized. An intragenic 32.1 kb interval in BRCA2 that was likely hemizygous was detected in one patient. We also identified 4 stop-gain variants and 3 reading-frame altering exonic insertions/deletions (indels). CONCLUSIONS: We have presented a strategy for complete gene sequence analysis followed by a unified framework for interpreting non-coding variants that may affect gene expression. This approach distills large numbers of variants detected by NGS to a limited set of variants prioritized as potential deleterious changes.

DNA methylation of tumor suppressor protein-coding and non-coding genes in multiple myeloma.

Multiple myeloma is an incurable hematological malignancy arising from immortalized plasma cells in the bone marrow. DNA methylation refers to the catalytic addition of a methyl group to the cytosine ring of a CpG dinucleotide. Methylation of a promoter-associated CpG island, a cluster of CpG dinucleotides, may lead to silencing of the associated gene. In carcinogenesis, methylation of protein-coding or non-coding tumor suppressor genes/miRNAs is associated with transcriptional silencing, loss of tumor suppressor function and prognostic significance. This review first introduces pathogenesis of myeloma and DNA methylation in cancer. Then, it summarizes methylation of protein-coding tumor suppressor genes, especially, the latest genome-wide methylation studies in myeloma, followed by the latest findings of methylation of non-coding tumor suppressor miRNAs in myeloma.

Efficient expression of stabilized mRNA PEG-peptide polyplexes in liver.

The expression efficiency in liver following hydrodynamic delivery of in vitro transcribed mRNA was improved 2000-fold using a codon-optimized mRNA luciferase construct with flanking 3' and 5' human ß-globin untranslated regions (UTR mRNA) over an unoptimized mRNA without ß-globin UTRs. Nanoparticle UTR mRNA polyplexes were formed using a novel polyacridine polyethylene glycol (PEG) peptide, resulting in an additional 15-fold increase in expression efficiency in the liver. The combined increase in expression for UTR mRNA PEG-peptide polyplexes was 3500-fold over mRNA lacking UTRs and PEG-peptide. The expression efficiency of UTR mRNA polyplex was 10-fold greater than the expression from an equivalent 1 µg dose of pGL3. Maximal expression was maintained from 4 to 24 h. Serum incubation established the unique ability of the polyacridine PEG-peptide to protect UTR mRNA polyplexes from RNase metabolism by binding to double-stranded regions. UTR mRNA PEG-peptide polyplexes are efficient nonviral vectors that circumvent the need for a nuclear uptake, representing an advancement toward the development of a targeted gene delivery system to transfect liver hepatocytes.

Molecular Polymorphism and Divergence of Duplicated Genes in Tetraploid African Clawed Frogs (Xenopus).

Genome duplication creates redundancy in proteins and their interaction networks, and subsequent smaller-scale gene duplication can further amplify genetic redundancy. Mutations then lead to the loss, maintenance or functional divergence of duplicated genes. Genome duplication occurred many times in African clawed frogs (genus Xenopus), and almost all extant species in this group evolved from a polyploid ancestor. To better understand the nature of selective constraints in a polyploid genome, we examined molecular polymorphism and divergence of duplicates and single-copy genes in 2 tetraploid African clawed frog species, Xenopus laevis and X. victorianus. We found that molecular polymorphism in the coding regions of putative duplicated genes was higher than in singletons, but not significantly so. Our findings also suggest that transcriptome evolution in polyploids is influenced by variation in the genome-wide mutation rate, and do not reject the hypothesis that gene dosage balance is also important.

A screen of a large Czech cohort of oligodontia patients implicates a novel mutation in the PAX9 gene.

Tooth agenesis is one of the most common developmental anomalies in humans. To date, many mutations involving paired box 9 (PAX9), msh homeobox 1 (MSX1), and axin 2 (AXIN2) genes have been identified. The aim of the present study was to perform screening for mutations and/or polymorphisms using the capillary sequencing method in the critical regions of PAX9 and MSX1 genes in a group of 270 individuals with tooth agenesis and in 30 healthy subjects of Czech origin. This screening revealed a previously unknown heterozygous g.9527G>T mutation in the PAX9 gene in monozygotic twins with oligodontia and three additional affected family members. The same variant was not found in healthy relatives. This mutation is located in intron 2, in the region recognized as the splice site between exon 2 and intron 2. We hypothesize that the error in pre-mRNA splicing may lead to lower expression of PAX9 protein and could have contributed to the development of tooth agenesis in the affected subjects.

Identification of novel GRM1 mutations and single nucleotide polymorphisms in prostate cancer cell lines and tissues.

Metabotropic glutamate receptor 1 (GRM1) signaling has been implicated in benign and malignant disorders including prostate cancer (PCa). To further explore the role of genetic alterations of GRM1 in PCa, we screened the entire human GRM1 gene including coding sequence, exon-intron junctions, and flanking untranslated regions (UTRs) for the presence of mutations and single nucleotide polymorphisms (SNPs) in several PCa cell lines and matched tumor-normal tissues from Caucasian Americans (CAs) and African Americans (AAs). We used bidirectional sequencing, allele-specific PCR, and bioinformatics to identify the genetic changes in GRM1 and to predict their functional role. A novel missense mutation identified at C1744T (582 Pro > Ser) position of GRM1 gene in a primary AA-PCa cell line (E006AA) was predicted to affect the protein stability and functions. Another novel mutation identified at exon-intron junction of exon-8 in C4-2B cell line resulted in alteration of the GRM1 splicing donor site. In addition, we found missense SNP at T2977C (993 Ser > Pro) position and multiple non-coding mutations and SNPs in 3'-UTR of GRM1 gene in PCa cell lines and tissues. These novel mutations may contribute to the disease by alterations in GRM1 gene splicing, receptor activation, and post-receptor downstream signaling.