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1
Ouyang, Hongjia, Jiao Yu, Xiaolan Chen, Zhijun Wang, and Qinghua Nie. "A novel transcript of MEF2D promotes myoblast differentiation and its variations associated with growth traits in chicken." PeerJ 8 (February 4, 2020): e8351. http://dx.doi.org/10.7717/peerj.8351.
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Background Development of skeletal muscle is closely related to broiler production traits. The myocyte-specific enhancer binding factor (MEF) 2D gene (MEF2D) and its variant transcripts play important parts in myogenesis. Methods To identify the transcript variants of chicken MEF2D gene and their function, this study cloned chicken MEF2D gene and identified its transcript variants from different tissue samples. The expression levels of different transcripts of MEF2D gene in different tissues and different periods were measured, and their effects on myoblast proliferation and differentiation were investigated. Variations in MEF2D were identified and association analysis with chicken production traits carried out. Results Four novel transcript variants of MEF2D were obtained, all of which contained highly conserved sequences, including MADS-Box and MEF2-Domain functional regions. Transcript MEF2D-V4 was expressed specifically in muscle, and its expression was increased during embryonic muscle development. The MEF2D-V4 could promote differentiation of chicken myoblasts and its expression was regulated by RBFOX2. The single nucleotide polymorphism g.36186C > T generated a TAG stop codon, caused MEF2D-V4 to terminate translation early, and was associated with several growth traits, especially on early body weight. Conclusion We cloned the muscle-specific transcript of MEF2D and preliminarily revealed its role in embryonic muscle development.
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Ahmed Elnour, Abdalla Abdelrahman, and Mahdi H. A. Abdalla. "P210 and P190 BCR-ABL fusion transcripts variants frequencies among Philadelphia chromosome-positive chronic myeloid leukemia in Sudan." International Journal of Biomedical Research 9, no. 5 (May 29, 2018): 172. http://dx.doi.org/10.7439/ijbr.v9i5.4736.
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Breakpoint cluster region-abelson (BCR-ABL) leukemic fusion gene types in chronic myeloid leukemia (CML) correlate with the disease clinical course and outcome. There are variations in the reports of previous studies about the frequencies and distribution of BCR-ABL transcripts in chronic myelogenous leukaemia among Sudanese patients. This research aims to determine the frequencies of BCR-ABL fusion transcript variants in Sudan. One hundred (informed consent) Philadelphia positive chronic myeloid leukaemia patients, in chronic phase, were enrolled in this study. EDTA anticoagulated peripheral blood samples were collected from each participant, RNA was extracted from mononuclear cells by (TRIzol) reagent. BCR-ABL transcripts were detected by qRT-PCR technique with specific primers forP190 and P210 BCR-ABL transcript variants. The typical p210 BCR-ABL transcripts (b3a2 or b2a2) were detected in all patients (100%) the b3a2 transcript was detected in 96/100 (96%) and the b2a2 transcript was detected in 4/100 (4%).co-expression of p210/p190 (b2a3/e1a2) was detected in 6/100 (6%). p190 variant was not detected independently.
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Morales, Joannella, Shashikant Pujar, Jane E. Loveland, Alex Astashyn, Ruth Bennett, Andrew Berry, Eric Cox, et al. "A joint NCBI and EMBL-EBI transcript set for clinical genomics and research." Nature 604, no. 7905 (April 6, 2022): 310–15. http://dx.doi.org/10.1038/s41586-022-04558-8.
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AbstractComprehensive genome annotation is essential to understand the impact of clinically relevant variants. However, the absence of a standard for clinical reporting and browser display complicates the process of consistent interpretation and reporting. To address these challenges, Ensembl/GENCODE1 and RefSeq2 launched a joint initiative, the Matched Annotation from NCBI and EMBL-EBI (MANE) collaboration, to converge on human gene and transcript annotation and to jointly define a high-value set of transcripts and corresponding proteins. Here, we describe the MANE transcript sets for use as universal standards for variant reporting and browser display. The MANE Select set identifies a representative transcript for each human protein-coding gene, whereas the MANE Plus Clinical set provides additional transcripts at loci where the Select transcripts alone are not sufficient to report all currently known clinical variants. Each MANE transcript represents an exact match between the exonic sequences of an Ensembl/GENCODE transcript and its counterpart in RefSeq such that the identifiers can be used synonymously. We have now released MANE Select transcripts for 97% of human protein-coding genes, including all American College of Medical Genetics and Genomics Secondary Findings list v3.0 (ref. 3) genes. MANE transcripts are accessible from major genome browsers and key resources. Widespread adoption of these transcript sets will increase the consistency of reporting, facilitate the exchange of data regardless of the annotation source and help to streamline clinical interpretation.
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Cook, Taylor W., Amy M. Wilstermann, Jackson T. Mitchell, Nicholas E. Arnold, Surender Rajasekaran, Caleb P. Bupp, and Jeremy W. Prokop. "Understanding Insulin in the Age of Precision Medicine and Big Data: Under-Explored Nature of Genomics." Biomolecules 13, no. 2 (January 30, 2023): 257. http://dx.doi.org/10.3390/biom13020257.
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Insulin is amongst the human genome’s most well-studied genes/proteins due to its connection to metabolic health. Within this article, we review literature and data to build a knowledge base of Insulin (INS) genetics that influence transcription, transcript processing, translation, hormone maturation, secretion, receptor binding, and metabolism while highlighting the future needs of insulin research. The INS gene region has 2076 unique variants from population genetics. Several variants are found near the transcriptional start site, enhancers, and following the INS transcripts that might influence the readthrough fusion transcript INS–IGF2. This INS–IGF2 transcript splice site was confirmed within hundreds of pancreatic RNAseq samples, lacks drift based on human genome sequencing, and has possible elevated expression due to viral regulation within the liver. Moreover, a rare, poorly characterized African population-enriched variant of INS–IGF2 results in a loss of the stop codon. INS transcript UTR variants rs689 and rs3842753, associated with type 1 diabetes, are found in many pancreatic RNAseq datasets with an elevation of the 3′UTR alternatively spliced INS transcript. Finally, by combining literature, evolutionary profiling, and structural biology, we map rare missense variants that influence preproinsulin translation, proinsulin processing, dimer/hexamer secretory storage, receptor activation, and C-peptide detection for quasi-insulin blood measurements.
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Rosa, Villegas-Ruíz, Caballero-Palacios, Pérez-López, Murata, Zapata-Tarres, Cárdenas-Cardos, Paredes-Aguilera, Rivera-Luna, and Juárez-Méndez. "Expression of ZNF695 Transcript Variants in Childhood B-Cell Acute Lymphoblastic Leukemia." Genes 10, no. 9 (September 16, 2019): 716. http://dx.doi.org/10.3390/genes10090716.
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B-cell acute lymphoblastic leukemia is the most commonly diagnosed childhood malignancy worldwide; more than 50% of these cases are diagnosed in Mexico. Although the five-year survival rate is >80%, 30% of patients experience relapse with poor prognosis. Cancer-associated gene expression profiles have been identified in several malignancies, and some transcripts have been used to predict disease prognosis. The human transcriptome is incompletely elucidated; moreover, more than 80% of transcripts can be processed via alternative splicing (AS), which increases transcript and protein diversity. The human transcriptome is divided; coding RNA accounts for 2%, and the remaining 98% is noncoding RNA. Noncoding RNA can undergo AS, promoting the diversity of noncoding transcripts. We designed specific primers to amplify previously reported alternative transcript variants of ZNF695 and showed that six ZNF695 transcript variants are co-expressed in cancer cell lines. The amplicons were sequenced and identified. Additionally, we analyzed the expression of these six transcript variants in bone marrow from B-cell acute lymphoblastic leukemia patients and observed that ZNF695 transcript variants one and three were the predominant variants expressed in leukemia. Moreover, our results showed the co-expression of coding and long noncoding RNA. Finally, we observed that long noncoding RNA ZNF695 expression predicted survival rates.
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Valenzuela-Palomo, Alberto, Lara Sanoguera-Miralles, Elena Bueno-Martínez, Ada Esteban-Sánchez, Inés Llinares-Burguet, Alicia García-Álvarez, Pedro Pérez-Segura, Susana Gómez-Barrero, Miguel de la Hoya, and Eladio A. Velasco-Sampedro. "Splicing Analysis of 16 PALB2 ClinVar Variants by Minigene Assays: Identification of Six Likely Pathogenic Variants." Cancers 14, no. 18 (September 19, 2022): 4541. http://dx.doi.org/10.3390/cancers14184541.
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PALB2 loss-of-function variants are associated with significant increased risk of breast cancer as well as other types of tumors. Likewise, splicing disruptions are a common mechanism of disease susceptibility. Indeed, we previously showed, by minigene assays, that 35 out of 42 PALB2 variants impaired splicing. Taking advantage of one of these constructs (mgPALB2_ex1-3), we proceeded to analyze other variants at exons 1 to 3 reported at the ClinVar database. Thirty-one variants were bioinformatically analyzed with MaxEntScan and SpliceAI. Then, 16 variants were selected for subsequent RNA assays. We identified a total of 12 spliceogenic variants, 11 of which did not produce any trace of the expected minigene full-length transcript. Interestingly, variant c.49-1G > A mimicked previous outcomes in patient RNA (transcript ∆(E2p6)), supporting the reproducibility of the minigene approach. A total of eight variant-induced transcripts were characterized, three of which (∆(E1q17), ∆(E3p11), and ∆(E3)) were predicted to introduce a premature termination codon and to undergo nonsense-mediated decay, and five (▼(E1q9), ∆(E2p6), ∆(E2), ▼(E3q48)-a, and ▼(E3q48)-b) maintained the reading frame. According to an ACMG/AMP (American College of Medical Genetics and Genomics/Association for Molecular Pathology)-based classification scheme, which integrates mgPALB2 data, six PALB2 variants were classified as pathogenic/likely pathogenic, five as VUS, and five as likely benign. Furthermore, five ±1,2 variants were catalogued as VUS because they produced significant proportions of in-frame transcripts of unknown impact on protein function.
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John, Miya, and Caroline E. Ford. "Pan-Tissue and -Cancer Analysis of ROR1 and ROR2 Transcript Variants Identify Novel Functional Significance for an Alternative Splice Variant of ROR1." Biomedicines 10, no. 10 (October 13, 2022): 2559. http://dx.doi.org/10.3390/biomedicines10102559.
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ROR1/2 are putative druggable targets increasing in significance in translational oncology. Expression of ROR1/2 mRNA and transcript variants has not been systematically examined thus far. ROR1/2 transcript variant sequences, signal peptides for cell surface localisation, and mRNA and transcript variant expression were examined in 34 transcriptomic datasets including 33 cancer types and 54 non-diseased human tissues. ROR1/2 have four and eight transcript variants, respectively. ROR1/2 mRNA and transcript variant expression was detected in various non-diseased tissues. Our analysis identifies predominant expression of ROR1 transcript variant ENST00000545203, which lacks a signal peptide for cell surface localisation, rather than the predicted principal variant ENST00000371079. ENST00000375708 is the predominantly expressed transcript variant of ROR2. ROR1/2 expression in healthy human tissues should be carefully considered for safety assessment of targeted therapy. Studies exploring the function and significance of the predominantly expressed ROR1 transcript variant ENST00000545203 are warranted.
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Germeshausen, Manuela, Magda Grudzien, Cornelia Zeidler, Hengameh Abdollahpour, Sevgi Yetgin, Nima Rezaei, Matthias Ballmaier, Bodo Grimbacher, Karl Welte, and Christoph Klein. "Novel HAX1 mutations in patients with severe congenital neutropenia reveal isoform-dependent genotype-phenotype associations." Blood 111, no. 10 (May 15, 2008): 4954–57. http://dx.doi.org/10.1182/blood-2007-11-120667.
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Abstract Homozygous mutations in HAX1 cause an autosomal recessive form of severe congenital neutropenia (CN). By screening 88 patients with CN, we identified 6 additional patients with HAX1 mutations carrying 4 novel mutations. Of these, 2 affect both published transcript variants of HAX1; the other 2 mutations affect only transcript variant 1. Analysis of the patients' genotypes and phenotypes revealed a striking correlation: Mutations affecting transcript variant 1 only were associated with CN (23 of 23 patients), whereas mutations affecting both transcript variants caused CN and neurologic symptoms, including epilepsy and neurodevelopmental delay (6 of 6 patients). In contrast to peripheral blood, transcript variant 2 was markedly expressed in human brain tissue. The clinical phenotype of HAX1 deficiency appears to depend on the localization of the mutation and their influence on the transcript variants. Therefore, our findings suggest that HAX1 isoforms may play a distinctive role in the neuronal system.
