Base Of Intron Research Articles

Abstract 4280: Improving the sensitivity of wide targeted cancer gene panels via novel genome analysis tools

Abstract In the recent years, wide targeted gene panels have gained popularity, due to their ability to interrogate many genes at the same time. In cancer, particularly, there is a set of ~600 genes known as Cancer Gene Census for which mutations have been causally implicated. Many of the wide cancer panels, therefore, target a subset of these genes. As targeted panels widen, the average coverage of them reduces, if the total budget of the DNA reads remains constant. Therefore, wide panels usually have a more modest coverage, as compared to narrow panels. The reduction in coverage, along with the biases that exist in targeted enrichment approaches make a challenging environment for genome assembly pipelines. We argue that, in such systems, false negatives are prone to increase to non-trivial values, primarily due to: 1) inefficiencies in the enrichment methods, especially if they are hybridization-based; 2) sporadic insufficiency of coverage; 3) biases in the analysis pipelines; 4) filtering operations that take place, in attempt to reduce false positives. As much of the losses are due to the dependency of the assays and the pipelines to the reference genome, we have approached this issue by designing a tandem set of tools: 1) A hybrid-denovo genome assembly pipeline, which has significantly less bias to the reference genome, as compared to the conventional align-to-reference methods. This pipeline can be used in lieu of or in combination with the conventional pipelines; 2) An in-silico verification (ISV) tool that provides the experts with a multifaceted visual and textual information, to gauge the evidence (or lack thereof) for the variants of interest. While not meant to necessarily be a replacement to wet validation, ISV provides the ability to identify the errors that may otherwise seep through the system. In order to show feasibility of this approach, a small set of FFPE TruSight Tumor 170 samples including cases of lung and breast cancer was used. The comparisons were done for all RefSeq exonic bases on a hereditary cancer gene subset. Our preliminary analysis using this data shows that by using our proprietary genome analysis pipeline and ISV tool, the sensitivity of the reported variants could be improved from ~80% to ~98% or higher, without impairing the specificity. If we consider intronic bases which have reasonable coverage, including those involved in splicing, the sensitivity improvements could be even larger. Citation Format: Bahram G. Kermani, Evans L. Roberts, Theresa A. Boyle, Anthony M. Magliocco. Improving the sensitivity of wide targeted cancer gene panels via novel genome analysis tools [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4280.

Cryptic splice activation but not exon skipping is observed in minigene assays of dystrophin c.9361+1G>A mutation identified by NGS

Next-generation sequencing (NGS) discloses nucleotide changes in the genome. Mutations at splicing regulatory elements are expected to cause splicing errors, such as exon skipping, cryptic splice site activation, partial exon loss or intron retention. In dystrophinopathy patients, prediction of splicing outcomes is essential to determine the phenotype: either severe Duchenne or mild Becker muscular dystrophy, based on the reading frame rule. In a Vietnamese patient, NGS identified a c.9361+1G>A mutation in the dystrophin gene and an additional DNA variation of A>G at +117 bases in intron 64. To ascertain the consequences of these DNA changes on dystrophin splicing, minigene constructs were prepared inserting dystrophin exon 64 plus various lengths of intron 64. Exon 64 skipping was observed in the minigene construct with 160 nucleotide (nt) of intron 64 sequence with both c.9361+1A and +117G. In contrast, minigene constructs with larger flanking intronic domains resulted in cryptic splice site activation rather than exon skipping. Meanwhile, the cryptic splice site activation was induced even in +117G when intron 64 was elongated to 272 nt and longer. It was expected that cryptic splice site activation is an in vivo splicing outcome.

The circling mutant Pcdh15roda is a new mouse model for hearing loss

Mouse mutagenesis is a key tool for studying gene function and several mutant alleles have been described and constitute mouse models for human hereditary diseases. Genetic hearing loss represents over 50% of all hearing loss cases in children and, due to the heterogeneity of the disorder, there is still a demand for the isolation and characterization of new genes and alleles.Here we report phenotypic and molecular characterization of a new mouse model for hereditary hearing loss. The mutant rodador, isolated by Massironi and colleagues in 2006, presents an autosomal recessive disorder characterized by deafness and balance dysfunction associated with abnormal stereocilia in the inner ear.The mutation was mapped to mouse chromosome 10, and characterization of the gene Pcdh15 revealed an AT-to-GC transition in intron 23 of mutant animals. The alteration led to the switch of a dinucleotide ApA for ApG, creating a novel intronic acceptor splice site, which leads to incorporation of eight intronic bases into the processed mRNA and alteration of the downstream reading frame. In silico analysis indicated that the mutated protein is truncated and lacks two cadherin domains, and the transmembrane and cytoplasmic domains. Real Time PCR analyses revealed a significantly reduced Pcdh15 mRNA level in the brain of mutant mice, which might be due to the mechanism of non-sense mediated decay.In man, mutations in the orthologue PCDH15 cause non-syndromic deafness and Usher Syndrome Type 1F, a genetic disorder characterized by hearing loss and retinitis pigmentosa. Rodador mouse constitutes a new model for studying deafness in these conditions and may help in the comprehension of the pathogeneses of the disease, as well as of the mechanisms involved in the morphogenesis and function of inner ear stereocilia. This is a new ENU-induced allele and the first isolated in a BALB/c background.

