Intragenic Position Research Articles

Multi-landmark alignment of genomic signals reveals conserved expression patterns across transcription start sites

The prevalent one-dimensional alignment of genomic signals to a reference landmark is a cornerstone of current methods to study transcription and its DNA-dependent processes but it is prone to mask potential relations among multiple DNA elements. We developed a systematic approach to align genomic signals to multiple locations simultaneously by expanding the dimensionality of the genomic-coordinate space. We analyzed transcription in human and uncovered a complex dependence on the relative position of neighboring transcription start sites (TSSs) that is consistently conserved among cell types. The dependence ranges from enhancement to suppression of transcription depending on the relative distances to the TSSs, their intragenic position, and the transcriptional activity of the gene. Our results reveal a conserved hierarchy of alternative TSS usage within a previously unrecognized level of genomic organization and provide a general methodology to analyze complex functional relationships among multiple types of DNA elements.

Abstract 2211: The landscape and implications of kinase-intergenic fusions in a large real-world lung cancer cohort

Abstract Background: Kinase gene fusions are strong driver mutations in neoplasia and have provided fundamental insights into the disease mechanisms that are involved in tumorigenesis. Into the era of next generation sequencing (NGS), numerous studies have described fusion events with breakpoints occurring at two intragenic positions that result in in-frame gene-gene fusions, but fusions that occur with intergenic regions are not uncommon. The clinical significance of such fusions needs to be carefully examined. Methods: We retrospectively reviewed the mutation profiles of over 30 000 lung cancer patients whose tissue and/or plasma samples underwent targeted NGS from June 2016 to July 2019. Clinical data of patients who had kinase-intergenic fusions were retrieved for analysis. RNA sequencing and immunohistochemical (IHC) assays were performed to verify chimeric fusion products on the transcriptional or protein level. Results: A total of 624 kinase-intergenic fusion events were identified in 538 unique lung cancer patients. Approximately 75% (404/538) of the entire cohort were non-small cell lung cancer (NSCLC), and 93% (375/404) of NSCLC were adenocarcinoma. The most frequently identified kinase genes included ALK, RET, ROS1, ERBB2/3, EGFR, FGFR1/2/3, and NTRK1/2/3. Our data showed that most (67%) kinase-intergenic gene rearrangements occurred on the same chromosome and kinase domains remained intact at 3' end. About 3% (19/624) of the kinase-intergenic fusions had one genomic breakpoint located in promoter regions, including 9 events involving ALK, RET, ROS1, EGFR, ERBB2, and FGFR3, which were fused to different gene promoters that may result in changes in kinase gene transcription. Importantly, 371 patients (69%) had concurrent oncogenic driver mutations or intragenic fusions, but the remaining 167 patients (31%) had kinase-intergenic fusions detected solely without other known oncogenic aberrations. Noteworthy, three patients who had ALK-intergenic fusions, but no other known oncogenic aberrations, were confirmed of EML4-ALK chimeric fusion product by RNA sequencing and/or ALK IHC results. Among them, one patient achieved a durable response of 14-month on crizotinib. An additional two patients who had ROS1 intergenic fusions demonstrated clinical benefit of 11 and 19 months from crizotinib, respectively, but the underlying functional fusion products remained to be further determined. Conclusion: This study investigated the role of kinase-intergenic fusions on a large-scale lung cancer population and revealed the potential oncogenic function of kinase-intergenic fusions. Our data also suggested that in addition to DNA sequencing, additional validation testing using RNA and/or protein assay should be performed in intergenic-breakpoint fusion cases to guide optimal treatment. Citation Format: Yutong Ma, Qiuxiang Ou, Xue Wu, Yang Shao. The landscape and implications of kinase-intergenic fusions in a large real-world lung cancer cohort [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2211.

Dysregulation of cancer genes by recurrent intergenic fusions

BackgroundGene fusions have been studied extensively, as frequent drivers of tumorigenesis as well as potential therapeutic targets. In many well-known cases, breakpoints occur at two intragenic positions, leading to in-frame gene-gene fusions that generate chimeric mRNAs. However, fusions often occur with intergenic breakpoints, and the role of such fusions has not been carefully examined.ResultsWe analyze whole-genome sequencing data from 268 patients to catalog gene-intergenic and intergenic-intergenic fusions and characterize their impact. First, we discover that, in contrast to the common assumption, chimeric oncogenic transcripts—such as those involving ETV4, ERG, RSPO3, and PIK3CA—can be generated by gene-intergenic fusions through splicing of the intervening region. Second, we find that over-expression of an upstream or downstream gene by a fusion-mediated repositioning of a regulatory sequence is much more common than previously suspected, with enhancers sometimes located megabases away. We detect a number of recurrent fusions, such as those involving ANO3, RGS9, FUT5, CHI3L1, OR1D4, and LIPG in breast; IGF2 in colon; ETV1 in prostate; and IGF2BP3 and SIX2 in thyroid cancers.ConclusionOur findings elucidate the potential oncogenic function of intergenic fusions and highlight the wide-ranging consequences of structural rearrangements in cancer genomes.