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Bueno-Martínez, Elena, Lara Sanoguera-Miralles, Alberto Valenzuela-Palomo, Víctor Lorca, Alicia Gómez-Sanz, Sara Carvalho, Jamie Allen, et al. "RAD51D Aberrant Splicing in Breast Cancer: Identification of Splicing Regulatory Elements and Minigene-Based Evaluation of 53 DNA Variants." Cancers 13, no. 11 (June 7, 2021): 2845. http://dx.doi.org/10.3390/cancers13112845.
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RAD51D loss-of-function variants increase lifetime risk of breast and ovarian cancer. Splicing disruption is a frequent pathogenic mechanism associated with variants in susceptibility genes. Herein, we have assessed the splicing and clinical impact of splice-site and exonic splicing enhancer (ESE) variants identified through the study of ~113,000 women of the BRIDGES cohort. A RAD51D minigene with exons 2–9 was constructed in splicing vector pSAD. Eleven BRIDGES splice-site variants (selected by MaxEntScan) were introduced into the minigene by site-directed mutagenesis and tested in MCF-7 cells. The 11 variants disrupted splicing, collectively generating 25 different aberrant transcripts. All variants but one produced negligible levels (<3.4%) of the full-length (FL) transcript. In addition, ESE elements of the alternative exon 3 were mapped by testing four overlapping exonic microdeletions (≥30-bp), revealing an ESE-rich interval (c.202_235del) with critical sequences for exon 3 recognition that might have been affected by germline variants. Next, 26 BRIDGES variants and 16 artificial exon 3 single-nucleotide substitutions were also assayed. Thirty variants impaired splicing with variable amounts (0–65.1%) of the FL transcript, although only c.202G>A demonstrated a complete aberrant splicing pattern without the FL transcript. On the other hand, c.214T>C increased efficiency of exon 3 recognition, so only the FL transcript was detected (100%). In conclusion, 41 RAD51D spliceogenic variants (28 of which were from the BRIDGES cohort) were identified by minigene assays. We show that minigene-based mapping of ESEs is a powerful approach for identifying ESE hotspots and ESE-disrupting variants. Finally, we have classified nine variants as likely pathogenic according to ACMG/AMP-based guidelines, highlighting the complex relationship between splicing alterations and variant interpretation.
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De la Cruz-Hernández, Erick, Alejandro García-Carrancá, Alejandro Mohar-Betancourt, Alfonso Dueñas-González, Adriana Contreras-Paredes, Enrique Pérez-Cardenas, Roberto Herrera-Goepfert, and Marcela Lizano-Soberón. "Differential splicing of E6 within human papillomavirus type 18 variants and functional consequences." Journal of General Virology 86, no. 9 (September 1, 2005): 2459–68. http://dx.doi.org/10.1099/vir.0.80945-0.
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Persistent infections of the uterine cervix with ‘high-risk’ human papillomavirus (HPV) are now recognized as necessary for the development of cervical cancer. Among them, HPV types 16 and 18 exhibit numerous variants associated with different risks for cervical cancer development. In this study, the questions of whether different HPV type 18 variants exhibit changes in early gene transcription and the molecular mechanisms underlying these differences were investigated. It was shown that, indeed, type 18 variants exhibited singular differences in E6 transcripts in vivo. Higher levels of the E6*I transcript were detected regularly in clones harbouring the African variant, as opposed to low levels of this transcript detected in clones containing the reference clone (Asian–Amerindian), where significantly higher levels of full-length E6 transcript were usually observed. As a direct consequence, higher levels of p53 protein were found in the presence of African E6, as opposed to the low levels of p53 observed with the Asian–Amerindian E6. These variations in consequence affected the levels of cellular proteins regulated by p53, such as Bax. Similar changes in the relative levels of E6 transcripts were observed when tumours containing type 18 E6 variants were analysed. The different ability of cells containing variant E6 genes to form tumours in nude mice was suggested by the fact that tumour volumes were considerably higher when cells expressed the Asian–Amerindian E6. Mutagenesis analysis of the reference clone showed that a C491A change reverts the phenotype. These results suggest that different splicing patterns of E6 within HPV type 18 variants may possibly have biological implications in viral tumorigenesis.
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Gelli, Elisa, Mara Colombo, Anna Pinto, Giovanna De Vecchi, Claudia Foglia, Sara Amitrano, Valeria Morbidoni, et al. "Usefulness and Limitations of Comprehensive Characterization of mRNA Splicing Profiles in the Definition of the Clinical Relevance of BRCA1/2 Variants of Uncertain Significance." Cancers 11, no. 3 (March 1, 2019): 295. http://dx.doi.org/10.3390/cancers11030295.
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Highly penetrant variants of BRCA1/2 genes are involved in hereditary predisposition to breast and ovarian cancer. The detection of pathogenic BRCA variants has a considerable clinical impact, allowing appropriate cancer-risk management. However, a major drawback is represented by the identification of variants of uncertain significance (VUS). Many VUS potentially affect mRNA splicing, making transcript analysis an essential step for the definition of their pathogenicity. Here, we characterize the impact on splicing of ten BRCA1/2 variants. Aberrant splicing patterns were demonstrated for eight variants whose alternative transcripts were fully characterized. Different events were observed, including exon skipping, intron retention, and usage of de novo and cryptic splice sites. Transcripts with premature stop codons or in-frame loss of functionally important residues were generated. Partial/complete splicing effect and quantitative contribution of different isoforms were assessed, leading to variant classification according to Evidence-based Network for the Interpretation of Mutant Alleles (ENIGMA) consortium guidelines. Two variants could be classified as pathogenic and two as likely benign, while due to a partial splicing effect, six variants remained of uncertain significance. The association with an undefined tumor risk justifies caution in recommending aggressive risk-reduction treatments, but prevents the possibility of receiving personalized therapies with potential beneficial effect. This indicates the need for applying additional approaches for the analysis of variants resistant to classification by gene transcript analyses.
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Al-Qahtani, Wedad, Mai Abduljabbar, Entissar AlSuhaibani, Anas Abdel Rahman, and Ahmad Aljada. "Quantification of the Lamin A/C Transcript Variants in Cancer Cell Lines by Targeted Absolute Quantitative Proteomics and Correlation with mRNA Expression." International Journal of Molecular Sciences 20, no. 8 (April 17, 2019): 1902. http://dx.doi.org/10.3390/ijms20081902.
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Lamin A/C proteins have key roles in nuclear structural integrity and chromosomal stability. Lamin A/C cumulative protein expression of all variants is reported by semi-quantitative Western blotting. To date, there have not been specific antibodies for the individual Lamin A/C transcript variants. We developed a mass spectrometric approach for the quantification of Lamin A/C transcript variants. A signature peptide for each specific splice variant of Lamin A/C was selected. A LC–MS/MS assay based on the selected signature peptides and their labeled internal standards was established to measure the expression of Lamin A/C transcript variant concentrations. The method validation was carried out according to Food and Drug Administration (FDA) guidelines. The expression levels of the Lamin A/C transcript variants were measured in samples derived from MCF7 and U937 cell lines. RT-qPCR assay was also used to quantitate and compare the mRNA expression of splice variants of Lamin A/C. The established and validated method showed a great linearity, sensitivity, and precision. The different expressed Lamin A/C variants in different cell lines were measured and their levels were in concordance with qRT-PCR results. The developed method is reproducible, reliable, and sensitive for measuring different Lamin A/C transcript variants in different cell lines.
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Hong, Jinyoung, Ji Hyun Kim, Se Hee Ahn, Hyunjung Gu, Suhwan Chang, Woochang Lee, Dae-Yeon Kim, Sail Chun, and Won-Ki Min. "Identification of a Splice Variant (c.5074+3A>C) of BRCA1 by RNA Sequencing and TOPO Cloning." Genes 12, no. 6 (May 26, 2021): 810. http://dx.doi.org/10.3390/genes12060810.
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Grading the pathogenicity of BRCA1/2 variants has great clinical importance in patient treatment as well as in the prevention and screening of hereditary breast and ovarian cancer (HBOC). For accurate evaluation, confirming the splicing effect of a possible splice site variant is crucial. We report a significant splicing variant (c.5074+3A>C) in BRCA1 in a patient with recurrent ovarian cancer. Next-generation sequencing (NGS) of BRCA1/2 from patient’s peripheral blood identified the variant, which was strongly suspected of being a splicing mutation based on in silico predictions. Direct RNA analysis yielded multiple transcripts, and TOPO cloning of the complementary DNA (cDNA) and Sanger sequencing revealed an aberrant transcript with an insertion of the first 153 bp of intron 17, and another transcript with the 153 bp insertion along with an exon 18 deletion. A premature termination codon was presumed to be formed by the 153 bp partial intron retention common to the two transcripts. Therefore, BRCA1 c.5074+3A>C was classified as a likely pathogenic variant. Our findings show that active use of functional studies of variants suspected of altered splicing are of great help in classifying them.
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Harris, Rebecca Louise, Carmen Wilma van den Berg, and Derrick John Bowen. "ASGR1 and ASGR2, the Genes that Encode the Asialoglycoprotein Receptor (Ashwell Receptor), Are Expressed in Peripheral Blood Monocytes and Show Interindividual Differences in Transcript Profile." Molecular Biology International 2012 (August 2, 2012): 1–10. http://dx.doi.org/10.1155/2012/283974.
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Background. The asialoglycoprotein receptor (ASGPR) is a hepatic receptor that mediates removal of potentially hazardous glycoconjugates from blood in health and disease. The receptor comprises two proteins, asialoglycoprotein receptor 1 and 2 (ASGR1 and ASGR2), encoded by the genes ASGR1 and ASGR2. Design and Methods. Using reverse transcription amplification (RT-PCR), expression of ASGR1 and ASGR2 was investigated in human peripheral blood monocytes. Results. Monocytes were found to express ASGR1 and ASGR2 transcripts. Correctly spliced transcript variants encoding different isoforms of ASGR1 and ASGR2 were present in monocytes. The profile of transcript variants from both ASGR1 and ASGR2 differed among individuals. Transcript expression levels were compared with the hepatocyte cell line HepG2 which produces high levels of ASGPR. Monocyte transcripts were 4 to 6 orders of magnitude less than in HepG2 but nonetheless readily detectable using standard RT-PCR. The monocyte cell line THP1 gave similar results to monocytes harvested from peripheral blood, indicating it may provide a suitable model system for studying ASGPR function in this cell type. Conclusions. Monocytes transcribe and correctly process transcripts encoding the constituent proteins of the ASGPR. Monocytes may therefore represent a mobile pool of the receptor, capable of reaching sites remote from the liver.
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Chin, Diana, Matthew A. Kutny, Jonathan Grim, Robert B. Gerbing, Kristen Miller, Jason E. Farrar, Jaime M. Guidry Auvil, et al. "Comprehensive Genomic and Transcript Profiling of CBL Gene in Childhood AML: A Report from Children's Oncology Group Studies AAML03P1, AAML0531 and COG/NCI Target AML Initiative." Blood 126, no. 23 (December 3, 2015): 170. http://dx.doi.org/10.1182/blood.v126.23.170.170.