Whole Exome Sequencing Reveals Uncommon Mutations in the Recently Identified Fanconi Anemia GeneSLX4/FANCP

Fanconi anemia (FA) is a rare genetic disorder characterized by congenital malformations, progressive bone marrow failure (BMF), and susceptibility to malignancies. FA is caused by biallelic or hemizygous mutations in one of 15 known FA genes, whose products are involved in the FA/BRCA DNA damage response pathway. Here, we report on a patient with previously unknown mutations of the most recently identified FA gene, SLX4/FANCP. Whole exome sequencing (WES) revealed a nonsense mutation and an unusual splice site mutation resulting in the partial replacement of exonic with intronic bases, thereby removing a nuclear localization signal. Immunoblotting detected no residual SLX4 protein, which was consistent with abrogated interactions with XPF/ERCC1 and MUS81/EME1. This cellular finding did not result in a more severe clinical phenotype than that of previously reported FA-P patients. Our study additionally exemplifies the versatility of WES for the detection of mutations in heterogenic disorders such as FA.

5′ Trans-Splicing Repair of the PLEC1 Gene

The efficient treatment of hereditary disorders, especially of those caused by dominant-negative mutations still remains an obstacle to be overcome. Allele specificity is a critical aspect that must be addressed by silencing therapies such as small interfering RNA, which has the potential risk of also reducing expression of the normal allele. To overcome this hurdle, we used spliceosome-mediated RNA trans-splicing (SMaRT) to replace mRNA exon segments in an in vitro disease model. In this model, a heterozygous insertion of a leucine codon into exon 9 of the plectin gene (PLEC1) leads to aggregation of plectin peptide chains and subsequent protein degradation recapitulating, together with a nonsense mutation on the other allele, the blistering skin disease epidermolysis bullosa simplex with muscular dystrophy (EBS-MD). Transient transfection of EBS-MD fibroblasts with a 5' pre-trans-splicing molecule encoding wild-type exons 2-9 led to specific replacement of the mutated 5' portion of the endogenous PLEC1 transcript through trans-splicing. This treatment reduced the levels of mutant mRNA and restored a wild-type pattern of plectin expression as revealed by immunofluorescence microscopy. When EBS-MD fibroblasts were transfected with retroviral constructs, the level of full-length plectin protein in the corrected fibroblasts increased by 58.7%. Thus, SMaRT may be a promising new tool for treatment of autosomal-dominant genetic diseases.

Mutations in exons 2 and 3 of the FOLR1 gene in demented and non-demented elderly subjects

We have previously reported six novel mutations in the 5'-UTR of the gene for folate receptor-alpha (FOLR1). In our search for additional mutations we screened patients, referred for investigation of suspected dementia (DGM subgroup) by SSCP and DNA sequencing from the end of exon 1 to the first bases of intron 3. We found 4 sequence variations, FOLR1 g.1314G>A, g.1816delC, g.1841G>A, and g.1928C>T. Pyrosequencing genotyping assays were developed for all of them, and 389 active seniors (AS subgroup) and the 202 DGM patients were genotyped for these mutations. The frequency q of the mutated allele was, among the AS subjects, 0.068, 0.0026, 0.0026, and 0.024 respectively, and among the DGM subjects, 0.067, 0.0076, 0.0078, and 0.023. The g.1816delC and g.1841G>A mutations thus were more frequent in the DGM than in the AS subgroup, but the difference did not reach statistical significance. The mutated alleles, FOLR1 1816(-) and 1841A, always occurred together in the same subjects, suggestive of a rare double-mutant haplotype. The two common polymorphisms, FOLR1 g. 1314G>A and g.1928C>T seemed not to raise tHcy plasma levels, whereas the double-mutated g.1816(-)-g.1841A haplotype may possibly have a slight tHcy-raising effect. Thus, so far 8 novel rare FOLR1 mutations with a combined prevalence of approximately 1.3% in Whites as well as two common polymorphisms with 5% and 13%, respectively, have been demonstrated. Only a few of the rare mutations may potentially be associated with raised plasma tHcy concentrations. No association with dementia was found.