Tandem inversion duplication within F8 Intron 1 associated with mild haemophilia A

In approximately 90% of mild haemophilia A (HA) patients, a missense mutation can be identified using complete gene sequencing. In this study, multiplex ligation-dependent probe amplification analysis was performed as a second step in 10 French-speaking Belgian with mild HA presenting no detectable causal mutation by complete sequencing of the factor VIII (FVIII) (F8) gene's 26 exons and its 1.2kb of contiguous promoter sequence. This gene dosage technique enabled the detection of exon 1 duplications of F8 in three apparently unrelated subjects. Using array-comparative genomic hybridization, breakpoint analysis delimited the duplication extent to 210kb in the F8 intron 1 and VBP1 gene intragenic position. We postulated that the rearrangement responsible for this duplication, never before reported, could be attributed to a symmetrical tandem inversion duplication, resulting in a large 233kb rearrangement of F8 intron 1. This rearranged intron should lead to the production of a small number of normal mRNA transcripts in relation to the mild HA phenotype. Our analysis of the entire F8 mRNA from index case 1, particularly the segment containing exons 1-9, revealed normal amplification and sequencing. Reduced plasma FVIII antigen levels caused by cross-reacting material is associated with a quantitative deficiency of plasma FVIII. Male patients were unresponsive to desmopressin (1-deamino-8-D-arginine vasopressin). All patients displayed identical F8 haplotypes, despite not being related, which suggests a possible founder effect caused by a 210kb duplication involving F8 exon 1.

High‐resolution analysis of DNaseI‐hypersensitive sites in a muscular dystrophy‐linked subtelomeric region of 4q

DNaseI hypersensitive (DH) sites are associated with nucleosome‐free chromatin subregions often in promoters, enhancers, and locus control regions. We used DNase‐chip to examine DH sites in two regions of special interest to facioscapulohumeral muscular dystrophy (FSHD). This dominant disease remains enigmatic even after five expression microarray studies. Custom tiling arrays for this study represented 4q35.2, including D4Z4 arrays of tandem 3.3‐kb repeats whose contraction is linked to FSHD, and 10q26.3 with its almost identical, but phenotypically neutral, D4Z4 arrays. In the center of the 4‐Mb 4q35.2, there was a low density of DH sites, consistent with its low density of known genes. Some 4q35.2 or 10q26.3 genes in three FSHD and three control myoblast cell populations had DH sites just in 5'gene regions (e.g., FRG1), and others had DH sites at many intragenic positions (e.g., FAT and INPP5A). DH sites were observed in sequences corresponding to hypothetical proteins (C10orf92 and C10orf93) and in subregions far from known genes. The 4q‐specific nature of FSHD is likely to be due to differences between the chromatin landscape of 4q35.2 and 10q26.3 that make only 4q D4Z4 contractions pathogenic. Our data illustrate the large‐scale differences in chromatin in these subtelomeric regions and suggest where to look for FSHD‐relevant long‐distance chromatin interactions. (Supported in part by NIH Grant NS048859)

Combining Models of Protein Translation and Population Genetics to Predict Protein Production Rates from Codon Usage Patterns

Genes are often biased in their codon usage. The degree of bias displayed often changes with expression level and intragenic position. Numerous indices, such as the codon adaptation index, have been developed to measure this bias. Although the expression level of a gene and index values are correlated, the heuristic nature of these metrics limits their ability to explain this relationship. As an alternative approach, this study integrates mechanistic models of cellular and population processes in a nested manner to develop a stochastic evolutionary model of a protein's production rate (SEMPPR). SEMPPR assumes that the evolution of codon bias is driven by selection to reduce the cost of nonsense errors and that this selection is counteracted by mutation and drift. Through the application of Bayes' theorem, SEMPPR generates a posterior probability distribution for the protein production rate of a given gene. Conceptually, SEMPPR's predictions are based on the degree of adaptation to reduce the cost of nonsense errors observed in the codon usage pattern of the gene. As an illustration, SEMPPR was parameterized using the Saccharomyces cerevisiae genome and its predictions tested using available empirical data. The results indicate that SEMPPR's predictions are as reliable index based ones. In addition, SEMPPR's output is more easily interpreted and its predictions could be improved through refinements of the models upon which it is built.