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Abstract The Casitas B-Lineage Lymphoma (CBL) gene encodes for an E3 ubiquitin ligase that targets activated receptor tyrosine kinases for degradation. Mutations of the CBL gene have been described in juvenile myelomonocytic leukemia (JMML) but less is known about mutations and variants of CBL in de novo AML. We previously reported that somatic mutations of CBL are rare in pediatric AML. In this report we present a comprehensive evaluation of genomic and transcript variants of CBL including novel deletion events as well as transcript variants which, in combination with somatic mutations, account for over 6% of pediatric AML with extreme association with inv(16) and favorable outcome. Initial assessment of CBL transcript in a cohort of 100 patients identified previously reported deletion of exon 8 (CBL ΔE8, N=2) associated with CBL splice mutations as well as a novel whole exon 8 and 9 deletion variant (CBL ΔE8+9, N=3) without identifiable underlying somatic alterations. Long distance PCR, as well as custom Nanostring CNV array evaluation revealed a genomic deletion underlying this transcript variant. Subsequent whole genome sequencing as part of COG/NCI TARGET AML initiative, identified discrete genomic deletions of 1998, 3588 and 6189 bp across exon 8 and 9, leading to the generation of this novel variant. We evaluated the functional consequence of the novel CBL ΔE8+9 deletion variant by expressing it in IL3-dependent Ba/F3 cell line. Compared to control cells, Ba/F3 cells expressing CBL ΔE8+9 demonstrated cytokine independent growth. A comprehensive profiling of CBL variants was conducted in 796 pediatric de novo AML patients by transcript profiling (transcript variants) or by exome capture sequencing (somatic mutations including point mutations and smaller indels). All patients were treated on Children's Oncology Group studies AAML03P1 (N=167) and AAML0531 (N=629) and presence of CBL variants was correlated with disease characteristics and clinical outcome. Of the 796 patient specimens tested, 50 patients (6.3%) had one of 3 distinct CBL variants; transcript variant (N=28), somatic mutation (N=14), or dual transcript variant and somatic mutation (N=8). All cases of CBL ΔE8+9 were associated with a corresponding genomic deletion. Out of 14 cases of CBL ΔE8 and 1 case of CBL ΔE9, only 4 cases (27%) had a splice site mutation identified as the underlying mechanism of splice variant. Presence of CBL variants was correlated with clinical characteristics and outcome. Those with CBL variants had a significantly higher prevalence of inv(16) compared with CBL wild type (WT) (37% vs. 13%, p<0.001). This association differed by CBL variant type; 44% transcript variants and 50% dual variants had inv(16) compared to 14% somatic mutations and 13% CBL WT (p<0.001). NPMc+ was more prevalent in those with CBL somatic mutations (29%) than transcript variant (4%), dual variant (0%) or CBL WT (8%) (p=0.035). Similarly, genetic risk groups differed between CBL variants vs. WT (Low risk 70% vs 39%, p=<0.001; Standard risk 22% vs. 46%, p=0.001; High risk 8% vs. 15%, p=0.196). Clinical characteristics including gender, age, race and ethnicity were not significantly different. FAB morphologic assessment revealed an enrichment for the M4 subtype in CBL variant vs. WT (53% vs. 23%, P<.001) which is likely accounted for by the association of inv(16) with this morphologic group. Patients with CBL variants had a 100% clinical remission rate by end of induction II compared to 89% for CBL WT patients (p=0.014). Survival from study entry was similar between CBL mutant vs. WT patients (5 year OS 72% vs. 66%, p=0.24; 5 year EFS 61% vs. 50%, p=0.11). Due to the strong association of CBL mutation with core binding factor leukemia, we assessed whether CBL variant was prognostic of outcome within this favorable risk group, but there was no significant difference in outcomes. Variants of the CBL gene in pediatric AML include genetic mutations with and without whole exon deletions. These CBL variants are highly associated with low risk AML but do not provide independent risk prognosis. The cooperating events of CBL variants in core binding factor leukemia deserve greater study. Our initial analysis of the transcript variants in a cell line model suggest that these large exon 8+9 deletions represent important oncogenic events. The authors would like to gratefully acknowledge the important contributions of the late Dr. Robert Arceci to the AML TARGET initiative. Disclosures No relevant conflicts of interest to declare.
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Chioda, Mariacristina, Fabio Spada, Ragnhild Eskeland, and Eric M. Thompson. "Histone mRNAs Do Not Accumulate during S Phase of either Mitotic or Endoreduplicative Cycles in the Chordate Oikopleura dioica." Molecular and Cellular Biology 24, no. 12 (June 15, 2004): 5391–403. http://dx.doi.org/10.1128/mcb.24.12.5391-5403.2004.
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ABSTRACT Metazoan histones are generally classified as replication-dependent or replacement variants. Replication-dependent histone genes contain cell cycle-responsive promoter elements, their transcripts terminate in an unpolyadenylated conserved stem-loop, and their mRNAs accumulate sharply during S phase. Replacement variant genes lack cell cycle-responsive promoter elements, their polyadenylated transcripts lack the stem-loop, and they are expressed at low levels throughout the cell cycle. During early development of some organisms with rapid cleavage cycles, replication-dependent mRNAs are not fully S phase restricted until complete cell cycle regulation is achieved. The accumulation of polyadenylated transcripts during this period has been considered incompatible with metazoan development. We show here that histone metabolism in the urochordate Oikopleura dioica does not accord with some key tenets of the replication-dependent/replacement variant paradigm. During the premetamorphic mitotic phase of development, expressed variants shared characteristics of replication-dependent histones, including the 3′ stem-loop, but, in contrast, were extensively polyadenylated. After metamorphosis, when cells in many tissues enter endocycles, there was a global downregulation of histone transcript levels, with most variant transcripts processed at the stem-loop. Contrary to the 30-fold S-phase upregulation of histone transcripts described in common metazoan model organisms, we observed essentially constant histone transcript levels throughout both mitotic and endoreduplicative cell cycles.
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Amin, Huma, and Suhaib Ahmed. "Characteristics of BCR–ABL gene variants in patients of chronic myeloid leukemia." Open Medicine 16, no. 1 (January 1, 2021): 904–12. http://dx.doi.org/10.1515/med-2021-0309.
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Abstract Background Depending on breakpoints of rearrangement different types of BCR–ABL fusion protein can be generated in patients of chronic myeloid leukemia (CML). The aim of this study is to observe frequencies of major transcripts in CML patients by reverse transcriptase polymerase chain reaction (RT-PCR) and their hematological features at the time of presentation. Materials and methods This cross sectional study was performed at Molecular Lab of Riphah International University, Islamabad from January to June 2019. Consecutive peripheral blood samples of 70 newly diagnosed CML patients in chronic phase were analyzed by RT-PCR to detect different BCR–ABL transcripts. Routine blood cell counts were assessed by an automated hematology analyzer. Results All samples expressed typical BCR–ABL rearrangement. Expression of either e14a2 or e13a2 transcript was detected in 38 (54%) and 30 (43%) patients, respectively. Coexpression of e13a2 + e14a2 was found in 2 (3%) patients. The mean total leukocyte count was higher in group expressing e13a2 (P = 0.01). Higher mean platelet count was noted in patients with e14a2 transcript, but this difference was statistically insignificant (P = 0.1). The association of male gender was observed with the group exhibiting e14a2 (P = 0.01). There was no statistically significant association between transcript type and different ranges of age, hemoglobin levels, and platelet and total leukocyte counts (P > 0.05). Conclusion e14a2 transcript was most common transcript in CML patients. Patients exhibiting e13a2 subgroup presented with significantly higher mean white blood cell count at the time of presentation. Significantly higher proportion of male patients was found to express e14a2 transcript over e13a2.
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Park, Joohyun, Annemarie Reilaender, Jan N. Petry-Schmelzer, Petra Stöbe, Isabell Cordts, Florian Harmuth, Maren Rautenberg, et al. "Transcript-Specific Loss-of-Function Variants in VPS16 Are Enriched in Patients With Dystonia." Neurology Genetics 8, no. 1 (December 7, 2021): e644. http://dx.doi.org/10.1212/nxg.0000000000000644.
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Background and ObjectivesOur objective was to improve rare variant interpretation using statistical measures as well as publicly accessible annotation of expression levels and tissue specificity of different splice isoforms. We describe rare VPS16 variants observed in patients with dystonia and patients without dystonia, elaborate on our interpretation of VPS16 variants affecting different transcripts, and provide detailed clinical description of the movement disorder caused by VPS16 variants.MethodsIn-house exome and genome data sets (n = 11,539) were screened for rare heterozygous missense and putative loss-of-function (pLoF) variants in VPS16. Using pext (proportion expressed across transcripts) values from the Genome Aggregation Database (gnomAD), we differentiated variants affecting weakly and highly expressed exons/transcripts and applied statistical measures to systematically identify disease-associated genetic variation among patients with dystonia (n = 280).ResultsSix different heterozygous pLoFs in VPS16 transcripts were identified in 13 individuals. Three of these pLoFs occurred in 9 individuals with different phenotypes, and 3 pLoFs were identified in 4 unrelated individuals with early-onset dystonia. Although pLoFs were enriched in the dystonia cohort (n = 280; p = 2.04 × 10−4; 4/280 cases vs 9/11,259 controls; Fisher exact test), it was not exome-wide significant. According to the pext values in gnomAD, all 3 pLoFs observed in the patients with dystonia were located in the highly expressed canonical transcript ENST00000380445.3, whereas 2 of 3 pLoFs detected in 8 individuals without dystonia were located in the first exon of the noncanonical transcript ENST00000380443.3 that is weakly expressed across all tissues. Taking these biological implications into account, pLoFs involving the canonical transcript were exome-wide significantly enriched in patients with dystonia (p = 1.67 × 10−6; 4/280 cases vs 1/11,259 controls; Fisher exact test). All VPS16 patients showed mild progressive dystonia with writer's cramp as the presenting symptom between age 7 and 34 years (mean 20 years) that often progressed to generalized dystonia and was even accompanied by hyperkinetic movements and myoclonus in 1 patient.DiscussionOur data provide strong evidence for VPS16 pLoFs to be implicated in dystonia and knowledge on exon resolution expression levels as well as statistical measures proved to be useful for variant interpretation.
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Palma, Marzia, Parviz Kokhaei, Lotta Hansson, Mohammad Hojat-Farsangi, Aniruddha Raja Choudhury, Anders Osterborg, and Håkan Mellstedt. "Expression of Human Telomerase Reverse Transcriptase (hTERT) Splice Variants In Chronic Lymphocytic Leukemia (CLL)." Blood 116, no. 21 (November 19, 2010): 2413. http://dx.doi.org/10.1182/blood.v116.21.2413.2413.
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Abstract Abstract 2413 High telomerase activity and telomere length are prognostic markers in CLL. In a previous study we described hTERT as potential CLL-specific antigen. The rate-limiting component of telomerase is telomerase reverse transcriptase (hTERT), for which multiple transcripts exist. In particular two splicing sites named α and β have been described, which generate deleted transcripts. The existence of multiple hTERT transcripts suggests that telomerase activity may be regulated by transcriptional control mechanisms as well as alternative splicing. In this study, we validated real-time RT-PCR assays for quantification of the hTERT transcripts with either α deletion (del-α, α-β+), or β deletion (del-β, α+β-) or both (del-αβ, α-β-). Only the full-length (FL, α+β+) transcript translates into a functional protein; the biological role of the other splice variants is still to be clarified. The aim of this work was to completely characterize hTERT splice variants in CLL, as well as study all hTERT splice variants in relation with disease activity. The splice variant pattern was studied in 68 CLL cases, 6 healthy controls and two CD34+ cell samples. Fourteen of 68 pts (20%) did not express any of the splice variants. FL (α+β+) was five-fold more expressed in patients with unmutated than with mutated IgVH genes (mean normalized expression 1.68×10−03 vs 3.36×10−04, p=0.0004). FL levels correlated with the percentage of IgVH homology (r=0.36, p=0.005). The del-α and the del-β transcripts were also expressed at higher levels in IgVH unmutated patients than mutated patients (2.77×10−03 vs 4.45×10−04, p=0.0004; and 8.78×10−04 vs 6.92×10−04, p=0.002, respectively). The expression of all four transcripts was higher in progressive compared to non-progressive patients. For FL average expression levels in progressive and non-progressive patients were 1.55×10−03 vs 3.36×10−04 (p<0.0001), respectively; for del-α 2.5×10−03 vs 5.5×0−04 (p=0.0003), respectively; for del-β 4.89×10−04 vs 4.2×10−04 (p= 0.01), respectively and for del-αβ 1.43×10−03 vs 2.16×10−03 (p<0.0001), respectively. FL expression was also significantly higher in progressive CLL patients compared to healthy controls (1.55×10−03 vs 2.04×10−04, p=0.03), while no difference was seen with regard to the other variants. Subsequently, we analyzed IgVH mutated and unmutated patients separately. Notably, the difference in FL expression levels between progressive and non-progressive patients was found only in the unmutated subgroup (2.19×10−03 and 4.85×10−04, respectively, p=0.04), whereas in patients with mutated CLL no difference could be identified. Overall, the levels of expression of FL transcript significantly correlated with those of the other splice variants: del-α (r=0.52, p<0.0001), del-β (r=0.50, p<0.0001), del-αβ (r=0.47, p<0.0001). Two patients (both IgVH mutated) were tested repeatedly in different disease phases and at disease progression an increase in FL transcript levels was seen, while the del-α transcript decreased. The levels of FL transcript detected in CD34+ cells and in the K562 cell line were 4.09×10−04 and 1.85×10−03, respectively, while for the del-α transcript they were 5.36×10−04 and 1.73×10−03, respectively and for the del-αβ transcript 1.67×10−03 and 4.93×10−03, respectively. The del-β transcript was not detected in CD34+ cells, while the expression in the K562 cell line was 2.7×10−03. This study provides a detailed insight into the hTERT transcript pattern in patients with CLL and shows that the expression of hTERT-FL is independent of disease phase in IgVH mutated but not in unmutated patients. Our results highlight the necessity of focusing on the functional transcript when analyzing hTERT expression in CLL patients and of interpreting the results in various phases of the disease in relation to the IgVH mutational status. The possibility that hTERT-FL mRNA gives rise to CLL-specific antigens that may be targeted with immunotherapy will be the object of further studies. Disclosures: No relevant conflicts of interest to declare.
