Academic literature on the topic 'Intron-exon boundary prediction'

Abstract Abstract 1223 Introduction/Background: Hemophilia A is a congenital X-linked bleeding disorder caused by various mutations in the coagulation factor VIII gene (F8). However, recent studies have described that no genetic mutation could be found in the F8 of about 2% of hemophilia A patients, even after nucleotide sequencing including the entire coding region, exon/intron boundaries, and the 5'- and 3'-untranslated region (Vidal et al, 2001; Klopp et al, 2002). Factor VIII deficient mechanisms underlying this phenomenon remain unexplained. To further elucidate the mechanisms causing hemophilia A in these patients, we performed a detailed analysis of F8 mRNA. Materials and methods: F8 mRNA from a Japanese hemophilia A patient with undetectable mutations was analyzed. Total RNA was isolated from peripheral blood cells using a QIAamp® RNA Blood Mini Kit (Qiagen) or PAXgene® Blood RNA Kit (Qiagen). Both preparations were performed following the manufacturer's instructions. In order to analyze the F8 mRNA, we performed the cDNA-amplification in two rounds of PCR using the nested approach reported by El-Maarri et al (2005). The nucleotide sequences of primer used followed those of their report. OneStep RT-PCR Kit (Qiagen) and TaKaRa LA Taq ™ (TaKaRa) were used for first and second round PCR amplification, respectively. Ectopic F8 mRNA expression level was relatively quantified by a real-time PCR technique using 4 TaqMan gene expression assays (Hs00240767_m1 amplify exon 1–2 boundary, Hs01109548_m1 amplify exon 6–7 boundary, Hs01109541_m1 amplify exon 14–15 boundary, Hs01109543_m1 amplify exon 20–21 boundary; Applied Biosystems). Results: Because the size of the F8 mRNA is very large ∼9kb, the entire F8 cDNA was divided into four different regions: exons 1–8 (region A); exons 8–14 (region B); exons 14–22 (region C); and exons 19–26 (region D) and amplified in the first round. Then, each of four regions were further divided into two different regions (a total of 8 overlapping regions; region 1–8), and amplified in the second round. An abnormality was observed in the amplification. Although the PCR products of regions 1 and 2, (region A), were obtained, the products remaining in all later regions (regions 3–8) were not. A similar phenomenon was also confirmed in the semi-quantification of the mRNA. Though we were able to quantify the mRNA by using both exon 1–2 and 5–6 boundary amplifications, we were not able to quantify the mRNA using the 14–15 and 20–21 boundaries. These results suggested that the quantity of the mRNA decrease remarkably in the vicinity of exon 8 as a boundary. Further analysis of the mRNA showed that quantity of the mRNA is normal from exon 1 through 9. Nucleotide sequencing of intron 9 revealed a single nucleotide substitution, adenine to guanine transition, at 602bp downstream from the 3' end of exon 9. This transition has not been registered in any international database as a mutation or a polymorphism and was not found in the F8 from 124 Japanese. These results strongly suggest that the transition is very rare and may be involved in factor VIII deficiency in these patients. Analysis of the nucleotide sequence of the substitution by splicing site prediction software predicted the formation of a new acceptor splice site. This result suggested the existence of splice abnormality. However, further characterization is needed to elucidate the mechanism that causes the decrease in mRNA in the middle of the gene. Conclusion: The mechanism behind factor VIII deficiency in hemophilia A patients with undetectable mutations is very interesting and various possibilities are conceivable. This study provides the possibility that some causative genetic abnormality remains in a further unanalyzed F8 region, most likely deep inside the intron, of these patients. Disclosures: No relevant conflicts of interest to declare.