CDKN2A Germline Mutations in Individuals with Cutaneous Malignant Melanoma

Cyclin-dependent kinase inhibitor type 2A (CDKN2A) has been identified as a major melanoma susceptibility gene based on the presence of germline mutations in high-risk melanoma families. In this study, we sought to identify and characterize the spectrum of CDKN2A mutations affecting p16 inhibitor of cyclin-dependent kinase type 4 (INK4a) in individuals with melanoma using a population-based study design. DNA samples from 1189 individuals with incident multiple primary melanoma (MPM) and 2424 with incident single primary melanoma unselected for family history of melanoma were available for screening of CDKN2A (p16INK4a) mutations. Variants were classified for functional impact based on intragenic position, existing functional data, sequence, and structural analysis. The impact of individual mutations and functional groupings was assessed by comparing frequencies in cases of MPM versus cases with a single first primary melanoma, and by comparing the reported incidence rates in first-degree relatives. Our results show that mutations occur infrequently in these high-risk groups, and that they occur mainly in exons 1alpha and 2. Rare coding variants with putative functional impact are observed to increase substantially the risk of melanoma. With the exception of the variant in position -34 of CDKN2A of known functional consequence, the remaining rare variants in the non-coding region have no apparent impact on risk.

Distribution of Menin-Occupied Regions in Chromatin Specifies a Broad Role of Menin in Transcriptional Regulation

Menin is the protein product of the MEN1 tumorsuppressor gene; one allele of MEN1 is inactivated in the germ line of patients with “multiple endocrine neoplasia type 1” (MEN1) cancer syndrome. Menin interacts with several proteins involved in transcriptional regulation. RNA expression analyses have identified several menin-regulated genes that could represent proximal or distal interaction sites for menin. This report presents a substantial and unbiased sampling of menin-occupied chromatin regions using Serial Analysis of Chromatin Occupancy; this method combines chromatin immunoprecipitation with Serial Analysis of Gene Expression. Hundreds of menin-occupied genomic sites were identified in promoter regions (32% of menin-occupied loci), near the 3' end of genes (14%), or inside genes (21%), extending other data about menin recruitments to many sites of transcriptional activity. A large number of meninoccupied sites (33%) were located outside known gene regions. Additional annotation of the human genome could help in identifying genes at these loci, or these might be gene-free regions of the genome where menin occupancy could play some structural or regulatory role. Menin occupancy at many intragenic positions distant from the core promoter reveals an unexpected type of menin target region at many loci in the genome. These unbiased data also suggest that menin could play a broad role in transcriptional regulation.

The relationship between palindrome avoidance and intragenic codon usage variations: a Monte Carlo study

Several studies have shown that codon usage within genes varies, as it seems dependent on both codon context and codon position within the gene. Given that palindromes in addition often are avoided in genomes, this study aimed at finding out if intragenic variations in codon usage may be a way to control the amount and location of palindromes. A Monte Carlo algorithm was written which resampled the codons in genes while keeping the amino acid sequence of the translation product constant. On the resampled sequences, palindromes were counted and their intragenic positions mapped. Escherichia coli K12 uses type II restriction–modification systems and displays pronounced codon usage phenomena. Using this as a reference organism it was clearly shown that the number of palindromes in genes is generally lower than the amount of palindromes in resampled genes; thus, the succession of codons seems to be a way to decrease the number of palindromes. The intragenic position of palindromes in resampled sequences, however, was largely equal to the position in the native genes, so codon usage phenomena are unlikely to be a way to control the intragenic position of palindromes. The analysis was repeated on two bacteriophages and gave similar same results, even though the virus genomes are much smaller. Studies on the endosymbionts Buchnera sp. APS and Wigglesworthia sp., which seemingly have no type II restriction–modification systems, showed that in these species there is only weak evidence for codon usage acting to control the number of palindromes.