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Mancini, F., G. Conza, and F. Moretti. "MDM4 (MDMX) and its Transcript Variants." Current Genomics 10, no. 1 (March 1, 2009): 42–50. http://dx.doi.org/10.2174/138920209787581280.
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Lai, John, Jiyuan An, Srilakshmi Srinivasan, Judith A. Clements, and Jyotsna Batra. "A computational analysis of the genetic and transcript diversity at the kallikrein locus." Biological Chemistry 397, no. 12 (December 1, 2016): 1307–13. http://dx.doi.org/10.1515/hsz-2016-0161.
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Abstract The kallikrein related peptidase gene family (KLKs) comprises 15 genes located between 19q13.3-13.4. KLKs have chymotrypsin and/or trypsin like activity, but the tissue/organ expression profile of each KLK varies considerably. Thus, the role of KLKs in human biology is also very diverse, and the deregulation of their function results in a wide-range of diseases. Here, we have cataloged the transcript (variants and fusions) and genetic (single nucleotide polymorphisms, small insertions/deletions, copy number variations (CNVs), and short tandem repeats) diversity at the KLK locus, providing a data set for researchers to explore the mechanisms through which KLK function may be deregulated. We reveal that the KLK locus hosts 85 fusion transcripts, and 80 variant transcripts. Interestingly, some fusion transcripts comprise up to 6 KLK genes. Our analysis of genetic variations of 2504 individuals from the 1000 Genome Project indicated that the KLK locus is rich in genetic diversity, with some fusion transcripts harboring over 1000 single nucleotide variations. We also found evidence from the literature linking 2387 KLK genetic variants with many types of diseases. Finally, genotyping data from the 131 KLK genetic variants in the NCI-60 cancer cell lines is provided as a resource for the cancer and KLK field.
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Deynichenko, K. A., K. G. Ptitsyn, S. P. Radko, L. K. Kurbatov, I. V. Vakhrushev, I. V. Buromski, S. S. Markin, A. I. Archakov, A. V. Lisitsa, and E. A. Ponomarenko. "Splice variants of mRNA of cytochrome P450 genes: analysis by the nanopore sequencing method in human liver tissue and HepG2 cell line." Biomeditsinskaya Khimiya 68, no. 2 (2022): 117–25. http://dx.doi.org/10.18097/pbmc20226802117.
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The analysis of cytochrome P450 transcripts was carried out by the nanopore sequencing in liver tissue samples of three donors and HepG2 line cells. It has been demonstrated that direct mRNA sequencing with a MinION nanopore sequencer (Oxford Nanopore Technologies) allows one to obtained quantitative profiles for transcripts (and their splice variants) of cytochrome P450 superfamily genes encoding isoforms involved in metabolism of the large (~80%) part of drugs. The splice variant profiles substantially differ for donors. The cytochrome P450 gene expression at the transcript level is significantly weaker in cells of the HepG2 line compared with that in the normal liver tissue. This limits the capability of the direct mRNA nanopore sequencing for studying alternative splicing of cytochrome P450 transcripts in HepG2 cells. Both quantitative and qualitative profiles of the cytochrome P450 gene expression at the transcript level are notably differ in human liver tissue and HepG2 cells.
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de Bruijn, Suzanne E., Sanne K. Verbakel, Erik de Vrieze, Hannie Kremer, Frans P. M. Cremers, Carel B. Hoyng, L. Ingeborgh van den Born, and Susanne Roosing. "Homozygous variants in KIAA1549, encoding a ciliary protein, are associated with autosomal recessive retinitis pigmentosa." Journal of Medical Genetics 55, no. 10 (August 17, 2018): 705–12. http://dx.doi.org/10.1136/jmedgenet-2018-105364.
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BackgroundRetinitis pigmentosa (RP) shows substantial genetic heterogeneity. It has been estimated that in approximately 60%–80% of RP cases, the genetic diagnosis can be found using whole exome sequencing (WES). In this study, the purpose was to identify causative variants in individuals with genetically unexplained retinal disease, which included one consanguineous family with two affected siblings and one case with RP.MethodsTo identify the genetic defect, WES was performed in both probands, and clinical analysis was performed. To obtain insight into the function of KIAA1549 in photoreceptors, mRNA expression, knockdown and protein localisation studies were performed.ResultsThrough analysis of WES data, based on population allele frequencies, and in silico prediction tools, we identified a homozygous missense variant and a homozygous frameshift variant in KIAA1549 that segregate in two unrelated families. Kiaa1549 was found to localise at the connecting cilium of the photoreceptor cells and the synapses of the mouse retina. Both variants affect the long transcript of KIAA1549, which encodes a 1950 amino acid protein and shows prominent brain expression. The shorter transcript encodes a 734 amino acid protein with a high retinal expression and is affected by the identified missense variant. Strikingly, knockdown of the long transcript also leads to decreased expression of the short transcript likely explaining the non-syndromic retinal phenotype caused by the two variants targeting different transcripts.ConclusionIn conclusion, our results underscore the causality of segregating variants in KIAA1549 for autosomal recessive RP. Moreover, our data indicate that KIAA1549 plays a role in photoreceptor function.
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Song, Tongxing, Jie Peng, Jiao Ren, Hong-kui Wei, and Jian Peng. "Cloning and Characterization of Spliced Variants of the Porcine G Protein Coupled Receptor 120." BioMed Research International 2015 (2015): 1–10. http://dx.doi.org/10.1155/2015/813816.
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The polyunsaturated fatty acids (PUFAs) receptor GPR120 exerts a significant impact on systemic nutrient homeostasis in human and rodents. However, the porcine GPR120 (pGPR120) has not been well characterized. In the current study, we found thatpGPR120had 3 spliced variants. Transcript 1 encoded 362-amino acids (aa) wild type pGPR120-WT, which shared 88% homology with human short form GPR120. Transcript 1 was the mainly expressed transcript ofpGPR120. It was expressed predominantly in ileum, jejunum, duodenum, spleen, and adipose. Transcript 3 (coding 320-aa isoform) was detected in spleen, while the transcript 2 (coding 310-aa isoform) was only slightly expressed in spleen. A selective agonist for human GPR120 (TUG-891) and PUFAs activated SRE-luc and NFAT-luc reporter in HEK293T cells transfected with construct for pGPR120-WT but not pGPR120-V2. However, 320-aa isoform was not a dominant negative isoform. The extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation levels in cells transfected with construct for pGPR120-WT were well activated by PUFAs, especially n-3 PUFA. These results showed that although pGPR120 had 3 transcripts, transcript 1 which encoded pGPR120-WT was the mainly expressed transcript. TUG-891 and PUFAs, especially n-3 PUFA, well activated pGPR120-WT. The current study contributed to dissecting the molecular regulation mechanisms of n-3 PUFA in pigs.
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Hartmann, Katherine, Michał Seweryn, and Wolfgang Sadee. "Interpreting coronary artery disease GWAS results: A functional genomics approach assessing biological significance." PLOS ONE 17, no. 2 (February 22, 2022): e0244904. http://dx.doi.org/10.1371/journal.pone.0244904.
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Genome-wide association studies (GWAS) have implicated 58 loci in coronary artery disease (CAD). However, the biological basis for these associations, the relevant genes, and causative variants often remain uncertain. Since the vast majority of GWAS loci reside outside coding regions, most exert regulatory functions. Here we explore the complexity of each of these loci, using tissue specific RNA sequencing data from GTEx to identify genes that exhibit altered expression patterns in the context of GWAS-significant loci, expanding the list of candidate genes from the 75 currently annotated by GWAS to 245, with almost half of these transcripts being non-coding. Tissue specific allelic expression imbalance data, also from GTEx, allows us to uncover GWAS variants that mark functional variation in a locus, e.g., rs7528419 residing in the SORT1 locus, in liver specifically, and rs72689147 in the GUYC1A1 locus, across a variety of tissues. We consider the GWAS variant rs1412444 in the LIPA locus in more detail as an example, probing tissue and transcript specific effects of genetic variation in the region. By evaluating linkage disequilibrium (LD) between tissue specific eQTLs, we reveal evidence for multiple functional variants within loci. We identify 3 variants (rs1412444, rs1051338, rs2250781) that when considered together, each improve the ability to account for LIPA gene expression, suggesting multiple interacting factors. These results refine the assignment of 58 GWAS loci to likely causative variants in a handful of cases and for the remainder help to re-prioritize associated genes and RNA isoforms, suggesting that ncRNAs maybe a relevant transcript in almost half of CAD GWAS results. Our findings support a multi-factorial system where a single variant can influence multiple genes and each genes is regulated by multiple variants.
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Germain, Claire, Franck Bihl, Stefan Zahn, Gwenola Poupon, Marie-Jeanne Dumaurier, Hariniaina Henintsoa Rampanarivo, Søren Berg Padkjær, Pieter Spee, and Veronique M. Braud. "Characterization of Alternatively Spliced Transcript Variants ofCLEC2DGene." Journal of Biological Chemistry 285, no. 46 (September 14, 2010): 36207–15. http://dx.doi.org/10.1074/jbc.m110.179622.
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Javed, Zaineb, Dong-Hui Shin, Weihua Pan, Amal Taher Elhaw, Priscilla Tang, Rebecca Phaeton, Mohamed Trebak, Vonn Walter, and Nadine Hempel. "Abstract 3781: Expression of ovarian cancer specific Drp1 splice variants regulate mitochondrial heterogeneity and cell plasticity during tumor progression." Cancer Research 82, no. 12_Supplement (June 15, 2022): 3781. http://dx.doi.org/10.1158/1538-7445.am2022-3781.