20027 Background: The considerably high costs of BRCA1/2 mutation screening together with the expanding request for inherited breast/ovarian cancer genetic counselling and testing, dictates the need for more accurate selection of the appropriate candidates for analysis. The aim of the present study was to evaluate, the impact that careful selection of appropriate candidates by means of BRCAPRO, Myriad tables and IC software prediction models might exert on reduction of societal costs. Methods: 86 unrelated high risk breast/ovarian cancer families were selected for mutation screening; BRCA1/2 entire coding sequences and each intron/exon boundary were screened by direct sequencing (PTT limited to BRCA1 exon 11). The a priori probability of carrying a pathologic BRCA1/2 germline mutation was calculated by either BRCAPRO or IC software (country-customized versions of BRCAPRO), or by the Myriad prevalence mutation tables (2004). We classified our index cases as “carrier-positive” when their carrier probability exceeds 10%. According to recent literature, our analysis assumed a cost of $1,713 for genes sequencing of each proband. Results: We observed 19 pathogenic germline mutations with an overall detection rate of 22.1%. IC software and Myriad tables were more efficient in predicting mutated cases, showing higher sensitivity (95% and 84%, respectively) and a better negative predictive value (NPV, 96% and 91%, respectively) compared to BRCAPRO (sensitivity 74% and NPV 87%). Particularly, the IC software misclassified a pathogenic mutation carrier in only one case. The costs afforded for genetic testing on the whole sample was $147,318. The family selection assisted by the IC software, BRCAPRO, Myriad tables could result in a total cost of $104,493, $78,798 and $90,789, respectively. Even maintaining high accuracy in carrier status prediction, the country-customized version of BRCAPRO imparted more than 30% cost reduction. Conclusions: Considering a balance between economical advantages and the different performances in mutated status prediction, our analysis suggest that the country-customized versions of BRCAPRO and Myriad Tables allow efficient selection of candidates for BRCA1/2 mutation analysis and a significant impact on societal costs. No significant financial relationships to disclose.

Abstract Antigens of the Rh blood group system are encoded by 2 homologous genes, RHD and RHCE, that produce 2 red cell membrane proteins. The D-negative phenotype is considered to result, almost invariably, from homozygosity for a complete deletion ofRHD. The basis of all PCR tests for predicting fetal D phenotype from DNA obtained from amniocytes or maternal plasma is detection of the presence of RHD. These tests are used in order to ascertain the risk of hemolytic disease of the newborn. We have identified an RHD pseudogene (RHD ψ) in Rh D-negative Africans. RHDψ contains a 37 base pair (bp) insert in exon 4, which may introduce a stop codon at position 210. The insert is a sequence duplication across the boundary of intron 3 and exon 4.RHDψ contains another stop codon in exon 6. The frequency ofRHDψ in black South Africans is approximately 0.0714. Of 82 D-negative black Africans, 66% hadRHDψ, 15% had the RHD-CE-D hybrid gene associated with the VS+ V– phenotype, and only 18% completely lackedRHD. RHDψ is present in about 24% of D-negative African Americans and 17% of D-negative South Africans of mixed race. No RHD transcript could be detected in D-negative individuals with RHDψ, probably as a result of nonsense-mediated mRNA decay. Existing PCR-based methods for predicting D phenotype from DNA are not suitable for testing Africans or any population containing a substantial proportion of people with African ethnicity. Consequently, we have developed a new test that detects the 37 bp insert in exon 4 of RHDψ. (Blood. 2000; 95:12-18)

Antigens of the Rh blood group system are encoded by 2 homologous genes, RHD and RHCE, that produce 2 red cell membrane proteins. The D-negative phenotype is considered to result, almost invariably, from homozygosity for a complete deletion ofRHD. The basis of all PCR tests for predicting fetal D phenotype from DNA obtained from amniocytes or maternal plasma is detection of the presence of RHD. These tests are used in order to ascertain the risk of hemolytic disease of the newborn. We have identified an RHD pseudogene (RHD ψ) in Rh D-negative Africans. RHDψ contains a 37 base pair (bp) insert in exon 4, which may introduce a stop codon at position 210. The insert is a sequence duplication across the boundary of intron 3 and exon 4.RHDψ contains another stop codon in exon 6. The frequency ofRHDψ in black South Africans is approximately 0.0714. Of 82 D-negative black Africans, 66% hadRHDψ, 15% had the RHD-CE-D hybrid gene associated with the VS+ V– phenotype, and only 18% completely lackedRHD. RHDψ is present in about 24% of D-negative African Americans and 17% of D-negative South Africans of mixed race. No RHD transcript could be detected in D-negative individuals with RHDψ, probably as a result of nonsense-mediated mRNA decay. Existing PCR-based methods for predicting D phenotype from DNA are not suitable for testing Africans or any population containing a substantial proportion of people with African ethnicity. Consequently, we have developed a new test that detects the 37 bp insert in exon 4 of RHDψ. (Blood. 2000; 95:12-18)