Frequency Analysis of the Splice Site Regions in Different Organisms

Summary We have carried out a comparative analysis of the sub-sequences of size six| ten at the (donor| acceptor) splice site regions of five different organisms. The frequency analysis of the unique sub-sequences at the donor and acceptor regions suggests that the distribution of their occurrence is approximately exponential. We have observed that the number of unique sub-sequences (occurring with different frequencies) at the donor region are less than at the acceptor, suggesting that the sub-sequences at the acceptor region are more variable. The sub-sequences with high percentage of occurrence (uniqueness) are considered to be highly involved in splicing. Our analysis suggests that sub-sequences of length ~6-8 nucleotides (nt) at the splice sites – with six bases in intron (including the two central, conserved dinucleotides) and two bases in exon are optimal for the efficient assembly and binding of the spliceosomal complex during the process of splicing. The score pattern obtained by the alignment of the nucleotides at the donor region with the acceptor and vice-versa also suggests that a single sub-sequence at the donor region have different degree of similarity with sub-sequences at the acceptor thus determining that the donor sub-sequences are more crucial in pairing with the corresponding acceptor sub-sequences during the process of splicing.

Cancer gene mutation discovery and detection using array-based resequencing

We set out to determine the feasibility of using microarray-based resequencing for cancer gene mutation screening by designing GeneChip CustomSeq Resequencing arrays (Affymetrix, Santa Clara, CA, USA) for interrogation of ARAF, BRAF, CDK4, CDK6, CDKN2A, KLF6, HRAS, KRAS, MET, NRAS, PTEN, RAF1, RB1, RET and TP53 (164 exons in total). Arrays also included four intronic bases on either side of the exons to cover splice sites, thus the arrays covered a total of 23,966 bases. Overall performance was very good, with accuracy >99.99% and coverage ~97.5%. Twenty NSCLC samples were analyzed using the arrays, and several well-characterized somatic mutations and germline variants were found. The most significant novel finding was the detection of a transforming MET mutation (T1010I) in a NSCLC patient.

A Comparison on Polymorphism of Beta-lactoglobulin Gene in Bos indicus, Bos taurus and Indicine×Taurine Crossbred Cattle

The study was carried out on eight Bos indicus cattle breeds namely, Sahiwal, Tharparkar, Nimari, Khilari, Deoni, Amritmahal, Hariana and Hilly cattle; two Bos taurus cattle breeds namely, Jersey and Holstein Friesian and Indicine×Taurine crossbred cattle to find out the polymorphic pattern of beta-lactoglobulin gene. The polymorphism at beta-lactoglobulin gene was detected by conducting PCR-RFLP studies on 398 bp fragment spanning over 104 bases of exon IV and 294 bases of intron IV. Two alleles A and B and three genotypes AA, AB and BB were observed in all the cattle breeds. The frequency of B allele was comparatively higher than that of A allele. The allelic frequency of A varied from 0.20 to 0.30 in Bos indicus cattle breeds and 0.19 to 0.34 in Bos taurus breeds while in crossbred cattle the frequency was estimated as 0.21. The weighted frequency of A allele was highest in Indian cattle and lowest in crossbred cattle while the frequency in taurine cattle was found to be in between indicus and crossbred cattle. The non-significant differences of allelic frequency amongst Bos indicus, Bos taurus and crossbred cattle was observed. The effect of genotype on fat percentage was also found to be non-significant in cattle. (Asian-Aust. J. Anim. Sci. 2004. Vol 17, No. 6 : 733-736)

The Intertidal Harpacticoid CopepodTigriopus japonicus(Crustacea: Copepoda) β-actin Gene: Cloning, Sequence and Intraspecies Variation

The Tigriopus japonicus beta-actin genes were amplified from genomic DNA by polymerase chain reaction (PCR) and cloned into pCRII vector. Several clones of the T. japonicus beta-actin gene spanned 1662-1676 bp with gains or losses of some bases in intron 3 or 4 but they consisted generally of 5 exons and 4 introns with very high homology, implying polymorphism of this gene. The exon and intron boundaries were matched with the GT/AG rule. The T. japonicus beta-actin gene showed high homology to the fish (Rivulus marmoratus) and human genes, 99.2 and 98.4%, respectively at the amino acid sequence level. The phylogenetic implications inferred from the T. japoncius beta-actin gene are discussed.