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Abstract Mitochondrial shape is integral for its proper function and is maintained by a dynamic balance between the events of fission and fusion. Hence, a disruption in the balance is detrimental and has been associated with multiple pathologies including tumorigenesis. We noticed significant heterogeneity in mitochondrial morphology and function in ovarian cancer, which remains the deadliest gynecologic malignancy to date. We discovered that heterogenous mitochondrial dynamics in ovarian cancer cells were associated with specific transcript variant signatures of the fission protein Drp1 (encoded by the gene DNM1L), the primary GTPase responsible for mitochondrial fission. While several Drp1 splice variants have been reported, few studies have linked expression and potential interplay of splice variants of Drp1 on mitochondrial dynamics and function with pathophysiology especially in ovarian cancer. We used 3’RACE, western blotting and LC-MS/MS proteomics analysis to establish the identity of the major Drp1 splice variants expressed in ovarian cancer. We found ovarian cancer cell lines as well as patient-ascites derived cells, predominantly express two Drp1 variants: a transcript including both exons 16 and 17 (16/17) and a transcript lacking exon 16 (-/17). We also validated our findings in TCGA ovarian cancer specimens by analyzing Drp1 splice variant transcripts following annotation of TCGA raw RNAseq data and Salmon expression analysis. Our TCGA analysis of these variants highlighted significant difference in overall survival of ovarian cancer patients. Samples with high Drp1(-/17) expression were associated with poorer overall survival compared to those predominantly expressing Drp1(16/17). Furthermore, carrying out gene set enrichment analysis (GSEA) on TCGA specimens split by high expression of these two variants showed enrichment of distinct gene expression signatures. Overexpression and splice variant specific siRNA knockdown studies demonstrated that Drp1 variants have unique localization and effects on mitochondrial morphology and function. Furthermore, metabolic profiling and 13C metabolic flux analysis highlighted variant specific alterations in mitochondrial metabolic pathways and the TCA cycle. Drp1(-/17) expression enhanced mitochondrial respiratory function and as previously shown, Drp1(-/17) associated with both mitochondria and microtubules, potentially implying a more regulated fission activity as a consequence of controlled subcellular localization. Additionally, Drp1(-/17) was enriched and associated with quiescent phenotype compared to more proliferative phenotype of Drp1(16/17). Hence, expression of distinct Drp1 splice variants may be a novel mechanism to regulate mitochondrial fission, and integral to ovarian cancer cell plasticity under different selection pressures during tumor progression. Citation Format: Zaineb Javed, Dong-Hui Shin, Weihua Pan, Amal Taher Elhaw, Priscilla Tang, Rebecca Phaeton, Mohamed Trebak, Vonn Walter, Nadine Hempel. Expression of ovarian cancer specific Drp1 splice variants regulate mitochondrial heterogeneity and cell plasticity during tumor progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3781.
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Shamsani, Jannah, Stephen H. Kazakoff, Irina M. Armean, Will McLaren, Michael T. Parsons, Bryony A. Thompson, Tracy A. O’Mara, Sarah E. Hunt, Nicola Waddell, and Amanda B. Spurdle. "A plugin for the Ensembl Variant Effect Predictor that uses MaxEntScan to predict variant spliceogenicity." Bioinformatics 35, no. 13 (November 23, 2018): 2315–17. http://dx.doi.org/10.1093/bioinformatics/bty960.
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Abstract Summary Assessing the pathogenicity of genetic variants can be a complex and challenging task. Spliceogenic variants, which alter mRNA splicing, may yield mature transcripts that encode non-functional protein products, an important predictor of Mendelian disease risk. However, most variant annotation tools do not adequately assess spliceogenicity outside the native splice site and thus the disease-causing potential of variants in other intronic and exonic regions is often overlooked. Here, we present a plugin for the Ensembl Variant Effect Predictor that packages MaxEntScan and extends its functionality to provide splice site predictions using a maximum entropy model. The plugin incorporates a sliding window algorithm to predict splice site loss or gain for any variant that overlaps a transcript feature. We also demonstrate the utility of the plugin by comparing our predictions to two mRNA splicing datasets containing several cancer-susceptibility genes. Availability and implementation Source code is freely available under the Apache License, Version 2.0: https://github.com/Ensembl/VEP_plugins. Supplementary information Supplementary data are available at Bioinformatics online.
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Yuda, Junichiro, Toshihiro Miyamoto, Jun Odawara, Yoshikane Kikushige, Yasuyuki Ohkawa, and Koichi Akashi. "Persistence of Abnormally-Spliced, Functionally-Dead BCR-ABL Variants Is a Critical Obstacle to Achieve Sustained Complete Molecular Response in CML Patients: Results of a Quantitative, Highly-Sensitive, Deep Sequencing Study." Blood 124, no. 21 (December 6, 2014): 4525. http://dx.doi.org/10.1182/blood.v124.21.4525.4525.
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Abstract Background: Tyrosine kinase inhibitors (TKI) have dramatically changed the treatment algorithm of chronic myeloid leukemia (CML). Despite improved outcomes in the TKI era, mutations in the BCR-ABL kinase domain are one of the major mechanisms of resistance to TKIs. In addition, recent studies have suggested that abnormally-spliced (AS) BCR-ABL transcript variants, such as retention of 35bp intronic nucleotides at Exon8/9 splice junction (BCR-ABL Ins 35bp) is one of the main obstacles for patients to exhibit "optimal response" to TKI (Gaillard JB, et al. Mol Cancer Ther 2010; T O'Hare, et al. Blood 2011). These BCR-ABL variants have a stop codon in kinase domain residues, resulting in generation of "function dead" BCR-ABL. We hypothesized that emergence of such functionally-dead BCR-ABL variants should render such CML clones insensitive to TKI, contributing to persistence of CML clone, even at molecular remission phases. Here, by combining a long-range nested PCR-amplification method and an analysis by high-throughput next generation sequencer (NGS), we have established a highly-sensitive assay system to track minimal amounts of AS BCR-ABL transcript variants. Methods: We analyzed 294 samples from 62 CML patients. Twenty-six patients were treated with Imatinib and Dasatinib, 13 with Imatinib alone, 9 with Dasatinib alone, 6 with Nilotinib alone, 6 with Imatinib and Nilotinib, and 2 with Dasatinib and Nilotinib. cDNA was synthesized from extracted RNA samples, and BCR-ABL was amplified by long-range nested PCR method. Splicing forms of BCR-ABL were then deep-sequenced by using HiSeq 1500 (illumina). Samples capable of achieving>10,000 read counts on ABL-1 Exon 8 were regarded as reliable to enroll into further statistical analysis. Results: In all 7 patients analyzed at diagnosis, CML cells have wild-type (WT) BCR-ABL but also have extremely small amount of AS BCR-ABL transcript variants. After TKI treatment, mainly two type of splicing variants became evident; the BCR-ABL Ins 35bp and Intron8-retained BCR-ABL that possesses inappropriately spliced Intron8 of ABL-1. In both cases, such transcripts fail to form functional BCR-ABL. As shown in Figure 1, along with TKIs treatment for 6 months, total amount of BCR-ABL quickly decreased, whereas percentages of AS BCR-ABL variants (BCR-ABL Ins 35bp and Intron8-retained BCR-ABL) gradually increased, suggesting that clones possessing AS BCR-ABL variants might become dominant by clonal selection through TKI treatment. In 55 patients with deep molecular response under TKI treatment for >1 year, 29 out of 55 (52%) patients achieved MR4.5, whereas the remaining 26 (48%) patients retained MR3.0, failing to achieve MR4.5. In 29 patients who have reached MR4.5, BCR-ABL variants were detected in 11 (38%) patients in any points during treatment. In contrast, within 26 patients who retained only MR3.0, 22 (85%) patients possessed BCR-ABL variants. Thus, emergence of AS BCR-ABL variants are significantly more frequent in patients retaining only MR3.0 (p<0.05). In these patients, BCR-ABL transcript variants were continually detected at several points of analysis. A representative patient who could not achieved MR4.5 is shown in Figure 2. After switching from Imatinib to Dasatinib, total amount of BCR-ABL gradually decreased, while AS BCR-ABL transcript variants did not, resulting in the poor response to Dasatinib. Discussion: Our highly-sensitive quantitation system for AS BCR-ABL variants revealed that the majority of major molecular responders (≥MR3.0) possess BCR-ABL variants, but these are more frequently found in patients failed to achieve MR4.5 (P<0.05). These data strongly suggest that selective persistence of AS BCR-ABL variants is one of the key mechanisms of TKI resistance in major molecular responders. CML clones with kinase-dead BCR-ABL should not be sensitive to TKI, should not be addicted to ABL kinase activity, and should therefore be difficult to be killed by TKI alone. Thus, quantitation of AS BCR-ABL variants might be critical to predict clinical outcomes of CML patients, for example, whether or not they relapse after TKI cessation. Figure 1. Molecular kinetics in early reduction of BCR-ABL after TKIs. Figure 1. Molecular kinetics in early reduction of BCR-ABL after TKIs. Figure 2. Transition of AS BCR-ABL transcript variant in case who achieved MR3.0 but not MR4.5 under TKIs treatment for more than 1-year. Figure 2. Transition of AS BCR-ABL transcript variant in case who achieved MR3.0 but not MR4.5 under TKIs treatment for more than 1-year. Disclosures No relevant conflicts of interest to declare.
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Doll, Julia, Susanne Kolb, Linda Schnapp, Aboulfazl Rad, Franz Rüschendorf, Imran Khan, Abolfazl Adli, et al. "Novel Loss-of-Function Variants in CDC14A are Associated with Recessive Sensorineural Hearing Loss in Iranian and Pakistani Patients." International Journal of Molecular Sciences 21, no. 1 (January 2, 2020): 311. http://dx.doi.org/10.3390/ijms21010311.
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CDC14A encodes the Cell Division Cycle 14A protein and has been associated with autosomal recessive non-syndromic hearing loss (DFNB32), as well as hearing impairment and infertile male syndrome (HIIMS) since 2016. To date, only nine variants have been associated in patients whose initial symptoms included moderate-to-profound hearing impairment. Exome analysis of Iranian and Pakistani probands who both showed bilateral, sensorineural hearing loss revealed a novel splice site variant (c.1421+2T>C, p.?) that disrupts the splice donor site and a novel frameshift variant (c.1041dup, p.Ser348Glnfs*2) in the gene CDC14A, respectively. To evaluate the pathogenicity of both loss-of-function variants, we analyzed the effects of both variants on the RNA-level. The splice variant was characterized using a minigene assay. Altered expression levels due to the c.1041dup variant were assessed using RT-qPCR. In summary, cDNA analysis confirmed that the c.1421+2T>C variant activates a cryptic splice site, resulting in a truncated transcript (c.1414_1421del, p.Val472Leufs*20) and the c.1041dup variant results in a defective transcript that is likely degraded by nonsense-mediated mRNA decay. The present study functionally characterizes two variants and provides further confirmatory evidence that CDC14A is associated with a rare form of hereditary hearing loss.
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Frojmark, Anne-Sophie, Jitendra Badhai, Edward J. Davey, and Niklas Dahl. "Diamond Blackfan Anemia: An RPS19 Transcript Variant Specifically Interacts with Nuclear Proteins." Blood 106, no. 11 (November 16, 2005): 1032. http://dx.doi.org/10.1182/blood.v106.11.1032.1032.
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Abstract Diamond Blackfan Anemia (DBA) is a rare congenic anemia characterized by reduced erythrocyte precursors. Mutations in the ribosomal protein S19 (RPS19) gene are found in about 20% of patients, although it is unclear how this relates to the pathophysiology of the disease. Two transcripts have been reported for human RPS19 in the NCBI database (BC000023 and BC018616), which only differ in the length of the 5′UTR. Both transcripts predict translation to the same protein product and we therefore speculate that the extended 5′UTR may be of importance. While the short (569bp) transcript is well studied, the long (873bp) transcript has not been investigated. We determined the size and tissue distribution as a basic characterization of the long RPS19 transcript. Furthermore, we examined proteins interacting with the different RPS19 transcript variants. Two methods were used determine the size of long RPS19 transcript. Ribonuclease protection assays and 5′-RACE indicate a transcript length consistent with the reported sequence with a 5′UTR of 372bp. Interestingly the 5′UTR predicted in the long transcript contains sequence for a previously reported promoter, suggesting a second novel promoter. Tissue distribution was determined using reverse transcriptase PCR on isolated RNA from a panel of tissues including heart, bone marrow, fetal liver, liver, brain, fetal brain, muscle, kidney, uterus, adrenal gland, lungs, placenta, prostate testes, ovary, thymus, colon, lymphocytes and from K562 and HeLa cell lines. Analysis indicated a ubiquitously expression of the long RPS19 transcript. In order to investigate interactions between the RPS19 transcripts and regulatory proteins, a UV cross-linking assay was performed using in vitro transcribed RNA representing both short and long transcripts. A protein of 55 kDa from nuclear fractions prepared from the erythroid cell lines K562, UT-7 and Cos-1 cells was found to bind to the long RPS19 transcript but not to the short RPS19 transcript. Our results provide evidence for an additional long, RPS19 transcript containing an extended 5′UTR that is ubiquitously transcribed and indicates the existence of a second promoter for RPS19. We further show that a nuclear protein, present in two different erythroid cell lines, interacts with the long RPS19 transcript indicating a function for the extended 5′UTR. This data suggests additional regulatory pathways involving RPS19 mRNA which may be of relevance to the understanding of the molecular mechanisms behind DBA.