Biallelic variants in USH2A are associated with retinitis pigmentosa (RP) and Type 2 Usher Syndrome (USH2), leading to impaired vision and, additionally, hearing loss in the latter. Although the introduction of next-generation sequencing into clinical diagnostics has led to a significant uplift in molecular diagnostic rates, many patients remain molecularly unsolved. It is thought that non-coding variants or variants of uncertain significance contribute significantly to this diagnostic gap. This study aims to demonstrate the clinical utility of the reverse transcription–polymerase chain reaction (RT-PCR)–Oxford Nanopore Technology (ONT) sequencing of USH2A mRNA transcripts from nasal epithelial cells to determine the splice-altering effect of candidate variants. Five affected individuals with USH2 or non-syndromic RP who had undergone whole genome sequencing were recruited for further investigation. All individuals had uncertain genotypes in USH2A, including deep intronic rare variants, c.8682-654C>G, c.9055+389G>A, and c.9959-2971C>T; a synonymous variant of uncertain significance, c.2139C>T; p.(Gly713=); and a predicted loss of function duplication spanning an intron/exon boundary, c.3812-3_3837dup p.(Met1280Ter). In silico assessment using SpliceAI provided splice-altering predictions for all candidate variants which were investigated using ONT sequencing. All predictions were found to be accurate; however, in the case of c.3812-3_3837dup, the outcome was a complex cryptic splicing pattern with predominant in-frame exon 18 skipping and a low level of exon 18 inclusion leading to the predicted stop gain. This study detected and functionally characterised simple and complex mis-splicing patterns in USH2A arising from previously unknown deep intronic variants and previously reported variants of uncertain significance, confirming the pathogenicity of the variants.

Abstract Alternative splicing (AS), in higher eukaryotes, is one of the mechanisms of post-transcriptional regulation that generate multiple transcripts from the same gene. One particular mode of AS is the skipping event where an exon may be alternatively excluded or constitutively included in the resulting mature mRNA. Both transcript isoforms from this skipping event site, i.e. in which the exon is either included (inclusion isoform) or excluded (skipping isoform), are typically present in one cell, and maintain a subtle balance that is vital to cellular function and dynamics. However, how the prevailing conditions dictate which isoform is expressed and what biological factors might influence the regulation of this process remain areas requiring further exploration. In this study, we have developed a novel computational method, graph-based exon-skipping scanner (GESS), for de novo detection of skipping event sites from raw RNA-seq reads without prior knowledge of gene annotations, as well as for determining the dominant isoform generated from such sites. We have applied our method to publicly available RNA-seq data in GM12878 and K562 cells from the ENCODE consortium and experimentally validated several skipping site predictions by RT-PCR. Furthermore, we integrated other sequencing-based genomic data to investigate the impact of splicing activities, transcription factors (TFs) and epigenetic histone modifications on splicing outcomes. Our computational analysis found that splice sites within the skipping-isoform-dominated group (SIDG) tended to exhibit weaker MaxEntScan-calculated splice site strength around middle, ‘skipping’, exons compared to those in the inclusion-isoform-dominated group (IIDG). We further showed the positional preference pattern of splicing factors, characterized by enrichment in the intronic splice sites immediately bordering middle exons. Finally, our analysis suggested that different epigenetic factors may introduce a variable obstacle in the process of exon–intron boundary establishment leading to skipping events.