Single-nucleotide polymorphisms in the lipoprotein lipase gene associated with coronary heart disease in Chinese

Coronary heart disease is a complex disease reflecting the interaction of multiple genes with the environment (e.g. diet, life style). Lipoprotein lipase (LPL) plays an important role in lipid metabolism and the pathogenesis of coronary atherosclerosis. Recent associations between single-nucleotide polymorphisms in the LPL gene and heart disease have been reported, but little is known in Chinese. The LPL gene spans >26 kb, with an mRNA of 3549 bp. In the present study, we screened 5155 bp (565 bp of 5′ flanking region, nine exons and donor- and acceptor-splice sites, and some intronic bases) in 160 Chinese patients with confirmed coronary heart disease and 150 age- and gender-matched controls. Thirteen of the sixteen single-nucleotide polymorphisms that we found have not been previously reported. In males, significant ( P<0.05) differences between the coronary heart disease patients and controls were found for five single-nucleotide polymorphisms: −421G>A (5′ flanking region); +13,577C>A (intron 2); +16,052G>A, R192Q (exon 5); +16,173C>G and +16,177T>C (intron 5). In females, significant differences between the patients with coronary heart disease and controls were found for only the −421G>A and +16,052G>A (R192Q) mutations. Among the coronary heart disease males, significant ( P<0.05) associations were found between the low-HDL high-triglyceride (LHDL/HTG) phenotype and the non-LHDL/HTG trait for the 5′ flanking-421G, the intron 2+13,577C, and the exon 5+16,052G mutations, with odds–ratios (ORs)[confidence intervals] of 3.90[1.12–13.66], 3.38[1.22–9.40], and 3.22[1.04–10.01], respectively; no corresponding associations were found in females. There were 69, 51, 57 and 41 unphased haplotype patterns in male coronary heart disease, male control, female coronary heart disease and female control groups, respectively; the computer program PM-Plus found the heterogeneity model by far the best fit ( P<0.0001 in males, >0.01 in females). These data show that some single-nucleotide polymorphisms in the LPL gene among Chinese are associated with abnormal lipid and lipoprotein profiles and predisposition to coronary heart disease, a genetically heterogeneous complex disease, and that they are gender-specific.

Large Scale Sequencing and Analysis of AT Rich Eukaryote Genomes

Environmental pressures can direct genomes from a normal to a more or less pronounced imbalance in the base composition. These pressures seem to occur relatively often since genomes with a deviation from a normal base composition are widespread throughout lower eukaryotes. These genomes show altered codon usage and enrichment for the preferred bases in intron and intergenic regions. Techniques designed for large scale sequencing and assembly of genomes with normal base composition will fail with these unusual genomes. Additionally, the currently available analysis tools are mainly suitable for gene finding in genomes with normal base composition. In recent years some large scale genome analysis projects involving species with a skew directed to a very high AT content were initiated. These projects are encountered with sequencing, assembly, and gap closure problems due to the high AT content. These problems can only be overcome with additional techniques, which partly were developed and used in the ongoing projects. In this review some characteristic aspects of AT rich genomes, the progress of the Dictyostelium discoideum and the Plasmodium falciparum projects, as well as techniques specifically used for the sequencing of these genomes are highlighted. ANALYSIS OF GENOMES In recent years many bacteria, some archaea, and some eukaryotes were selected for genome sequencing and analysis projects. In a second step functional genomics then will provide insights into basic cellular processes (1). Economical value or expansion of knowledge was of decisive importance for the selection of distinct species. The analysis of genomes of economically interesting species like crop plants, soy bean, etc. may lead to an increase in food production (2). The knowledge of genomes of pathogens and parasites will probably help to develop novel drugs against defined targets in the pathogeneses. Other species chosen play a key role for the understanding of biological processes and phenomena. The comparison of whole genomes of species from different or consecutive evolutionary stages will elucidate the kind of key genes in evolutionary processes.

Antisense morpholino oligonucleotide analog induces missplicing of C-myc mRNA.