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Demars, Julie, Nathalie Iannuccelli, Valerio Utzeri, Gerard Auvinet, Juliette Riquet, Luca Fontanesi, and Daniel Allain. "New Insights into the Melanophilin (MLPH) Gene Affecting Coat Color Dilution in Rabbits." Genes 9, no. 9 (August 23, 2018): 430. http://dx.doi.org/10.3390/genes9090430.
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Coat color dilution corresponds to a specific pigmentation phenotype that leads to a dilution of wild type pigments. It affects both eumelanin and pheomelanin containing melanosomes. The mode of inheritance of the dilution phenotype is autosomal recessive. Candidate gene approaches focused on the melanophilin (MLPH) gene highlighted two variants associated with the dilution phenotype in rabbits: The c.111-5C>A variant that is located in an acceptor splice site or the c.585delG variant, a frameshift mutation. On the transcript level, the skipping of two exons has been reported as the molecular mechanism responsible for the coat color dilution. To clarify, which of the two variants represents the causal variant, (i) we analyzed their allelic segregation by genotyping Castor and Chinchilla populations, and (ii) we evaluated their functional effects on the stability of MLPH transcripts in skin samples of animals with diluted or wild type coat color. Firstly, we showed that the c.585delG variant showed perfect association with the dilution phenotype in contrast to the intronic c.111-5C>A variant. Secondly, we identified three different MLPH isoforms including the wild type isoform, the exon-skipping isoform and a retained intron isoform. Thirdly, we observed a drastic and significant decrease of MLPH transcript levels in rabbits with a coat color dilution (p-values ranging from 10−03 to 10−06). Together, our results bring new insights into the coat color dilution trait.
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Loraine, Ann E., Gregg A. Helt, Melissa S. Cline, and Michael A. Siani-Rose. "Exploring Alternative Transcript Structure in the Human Genome Using BLOCKS and InterPro." Journal of Bioinformatics and Computational Biology 01, no. 02 (July 2003): 289–306. http://dx.doi.org/10.1142/s0219720003000113.
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Understanding how alternative splicing affects gene function is an important challenge facing modern-day molecular biology. Using homology-based, protein sequence analysis methods, it should be possible to investigate how transcript diversity impacts protein function. To test this, high-quality exon-intron structures were deduced for over 8000 human genes, including over 1300 (17 percent) that produce multiple transcript variants. A data mining technique (DiffMotif) was developed to identify genes in which transcript variation coincides with changes in conserved motifs between variants. Applying this method, we found that 30 percent of the multi-variant genes in our test set exhibited a differential profile of conserved InterPro and/or BLOCKS motifs across different mRNA variants. To investigate these, a visualization tool (ProtAnnot) that displays amino acid motifs in the context of genomic sequence was developed. Using this tool, genes revealed by the DiffMotif method were analyzed, and when possible, hypotheses regarding the potential role of alternative transcript structure in modulating gene function were developed. Examples of these, including: MEOX1, a homeobox-containing protein; AIRE, involved in auto-immune disease; PLAT, tissue type plasminogen activator; and CD79b, a component of the B-cell receptor complex, are presented. These results demonstrate that amino acid motif databases like BLOCKS and InterPro are useful tools for investigating how alternative transcript structure affects gene function.
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Lee, Ja-Rang, Young-Hyun Kim, Sang-Je Park, Se-Hee Choe, Hyeon-Mu Cho, Sang-Rae Lee, Sun-Uk Kim, et al. "Identification of Alternative Variants and Insertion of the Novel PolymorphicAluYl17inTSEN54Gene during Primate Evolution." International Journal of Genomics 2016 (2016): 1–10. http://dx.doi.org/10.1155/2016/1679574.
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TSEN54encodes a subunit of the tRNA-splicing endonuclease complex, which catalyzes the identification and cleavage of introns from precursor tRNAs. Previously, we identified anAluSx-derived alternative transcript inTSEN54of cynomolgus monkey. Reverse transcription-polymerase chain reaction (RT-PCR) amplification andTSEN54sequence analysis of primate and human samples identified five novel alternative transcripts, including theAluSxexonized transcript. Additionally, we performed comparative expression analysis via RT-qPCR in various cynomolgus, rhesus monkey, and human tissues. RT-qPCR amplification revealed differential expression patterns. Furthermore, genomic PCR amplification and sequencing of primate and human DNA samples revealed thatAluSxelements were integrated in human and all of the primate samples tested. Intriguingly, in langur genomic DNA, an additionalAluYelement was inserted intoAluSxof intron eight ofTSEN54. The newAluYelement showed polymorphic insertion. Using standardized nomenclature forAlurepeats, the polymorphicAluYof the langurTSEN54was designated as being of theAluYl17subfamily. Our results suggest that integration of theAluSxelement inTSEN54contributed to diversity in transcripts and induced lineage- or species-specific evolutionary events such as alternative splicing and polymorphic insertion during primate evolution.
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Zhou, Xiaopu, Yu Chen, Kin Y. Mok, Qianhua Zhao, Keliang Chen, Yuewen Chen, John Hardy, et al. "Identification of genetic risk factors in the Chinese population implicates a role of immune system in Alzheimer’s disease pathogenesis." Proceedings of the National Academy of Sciences 115, no. 8 (February 5, 2018): 1697–706. http://dx.doi.org/10.1073/pnas.1715554115.
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Alzheimer’s disease (AD) is a leading cause of mortality among the elderly. We performed a whole-genome sequencing study of AD in the Chinese population. In addition to the variants identified in or around the APOE locus (sentinel variant rs73052335, P = 1.44 × 10−14), two common variants, GCH1 (rs72713460, P = 4.36 × 10−5) and KCNJ15 (rs928771, P = 3.60 × 10−6), were identified and further verified for their possible risk effects for AD in three small non-Asian AD cohorts. Genotype–phenotype analysis showed that KCNJ15 variant rs928771 affects the onset age of AD, with earlier disease onset in minor allele carriers. In addition, altered expression level of the KCNJ15 transcript can be observed in the blood of AD subjects. Moreover, the risk variants of GCH1 and KCNJ15 are associated with changes in their transcript levels in specific tissues, as well as changes of plasma biomarkers levels in AD subjects. Importantly, network analysis of hippocampus and blood transcriptome datasets suggests that the risk variants in the APOE, GCH1, and KCNJ15 loci might exert their functions through their regulatory effects on immune-related pathways. Taking these data together, we identified common variants of GCH1 and KCNJ15 in the Chinese population that contribute to AD risk. These variants may exert their functional effects through the immune system.
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Cron, Randy Q., Grant S. Schulert, Mingce Zhang, Ammar Husami, Ndate Fall, Hermine Brunner, Kejian Zhang, and Alexei A. Grom. "Novel UNC13D intronic variant disrupting a NF κB enhancer in a patient with recurrent macrophage activation syndrome and systemic juvenile idiopathic arthritis." Journal of Immunology 200, no. 1_Supplement (May 1, 2018): 45.21. http://dx.doi.org/10.4049/jimmunol.200.supp.45.21.
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Abstract Macrophage activation syndrome (MAS) is a life-threatening complication of systemic juvenile idiopathic arthritis (SJIA), and has pathologic similarity to hemophagocytic lymphohistiocytosis (HLH). Intronic variants in UNC13D are found in patients with familial HLH-3 (FHL3), but the role of non-coding variants in MAS is unknown. The objective of this study was to identify deep intronic UNC13D variants in patients with MAS. A custom enrichment library was constructed to sequence approximately 1 MB flanking UNC13D in 24 patients with SJIA, recurrent MAS, and negative prior genetic (exon/coding) testing. Functional consequences of intronic variants were assessed using quantitative PCR on patient derived peripheral blood mononuclear cells (PBMC), electrophoretic mobility shift assay (EMSA), in vitro transcriptional enhancer assays, and in vitro NK cell degranulation assays. We report a patient with SJIA and recurrent episodes of MAS, found to have a novel functional intronic variant in UNC13D, c.117+143A>G. This variant occurs in a proposed regulatory region that drives an alternative, lymphocyte specific UNC13D transcript, and is associated with reduced transcript levels in patient PBMC. In support of this, the patient’s variant disrupts NFκB binding to a functional transcriptional enhancer, leading to reduced enhancer activity and NK cell degranulation in vitro. Another patient was identified with a previously described UNC13D intronic variant, for a total non-coding variant hit rate of 8.3% (2/24). These findings highlight that intronic variants in key regulatory regions may be associated with MAS in patients with SJIA, and support deep sequencing approaches when causative coding variants are not identified.
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Gorbenko, A. S., M. A. Stolyar, E. V. Vasiliev, M. A. Mikhalev, V. I. Bakhtina, T. I. Olkhovik, E. E. Mochalova, K. E. Orlova, and I. A. Olkhovskiy. "Use of the «BCR/ABL – multitest» kit in the algorithm of laboratory diagnostics of oncohematological diseases: economic aspects." Russian Clinical Laboratory Diagnostics 66, no. 9 (September 10, 2021): 571–76. http://dx.doi.org/10.51620/0869-2084-2021-66-9-571-576.
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Abnormal mRNAs of the hybrid BCR-ABL gene in the majority of cases initiate the synthesis of proteins with a mass of 210 kDa (p210), 190 kDa (p190), and 230 kDa (p230). Expression of the p210 variant is most common in CML (95% of cases), while the p190 and p230 variants are less common (1-4%). On the contrary, p190 predominates in ALL. Measurement of BCR/ABL gene expression is included in clinical guidelines for the diagnosis of CML and ALL as sequential tests in accordance with their occurrence. At the same time, in the context of primary patients testing with suspected hematological malignancies with a low prevalence of BCR-ABL positive patients in the cohort of examined individuals, sequential testing is associated with low cost-effectiveness. Purpose: approbation of a parallel algorithm for detecting all three (p210, p190 and p230) using the multiplex RT-PCR format implemented in the «BCR/ABL-MULTITEST» reagent kit. We used anonymized blood samples from patients with suspected CML, as well as samples from ALL patients before starting therapy. Testing of blood samples was carried out using two variants of the algorithm: sequential determination of individual BCR-ABL transcripts and parallel determination using the developed set of reagents «BCR/ABL-MULTITEST». To detect the p210 transcript, a commercial kit «AmpliSens® Leukemia Quantum M-bcr-FRT» (Central Research Institute of Epidemiology of Rospotrebnadzor, Russia) was used. Simultaneously, a test was used to detect all three variants of BCR-ABL transcripts using the «BCR/ABL - MULTITEST» reagent kit based on a monochrome multiplex reaction «in one test tube». Reverse transcription were carried out using the REVERTA-L reagent kit (Central Research Institute of Epidemiology of Rospotrebnadzor, Russia) in accordance with the manufacturer’s instructions. Using the reagent kits «BCR/ABL-MULTITEST» and «AmpliSens® Leukemia Quantum M-bcr-FRT» there is a high level of correlation of quantitative results of determining the chimeric transcript BCR-ABL р210 (r = 0.99). When using the proposed parallel algorithm with the primary use of the «BCR/ABL-MULTITEST» reagent kit, out of 95 patients with suspected CML, 9 samples with p210 transcript were identified, one with p190 BCR / ABL, and in one case a transcript variant characteristic of chronic neutrophilic leukemia - p230 BCR / ABL. The estimated cost for detecting one positive case of BCR-ABL when using the parallel diagnostic algorithm «BCR/ABL-MULTITEST» with a focused flow of studies is reduced by about 2 times due to a decrease in the amount of laboratory plastic used and the volume of the reaction mixture, as well as the absence of the need for repeated separate tests to detect p190 and p230. The use of the multiplex PCR-RT test system «BCR/ABL-MULTITEST» allows detecting in one test tube all three main variants of BCR-ABL transcripts - p210, p190, p230 and achieving significant resource savings when examining a cohort of patients with suspected CML and ALL and low frequency of positive samples.
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Erho, Nicholas, Christine Buerki, Timothy J. Triche, Elai Davicioni, and Ismael A. Vergara. "Transcriptome-Wide Detection of Differentially Expressed Coding and Non-Coding Transcripts and Their Clinical Significance in Prostate Cancer." Journal of Oncology 2012 (2012): 1–11. http://dx.doi.org/10.1155/2012/541353.