Expression, alternative splicing and haplotype analysis of transcribed testis specific protein (TSPY) genes

Testis specific protein (TSPY) is a human Y-chromosome derived gene with numerous functional and non-functional copies. Specific expression patterns in testis and testicular tumors, as in prostate cancer samples and cell lines led to the postulation of a potential role in cell proliferation, supported by the presence of a suppressor of variegation, enhancer of zeste and Trithorax/nucleosome assembling protein (nucleosome assembly protein) domain in the mature protein. Expression studies have now identified two transcripts of variable length, termed TSPY-S and -L, which differ in their 3′-translated region due to alternative splicing, and in the quantitative level of transcripts, with TSPY-S being at least 3–4-fold more abundant. In immunoblot experiments on human testis and LNCaP protein extracts using an anti-peptide-antiserum against the TSPY-L specific C-terminus TSPY-L was characterized as a functional variant on the protein level. As there are at least three intragenic positions differing between various TSPY genes and thus defining certain haplotypes, the alternatively spliced TSPY transcripts were analysed for their haplotypes in order to link them to well defined TSPY loci. Surprisingly, no evidence of a G-G-18 haplotype was found for the TSPY-L transcript, while this haplotype makes up almost 50% of all TSPY-S transcripts. This excludes the corresponding TSPY-1 locus from alternative splicing. The only significant differences between the TSPY-1 locus and eight other loci were identified in the promotor region as revealed by detailed sequence comparisons. Thus one might speculate that the alternative splicing could be influenced by elements binding to the promotor region.

Analysis of flanking sequences from dissociation insertion lines: a database for reverse genetics in Arabidopsis.

We have generated Dissociation (Ds) element insertions throughout the Arabidopsis genome as a means of random mutagenesis. Here, we present the molecular analysis of genomic sequences that flank the Ds insertions of 931 independent transposant lines. Flanking sequences from 511 lines proved to be identical or homologous to DNA or protein sequences in public databases, and disruptions within known or putative genes were indicated for 354 lines. Because a significant portion (45%) of the insertions occurred within sequences defined by GenBank BAC and P1 clones, we were able to assess the distribution of Ds insertions throughout the genome. We discovered a significant preference for Ds transposition to the regions adjacent to nucleolus organizer regions on chromosomes 2 and 4. Otherwise, the mapped insertions appeared to be evenly dispersed throughout the genome. For any given gene, insertions preferentially occurred at the 5' end, although disruption was clearly possible at any intragenic position. The insertion sites of >500 lines that could be characterized by reference to public databases are presented in a tabular format at http://www.plantcell. org/cgi/content/full/11/12/2263/DC1. This database should be of value to researchers using reverse genetics approaches to determine gene function.

Codon usage and intragenic position

Data on codon usage bias in E. coli are re-examined with respect to intragenic position. The bias is less extreme near the beginning than in the rest of the gene, particularly in highly expressed genes. This is contrary to the previous finding that there is a linear decline in codon usage bias with position along weakly expressed genes but little or no change in bias along highly expressed genes. The effect is not confined to genes coding for proteins with leader peptides, as suggested earlier (Burns and Beacham, 1985). There is some evidence of a similar but smaller effect in yeast.

Translation rate modification by preferential codon usage: Intragenic position effects

We present a model for calculating the protein production rate as a function of the translation rate. The model takes into account that the elongation rate along an mRNA molecule is non-uniform as a result of different tRNA availabilities for different codons. Initiation of ribosomes on an mRNA is normally the rate-limiting step in the translation process, and blocking of the initiation site can be avoided if the codons closest to this site allow fast translation by the ribosome. Hence, different selective forces may act on the choice of synonymous codons in the initiation region than elsewhere on a given mRNA. We show that the elongation rate along the whole mRNA influences the production rate of abundant proteins, whereas only the elongation rate in the initiation region is of importance for the production rate of rare proteins. We also present an analysis of the codon distribution along known mRNAs coding for abundant and rare proteins.

Characterization and intragenic position of mutations in the gene for galactoside permease of Escherichia coli.

To test certain models for the structure of the gene for galactoside permease and its product, appropriate mutants of E. coli were isolated after mutagenic treatment, partially characterized and mapped. The 69 mutants found to be in the permease gene were mapped by crosses with episomal deletions which divided the gene into 13 regions.

Classification and intragenic position of mutations in the beta-galactosidase gene of Escherichia coli.

Following treatment with N-methyl-N′-nitro-N-nitrosoguanidine, 1,257 mutants of Escherichia coli K12 were isolated on lactose-tetrazolium medium. Of these mutants, 345 were lactose-negative and lacked appreciable β-galactosidase activity. About half of these enzyme-deficient mutants had lost the whole lactose operon; the remainder (174) were point mutations within the β-galactosidase gene. With the exception of 42 which could not be classified, the mutations were identified either as chain-terminating (UAG 57, UAA 6, UGA 60) or missense (9). There were no mutations of the reading frame and no short deletions. The unclassifiable mutants do not form crossreacting protein and are probably a type of chain-terminating mutant.