A 28-mer morpholino oligonucleotide analog was designed to hybridize to 8 bases of intron 1 and extend 2 bases beyond the translation initiation codon in exon 2 of the unspliced c-myc RNA transcript. Delivery of this compound into human chronic myeloid leukemia KYO1 cells, by streptolysin O permeabilization, resulted in almost total ablation of the 65 kDa c-MYC protein expression for at least 24 hours after treatment. An unexpected band with SDS-PAGE electrophoretic mobility indicating a protein of about 47 kDa was apparent on the 24-hour western blots that were developed using antibodies that recognize MYC protein C terminal epitopes. No inhibition of the approximately 2400 nt c-myc mRNA expression was observed by northern hybridization, a result of the inability of morpholino analogs to direct the activity of ribonuclease H. In fact, high molecular weight c-myc RNA species were found to have accumulated in antisense-treated KYO1 cells. Control sense and scrambled antisense morpholino analogs did not inhibit MYC protein expression or induce the appearance of the anomalous RNA and protein bands. Molecular analyses by RT-PCR and sequencing revealed that the morpholino antisense effector had (1) inhibited splicing of the c-myc pre-mRNA, (2) induced missplicing of the pre-mRNA, and (3) inhibited translation of normal spliced c-myc mRNA. Identical results were obtained with acute promyelocytic leukemia, acute lymphoblastic leukemia, and histiocytic lymphoma cell lines.

Infrequent loss of heterozygosity and mutation of the p53 gene in immortal and transformed mouse embryo fibroblasts.

Some of the progeny of isolated mouse embryo fibroblasts acquire the ability to grow indefinitely during cultivation, presumably through some mutational events. The relevance of p53 mutations and loss of heterozygosity to the mechanism of such immortal growth capability remains controversial. Since four bases in intron 1 of the p53 gene in C3H/HeJ mice are replaced by 13 different bases in DBA/2J mice, it is possible to distinguish maternal and paternal p53 alleles in the cells of F1 hybrids of these strains (C3D2F1) by electrophoresis of polymerase chain reaction fragments including the region. We established 23 spontaneously immortalized fibroblast cell lines from C3D2F1 mouse embryos and 29 transformed cell lines induced from one of the immortal cell lines, either by treatment with chemical carcinogens or by transfection with the c-Ha-ras gene. Of these 52 cell lines, only one, derived from fibroblasts unpassaged for 4 mo, showed p53 gene loss of heterozygosity and a structural alteration in the remaining allele. Our results demonstrated that p53 mutations are not a strict requirement for immortalization and transformation of mouse embryo fibroblasts in vitro.

A reply: the human proacrosin gene

Vazquez-Levin et al. have published, in this edition of your journal, a paper on the molecular cloning, sequencing and restriction mapping of the genomic sequence encoding human proacrosin [I]. The gene was isolated by using our human proacrosin cDNA probe [2] which we sent to Dr Gordon in 1990. The results obtained by the authors confirm the nucleotide sequence and genomic organization of the proacrosin gene which we published in 1990 [3]. Vazquez-Levin et al. report two differences between their nucleotide sequence and ours, namely in 22 bases of intron 1 and in the localisation of a cytosine residue in exons 3 and 4. We would like to comment on these differences. 1. Vazquez-Levin et al. [l] report in the abstract of their paper that the 500 bases sequenced distal to the initial 54 bases at the 5’ end of intron 1 shows no similarity to our published sequence [3]. Such a conclusion is not justified because we have published only 76 bases of the 5’ end of intron 1. We have obtained this 76-bp sequence by sequencing the 1.2-kb PstIEcoRI genomic fragment containing exon 1, intron 1 and exon 2 from both ends (Fig. 1 in [3]). Therefore, we believe the explanation that an inversion is the reason for the 22-bp difference between our sequence and that of Vazquez-Levin et al. to be unlikely. 2. Vazquez-Levin et al. state that the boundaries of the third intron of the human proacrosin gene differed such that a cytosine residue reported by us in exon 3 [3] was found to be the first base of exon 4. This situation is true in the proacrosin gene of mouse [4], rat [5] and boar (unpublished results), but not in the human proacrosin gene. To exclude possible sequencing errors, we confirmed the results in the human proacrosin gene several times and the cytosine residue was always found in exon 3. Vazquez-Levin et al. [l] report that they have sequenced more than 500 bases of the promoter region of the human

Molecular structure of a naturally occurring alcoholdehydrogenase null activity allele inDrosophilamelanogaster

An alcohol dehydrogenase null activity allele, AdhnAH52, extracted from a natural population of Drosophila melanogaster has been cloned and sequenced. Compared with the wild-type consensus sequence, the nucleotide sequence of AdhnAH52 contains eight extra bases in intron 2, adjacent to the 5' splice site. It seems likely that the extra bases result from two structural changes, with a 10-base pair insertion at the same site as a 2-base pair deletion. The insertion includes an 8-base pair duplication of an adjoining region. This structural change alters transcription to give rise to an mRNA which is longer than normal and at 10% of the wild-type level.