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Prostate cancer is a clinically and biologically heterogeneous disease. Deregulation of splice variants has been shown to contribute significantly to this complexity. High-throughput technologies such as oligonucleotide microarrays allow for the detection of transcripts that play a role in disease progression in a transcriptome-wide level. In this study, we use a publicly available dataset of normal adjacent, primary tumor, and metastatic prostate cancer samples (GSE21034) to detect differentially expressed coding and non-coding transcripts between these disease states. To achieve this, we focus on transcript-specific probe selection regions, that is, those probe sets that correspond unambiguously to a single transcript. Based on this, we are able to pinpoint at the transcript-specific level transcripts that are differentially expressed throughout prostate cancer progression. We confirm previously reported cases and find novel transcripts for which no prior implication in prostate cancer progression has been made. Furthermore, we show that transcript-specific differential expression has unique prognostic potential and provides a clinically significant source of biomarker signatures for prostate cancer risk stratification. The results presented here serve as a catalog of differentially expressed transcript-specific markers throughout prostate cancer progression that can be used as basis for further development and translation into the clinic.
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Bosco, Luca, Daniela Leone, Laura Costa Comellas, Mauro Monforte, Marika Pane, Eugenio Mercuri, Enrico Bertini, Adele D’Amico, and Fabiana Fattori. "Novel Splicing Mutation in MTM1 Leading to Two Abnormal Transcripts Causes Severe Myotubular Myopathy." International Journal of Molecular Sciences 23, no. 18 (September 7, 2022): 10274. http://dx.doi.org/10.3390/ijms231810274.
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X-linked myotubular myopathy (XLMTM) is a severe form of centronuclear myopathy, characterized by generalized weakness and respiratory insufficiency, associated with pathogenic variants in the MTM1 gene. NGS targeted sequencing on the DNA of a three-month-old child affected by XLMTM identified the novel hemizygous MTM1 c.1261-5T>G intronic variant, which interferes with the normal splicing process, generating two different abnormal transcripts simultaneously expressed in the patient’s muscular cells. The first aberrant transcript, induced by the activation of a cryptic splice site in intron 11, includes four intronic nucleotides upstream of exon 12, resulting in a shift in the transcript reading frame and introducing a new premature stop codon in the catalytic domain of the protein (p.Arg421SerfsTer7). The second aberrant MTM1 transcript, due to the lack of recognition of the 3′ acceptor splice site of intron 11 from the spliceosome complex, leads to the complete skipping of exon 12. We expanded the genotypic spectrum of XLMTM underlying the importance of intron–exons boundaries sequencing in male patients affected by XLMTM.
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Poncet, Anaïs F., Olivier Grunewald, Veronika Vaclavik, Isabelle Meunier, Isabelle Drumare, Valérie Pelletier, Béatrice Bocquet, et al. "Contribution of Whole-Genome Sequencing and Transcript Analysis to Decipher Retinal Diseases Associated with MFSD8 Variants." International Journal of Molecular Sciences 23, no. 8 (April 13, 2022): 4294. http://dx.doi.org/10.3390/ijms23084294.
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Biallelic gene defects in MFSD8 are not only a cause of the late-infantile form of neuronal ceroid lipofuscinosis, but also of rare isolated retinal degeneration. We report clinical and genetic data of seven patients compound heterozygous or homozygous for variants in MFSD8, issued from a French cohort with inherited retinal degeneration, and two additional patients retrieved from a Swiss cohort. Next-generation sequencing of large panels combined with whole-genome sequencing allowed for the identification of twelve variants from which seven were novel. Among them were one deep intronic variant c.998+1669A>G, one large deletion encompassing exon 9 and 10, and a silent change c.750A>G. Transcript analysis performed on patients’ lymphoblastoid cell lines revealed the creation of a donor splice site by c.998+1669A>G, resulting in a 140 bp pseudoexon insertion in intron 10. Variant c.750A>G produced exon 8 skipping. In silico and in cellulo studies of these variants allowed us to assign the pathogenic effect, and showed that the combination of at least one severe variant with a moderate one leads to isolated retinal dystrophy, whereas the combination in trans of two severe variants is responsible for early onset severe retinal dystrophy in the context of late-infantile neuronal ceroid lipofuscinosis.
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Mason, Cayla, Frida Holm, Eva Hellqvist, Christian Barrett, Kelly A. Frazer, Anil Sadarangani, and Catriona HM Jamieson. "The Role Of CD44 Isoform Expression In Niche Resident Chronic Myeloid Leukemia Stem Cell Evolution." Blood 122, no. 21 (November 15, 2013): 4028. http://dx.doi.org/10.1182/blood.v122.21.4028.4028.
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Abstract Introduction Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm characterized by the presence of the BCR-ABL fusion oncogene whose protein product has greatly increased ABL1 kinase activity. Although specific BCR-ABL tyrosine kinase inhibitors (TKIs) and the second generation TKIs like Nilotinib and the dual specific SCR and ABL inhibitor Dasatinib/Sprycel have dramatically improved CML therapy and significantly slowed disease progression by eradicating the bulk of CML cells in the circulation, they frequently fail to eliminate quiescent leukemic stem cells residing in the protective bone marrow niche. Leukemia stem cells (LSC) are able to drive disease relapse and may eventually contribute to the emergence of TKI resistant blast crisis (BC) CML, which is the final phase in the evolution of CML with rapid progression and short survival. CD44 is an adhesion molecule that promotes retention in the niche through adhesion to extracellular matrix components, such as hyaluronic acid and osteopontin. It plays an important role in wound healing and cell migration as well as in tumor invasion and metastasis. Through alternative mRNA splicing several CD44 isoforms exist, some of which are frequently overexpressed by cancer stem cells, including LSCs. The CD44 variant expression pattern on human blast crisis CML LSC, however, had not been elucidated. In this study we aimed to investigate the CD44 transcript variant expression of human blast crisis CML LSC. Methods and Results We performed whole transcriptome RNA sequencing of FACS sorted CML LSCs (Lin-CD34+CD38+) from chronic phase (CP)(n=8) and blast crisis (BC)(n=8), CML patients as well as the normal counterpart from cord blood (CB) (n=3) and adult peripheral blood (NPB)(n=3). A number of CD44 transcript variants were detected: v3, v4 (CD44s), v5, v6, v7, v8 plus additional variants. Earlier data suggest a total of 16 different isoforms of CD44. We found a higher overall variant gene expression of CD44 in BC compared to CP. A higher expression of CD44 transcript variant 3 and CD44 transcript variant 5 was detected in both CP and BC compared to CB and NPB. Specific CD44 transcript variant expression patterns distinguished BC progenitors from CP samples. Using splice isoform specific PCR, we were able to confirm isoform variants that were upregulated in the BC CML samples. We also compared the expression of the CD44 transcript variants in young versus old bone marrow in order to exclude that LSC-specific isoform variant expression was expressed in aged patients. We could not detect such a correlation. Conclusions These observations suggest that unique CD44 isoform expression patterns predict progression from CP to BC as well as the generation of TKI resistant LSCs and may be used as biomarkers of response to LSC targeted therapy. Disclosures: Jamieson: J&J, Roche: Research Funding; Sanofi: Membership on an entity’s Board of Directors or advisory committees.
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Ścieżyńska, Aneta, Marta Soszyńska, Michał Komorowski, Anna Podgórska, Natalia Krześniak, Aleksandra Nogowska, Martyna Smolińska, et al. "Molecular Analysis of the ABCA4 Gene Mutations in Patients with Stargardt Disease Using Human Hair Follicles." International Journal of Molecular Sciences 21, no. 10 (May 13, 2020): 3430. http://dx.doi.org/10.3390/ijms21103430.
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ABCA4 gene mutations are the cause of a spectrum of ABCA4 retinopathies, and the most common juvenile macular degeneration is called Stargardt disease. ABCA4 has previously been observed almost exclusively in the retina. Therefore, studying the functional consequences of ABCA4 variants has required advanced molecular analysis techniques. The aim of the present study was to evaluate whether human hair follicles may be used for molecular analysis of the ABCA4 gene splice-site variants in patients with ABCA4 retinopathies. We assessed ABCA4 expression in hair follicles and skin at mRNA and protein levels by means of real-time PCR and Western blot analyses, respectively. We performed cDNA sequencing to reveal the presence of full-length ABCA4 transcripts and analyzed ABCA4 transcripts from three patients with Stargardt disease carrying different splice-site ABCA4 variants: c.5312+1G>A, c.5312+2T>G and c.5836-3C>A. cDNA analysis revealed that c.5312+1G>A, c.5312+2T>G variants led to the skipping of exon 37, and the c.5836-3C>A variant resulted in the insertion of 30 nucleotides into the transcript. Our results strongly argue for the use of hair follicles as a model for the molecular analysis of the pathogenicity of ABCA4 variants in patients with ABCA4 retinopathies.
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D'Agata, Velia, and Sebastiano Cavallaro. "Parkin Transcript Variants in Rat and Human Brain." Neurochemical Research 29, no. 9 (September 2004): 1715–24. http://dx.doi.org/10.1023/b:nere.0000035807.25370.5e.
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Koks, Sulev, Abigail L. Pfaff, Vivien J. Bubb, and John P. Quinn. "Transcript Variants of Genes Involved in Neurodegeneration Are Differentially Regulated by the APOE and MAPT Haplotypes." Genes 12, no. 3 (March 15, 2021): 423. http://dx.doi.org/10.3390/genes12030423.
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Genetic variations at the Apolipoprotein E (ApoE) and microtubule-associated protein tau (MAPT) loci have been implicated in multiple neurogenerative diseases, but their exact molecular mechanisms are unclear. In this study, we performed transcript level linear modelling using the blood whole transcriptome data and genotypes of the 570 subjects in the Parkinson’s Progression Markers Initiative (PPMI) cohort. ApoE, MAPT haplotypes and two SNPs at the SNCA locus (rs356181, rs3910105) were used to detect expression quantitative trait loci eQTLs associated with the transcriptome and differential usage of transcript isoforms. As a result, we identified 151 genes associated with the genotypic variations, 29 cis and 122 trans eQTL positions. Profound effect with genome-wide significance of ApoE e4 haplotype on the expression of TOMM40 transcripts was identified. This finding potentially explains in part the frequently established genetic association with the APOE e4 haplotypes in neurodegenerative diseases. Moreover, MAPT haplotypes had significant differential impact on 23 transcripts from the 17q21.31 and 17q24.1 loci. MAPT haplotypes had also the largest up-regulating (256) and the largest down-regulating (−178) effect sizes measured as β values on two different transcripts from the same gene (LRRC37A2). Intronic SNP in the SNCA gene, rs3910105, differentially induced expression of three SNCA isoforms. In conclusion, this study established clear association between well-known haplotypic variance and transcript specific regulation in the blood. APOE e4 and MAPT H1/H2 haplotypic variants are associated with the expression of several genes related to the neurodegeneration.
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Alfieri, Mariaevelina, Anna Li Santi, Luigia Meo, Valentina Giudice, Carmine Selleri, and Pia Ragno. "Identification of uPAR Variants Acting as ceRNAs in Leukaemia Cells." Cancers 14, no. 8 (April 14, 2022): 1980. http://dx.doi.org/10.3390/cancers14081980.
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The 3′untranslated region (3′UTR) of the urokinase (uPA) receptor (uPAR) mRNA can act as a competitive endogenous RNA (ceRNA) in acute myeloid leukaemia (AML) cells, promoting the expression of pro-tumoral targets, including uPAR. Here, we identified three variants of uPAR mRNA containing the 3′UTR, in KG1 and U937 leukaemia cells expressing low and high uPAR levels, respectively. Identified variants lack exon 5 (uPAR Δ5) or exon 6 (uPAR Δ6) or part of exon 6, exon 7 and part of 3′UTR (uPAR Δ6/7). uPAR Δ5 and uPAR Δ6 transcript levels were higher in U937 cells compared to KG1 cells. Both uPAR variants were expressed also in AML blasts, at higher levels as compared to CD34 hematopoietic cells from healthy donors. The presence of the 3′UTR conferred high instability to the uPAR Δ5 variant transcript, preventing its translation in protein. Overexpression of the uPAR Δ5-3′UTR variant regulated the expression of some pro-tumoral factors previously reported to be regulated by the 3′UTR of uPAR and increased KG1 cell adhesion, migration and proliferation. These results demonstrate the expression of uPAR mRNA variants containing the 3′UTR in AML cells and the ceRNA activity and the biological effects of the uPAR Δ5-3′UTR variant.