Abstract Background This study aims to analyze the pathogenic gene in a Chinese family with non-syndromic hearing loss and identify a novel mutation site in the TNC gene. Methods A five-generation Chinese family from Anhui Province, presenting with autosomal dominant non-syndromic hearing loss, was recruited for this study. By analyzing the family history, conducting clinical examinations, and performing genetic analysis, we have thoroughly investigated potential pathogenic factors in this family. The peripheral blood samples were obtained from 20 family members, and the pathogenic genes were identified through whole exome sequencing. Subsequently, the mutation of gene locus was confirmed using Sanger sequencing. The conservation of TNC mutation sites was assessed using Clustal Omega software. We utilized functional prediction software including dbscSNV_AdaBoost, dbscSNV_RandomForest, NNSplice, NetGene2, and Mutation Taster to accurately predict the pathogenicity of these mutations. Furthermore, exon deletions were validated through RT-PCR analysis. Results The family exhibited autosomal dominant, progressive, post-lingual, non-syndromic hearing loss. A novel synonymous variant (c.5247A > T, p.Gly1749Gly) in TNC was identified in affected members. This variant is situated at the exon–intron junction boundary towards the end of exon 18. Notably, glycine residue at position 1749 is highly conserved across various species. Bioinformatics analysis indicates that this synonymous mutation leads to the disruption of the 5' end donor splicing site in the 18th intron of the TNC gene. Meanwhile, verification experiments have demonstrated that this synonymous mutation disrupts the splicing process of exon 18, leading to complete exon 18 skipping and direct splicing between exons 17 and 19. Conclusion This novel splice-altering variant (c.5247A > T, p.Gly1749Gly) in exon 18 of the TNC gene disrupts normal gene splicing and causes hearing loss among HBD families.

Exon capture coupled to high-throughput sequencing constitutes a cost-effective technical solution for addressing specific questions in evolutionary biology by focusing on expressed regions of the genome preferentially targeted by selection. Transcriptome-based capture, a process that can be used to capture the exons of non-model species, is use in phylogenomics. However, its use in population genomics remains rare due to the high costs of sequencing large numbers of indexed individuals across multiple populations. We evaluated the feasibility of combining transcriptome-based capture and the pooling of tissues from numerous individuals for DNA extraction as a cost-effective, generic and robust approach to estimating the variant allele frequencies of any species at the population level. We designed capture probes for ~5 Mb of chosen de novo transcripts from the Asian ladybird Harmonia axyridis (5,717 transcripts). We called ~300,000 bi-allelic SNPs for a pool of 36 non-indexed individuals. Capture efficiency was high, and pool-seq was as effective and accurate as individual-seq for detecting variants and estimating allele frequencies. Finally, we also evaluated an approach for simplifying bioinformatic analyses by mapping genomic reads directly to targeted transcript sequences to obtain coding variants. This approach is effective and does not affect the estimation of SNP allele frequencies, except for a small bias close to some exon ends. We demonstrate that this approach can also be used to predict the intron-exon boundaries of targeted de novo transcripts, making it possible to abolish genotyping biases near exon ends.

AbstractBrain-specific angiogenesis inhibitor 1 (BAI1) belongs to the adhesion G-protein-coupled receptors, which exhibit large multi-domain extracellular N termini that mediate cell–cell and cell–matrix interactions. To explore the existence of BAI1 isoforms, we queried genomic datasets for markers of active chromatin and new transcript variants in the ADGRB1 (adhesion G-protein-coupled receptor B1) gene. Two major types of mRNAs were identified in human/mouse brain, those with a start codon in exon 2 encoding a full-length protein of a predicted size of 173.5/173.3 kDa and shorter transcripts starting from alternative exons at the intron 17/exon 18 boundary with new or exon 19 start codons, predicting two shorter isoforms of 76.9/76.4 and 70.8/70.5 kDa, respectively. Immunoblots on wild-type and Adgrb1 exon 2-deleted mice, reverse transcription PCR, and promoter-luciferase reporter assay confirmed that the shorter isoforms originate from an alternative promoter in intron 17. The shorter BAI1 isoforms lack most of the N terminus and are very close in structure to the truncated BAI1 isoform generated through GPS processing from the full-length receptor. The cleaved BAI1 isoform has a 19 amino acid extracellular stalk that may serve as a receptor agonist, while the alternative transcripts generate BAI1 isoforms with extracellular N termini of 5 or 60 amino acids. Further studies are warranted to compare the functions of these isoforms and examine the distinct roles they play in different tissues and cell types.