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Pogosova-Agadjanyan, Era L., Hana Lee, Crystal K. Cummings, Soheil Meshinchi, Jerald P. Radich, and Derek L. Stirewalt. "Methylation Regulates Expression of Novel Splice Variant and Wild Type Transcripts of IRF8." Blood 116, no. 21 (November 19, 2010): 3634. http://dx.doi.org/10.1182/blood.v116.21.3634.3634.
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Abstract Abstract 3634 Introduction: Interferon regulatory factor 8 (IRF8) is a transcription factor that plays a critical role in normal hematopoiesis. IRF8−/− transgenic mice develop a myeloproliferative syndrome that transforms to acute myeloid leukemia (AML). IRF8 expression varies dramatically in the blasts of AML patients. A number of biological processes, including epigenetic changes, mutations, or alternative splicing, may contribute to the variability of IRF8 expression in AML patients. We investigated the potential causes of the aberrant IRF8 expression in AML blasts. Wild-type transcript (IRF8-WT): The entire coding sequence of IRF8 was amplified using HiFi Taq polymerase and sequenced from 7 leukemic cell lines and 12 AML samples. We did not find any mutations associated with aberrant expression of IRF8. Splice Variants (IRF8-SVs): The initial studies examining IRF8 coding region suggested transcript deviation in the 5′ region. The GeneRacer kit was used to sequence 5′-capped mRNA. We identified 3 previously-undescribed transcript variants. In all 3 sequences, exon 1 was spliced out and replaced by nucleotides from the terminal end of intron 1 (Figure 1A). Some of the splice variants contained potential in-frame start codons. Expression of 5′ Splice Variants in Leukemic Cell Lines and AML Samples: We examined the expression levels of transcript variants in 12 leukemic cell lines, 246 AML samples, and hematopoietic subpopulations of cells from “healthy” adults. IRF8-SVs were expressed at significantly higher levels in some AML blasts than normal hematopoietic cells. In fact, AML cell lines with the highest IRF8 levels primarily expressed the IRF8-SVs rather than the IRF8-WT. Promoter Methylation: Interferon response element (pIRE) within the IRF8 promoter, which controls the expression of the gene, was examined by PCR after bisulfite conversion. Cell lines with very low IRF8 expression were often methylated at the pIRE locus. In addition, some cell lines with marked over-expression of IFR8-SVs also displayed methylated pIRE, suggesting that promoter methylation may also be playing a role in controlling the expression of the splice variants. pIRE methylation was also examined in a more limited number of AML samples, finding that blasts with low expression of IRF8 were methylated at this locus (Figure 1B). Reversal of Splice Variant Expression Pattern: Our previous studies suggested that methylation may play a role in controlling IRF8-SVs expression. Therefore, we examined the effect of demethylating agents on IRF8-SVs expression in U937 cells – a leukemic cell line with very high levels of IRF8-SVs. IRF8 transcripts (WT and SVs) were examined before and after exposure to therapeutic levels of 5-azacytidine (5-Aza). These studies suggested that 5-Aza exposure and subsequent demethylation of the pIRE locus was associated with restoration of the IRF8-WT and decreased IRF8-SVs expression (Figure 1C). This later data provides additional evidence that pIRE hypermethylation may regulate the expression of the novel IRF8-SVs. Conclusions: We have identified novel IRF8 transcript variants that are over-expressed in AML blasts. These novel IRF8-SVs introduce additional nucleotides at the 5′ region of the gene and may result in a new start codon. AML blasts with high levels of IRF8 transcript often over-express IRF8-SVs. Although the functional significance of these variants is unknown, methylation may play a role in regulating their expression, such that demethylating agents decrease IRF8-SVs and promote the IRF8-WT expression. Additional studies are planned to investigate the biology and clinical significance of IRF8-SVs. Initial studies suggests that over-expression of IRF8-SVs is associated with an inferior clinical outcome for adult AML patients. Disclosures: No relevant conflicts of interest to declare.
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Dmytrenko, O. O., I. V. Dmytrenko, Zh M. Minchenko, and I. S. Diahil. "АЛЕЛЬНИЙ ПОЛІМОРФІЗМ СИСТЕМИ HLA У ХВОРИХ НА ХРОНІЧНУ МІЄЛОЇДНУ ЛЕЙКЕМІЮ З е13а2 та е14а2 ТРАНСКРИПТАМИ ГЕНА BCR/ABL1." Scientific Issue Ternopil Volodymyr Hnatiuk National Pedagogical University. Series: Biology 76, no. 2 (July 26, 2019): 53–57. http://dx.doi.org/10.25128/2078-2357.19.2.9.
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To study the associative relation of polymorphic HLA gene variants and BCR/ABL1 transcript types in patients with chronic myeloid leukemia (CML) 87 CML patients were examined. 42 patients had e13a2 BCR/ABL1 transcript and 45 patients had e14a2 BCR/ABL1 transcript. The prevalence of the genes allelic variants of major histocompatibility complex was analyzed and the association coefficients for the disease risk depending on the presence of certain types of BCR/ABL1 transcripts were calculated.
Unconditional markers of increased risk of CML (HLA-DRB1*11) and markers of resistance to CML (HLA-A*03) were highlighted. The allele frequencies of HLA-A*03, HLA-A*68, HLA-B*08, HLA-B*15, HLA-B*40, HLA-DRB1*04, DQB1*06 were significantly reduced and allele frequencies of HLA-DRB1*12 and DRB1*11 were increased in patients with the e13a2 transcript compared to healthy people. The allele frequencies of HLA-A*03, HLA-A*11, HLA-B*08, HLA-B*14, HLA-B*40, HLA-DRB1*04 and DQB1* 03 were significantly reduced and the allele frequency of HLA-DRB1*11 was significantly increased in patients with e14a2 transcript compared to healthy persons. Thus, the individual analysis of the complex of fusion proteins e13a2 and e14a2 and HLA alleles in CML patients could indicate the additive effect of both molecular structures joint carrier for CML developing risk.
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Janecke, Andreas R., Xiaoqin Liu, Rüdiger Adam, Sumanth Punuru, Arne Viestenz, Valeria Strauß, Martin Laass, et al. "Pathogenic STX3 variants affecting the retinal and intestinal transcripts cause an early-onset severe retinal dystrophy in microvillus inclusion disease subjects." Human Genetics 140, no. 8 (May 11, 2021): 1143–56. http://dx.doi.org/10.1007/s00439-021-02284-1.
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AbstractBiallelic STX3 variants were previously reported in five individuals with the severe congenital enteropathy, microvillus inclusion disease (MVID). Here, we provide a significant extension of the phenotypic spectrum caused by STX3 variants. We report ten individuals of diverse geographic origin with biallelic STX3 loss-of-function variants, identified through exome sequencing, single-nucleotide polymorphism array-based homozygosity mapping, and international collaboration. The evaluated individuals all presented with MVID. Eight individuals also displayed early-onset severe retinal dystrophy, i.e., syndromic—intestinal and retinal—disease. These individuals harbored STX3 variants that affected both the retinal and intestinal STX3 transcripts, whereas STX3 variants affected only the intestinal transcript in individuals with solitary MVID. That STX3 is essential for retinal photoreceptor survival was confirmed by the creation of a rod photoreceptor-specific STX3 knockout mouse model which revealed a time-dependent reduction in the number of rod photoreceptors, thinning of the outer nuclear layer, and the eventual loss of both rod and cone photoreceptors. Together, our results provide a link between STX3 loss-of-function variants and a human retinal dystrophy. Depending on the genomic site of a human loss-of-function STX3 variant, it can cause MVID, the novel intestinal-retinal syndrome reported here or, hypothetically, an isolated retinal dystrophy.
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Villegas-Ruíz, Vanessa, Antonio Romo-Mancillas, Isabel Medina-Vera, Kattia Alejandra Castro-López, Josselene Carina Ramirez-Chiquito, Marco Antonio Fonseca-Montaño, Mercedes Edna García-Cruz, Roberto Rivera-Luna, Julieta Griselda Mendoza-Torreblanca, and Sergio Juárez-Méndez. "The Proliferating Cell Nuclear Antigen (PCNA) Transcript Variants as Potential Relapse Markers in B-Cell Acute Lymphoblastic Leukemia." Cells 11, no. 20 (October 12, 2022): 3205. http://dx.doi.org/10.3390/cells11203205.
Full textAbstract:
Leukemia is the most common childhood malignancy in Mexico, representing more than 50% of all childhood cancers. Although treatment leads to a survival of up to 90% in developing countries, in our country, it is less than 65%. Additionally, ~30% of patients relapse with poor prognosis. Alternative splicing plays an important role in transcriptome diversity and cellular biology. This mechanism promotes an increase in the assortment of proteins with potentially distinct functions from a single gene. The proliferating cell nuclear antigen (PCNA) gene encodes two transcripts for the same protein of 261 amino acids, which is associated with several important cellular processes and with several types of cancer. However, the diversity of the transcript variants expressed in this condition is not clear. Then, we used microarray gene expression to identify changes in the exon expression level of PCNA. The data were validated using RT‒PCR and Sanger sequencing, and three additional transcripts (PCNA_V3, PCNA_V4, and PCNA_V5) were identified. Computational analyses were used to determine the potential proteins resulting, their structure, and interactions with PCNA native protein and themselves. Additionally, the PCNA transcript variants were inhibited using specific siRNA, determining that their inhibition contributes to the malignant characteristics in vitro. Finally, we quantified the PCNA transcript variants in acute lymphoblastic leukemia samples and identified their expression in this disease. Based on the clinical characteristics, we determined that PCNA_V2 and PCNA_V4 are expressed at significantly low levels in relapsed B-ALL patients. We conclude that the low expression of PCNA_V2 and PCNA_V4 could be a potential molecular marker of relapse in acute lymphoblastic leukemia patients.
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Bowles, Bradley, Karl Clark, and Eric Klee. "95233 Analysis of 5'UTR Variation in Rare Disease Patients Reveals Variants of Potential Disease Relevance." Journal of Clinical and Translational Science 5, s1 (March 2021): 101. http://dx.doi.org/10.1017/cts.2021.659.
Full textAbstract:
ABSTRACT IMPACT: This work sheds diagnostic insight on patients with idiopathic rare disease and has the potential to further their care and treatment as a result. OBJECTIVES/GOALS: Correct diagnosis is imperative to treating patients with idiopathic, suspected genetic conditions, yet sequencing approaches leave up to 70% of these patients undiagnosed. We sought to improve diagnosis rates for a cohort patients referred for sequencing by characterizing deleterious variants within the 5’UTR. METHODS/STUDY POPULATION: We retrospectively analyzed whole exome sequencing (WES) data from 472 unsolved rare disease patients within the Mayo Clinic Center for Individualized Medicine to identify variants within the 5’UTR that affect the presence of upstream open reading frames (uORFs). uORFs are short regions (typically 30bp - 600bp) that typically influence downstream gene translation by sequestering ribosomes. We specifically searched for variants with the potential to disrupt existing uORFs or introduce new uORFs within the 5’UTR, and developed a pipeline to annotate these variants with information including GnomAD allele frequency and gene loss of function intolerance (pLI) score. To aid in variant interpretation, we applied two deep learning tools to predict variant impacts on transcript ribosome load (TITER and FramePool). RESULTS/ANTICIPATED RESULTS: Our pipeline identified a median of 21 variants per patient that were predicted to have a deleterious impact on the translational efficiency of protein coding transcripts, primarily by introducing new start codons within the 5’UTR or by altering the Kozak consensus of existing start codons. A median of 10 of these variants occur upstream of haploinsufficient genes with an existing disease association. We also identified a subset of variants that are predicted to introduce translationally active N-terminal extensions to protein coding transcripts, with the potential to disrupt protein localization and processing. DISCUSSION/SIGNIFICANCE OF FINDINGS: This work demonstrates that analysis of 5’UTR variants can be incorporated into existing WES pipelines, and identifies a group of variants with potential significance to patient disease. Further experimental evidence is necessary to ascertain the pathogenicity of these variants.