Flanking Exon Research Articles

De Novo Mutations Contributes Approximately 7% of Pathogenicity in Inherited Eye Diseases.

The purpose of this study was to describe genotype-phenotype associations and novel insights into genetic characteristics in a trio-based cohort of inherited eye diseases (IEDs). To determine the etiological role of de novo mutations (DNMs) and genetic profile in IEDs, we retrospectively reviewed a large cohort of proband-parent trios of Chinese origin. The patients underwent a detailed examination and was clinically diagnosed by an ophthalmologist. Panel-based targeted exome sequencing was performed on DNA extracted from blood samples, containing coding regions of 792 IED-causative genes and their flanking exons. All participants underwent genetic testing. All proband-parent trios were divided into 22 subgroups, the overall diagnostic yield was 48.67% (605/1243), ranging from 4% to 94.44% for each of the subgroups. A total of 108 IED-causative genes were identified, with the top 24 genes explaining 67% of the 605 genetically solved trios. The genetic etiology of 6.76% (84/1243) of the trio was attributed to disease-causative DNMs, and the top 3 subgroups with the highest incidence of DNM were aniridia (n = 40%), Marfan syndrome/ectopia lentis (n = 38.78%), and retinoblastoma (n = 37.04%). The top 10 genes have a diagnostic yield of DNM greater than 3.5% in their subgroups, including PAX6 (40.00%), FBN1 (38.78%), RB1 (37.04%), CRX (10.34%), CHM (9.09%), WFS1 (8.00%), RP1L1 (5.88%), RS1 (5.26%), PCDH15 (4.00%), and ABCA4 (3.51%). Additionally, the incidence of DNM in offspring showed a trend of correlation with paternal age at reproduction, but not statistically significant with paternal (P = 0.154) and maternal (P = 0.959) age at reproduction. Trios-based genetic analysis has high accuracy and validity. Our study helps to quantify the burden of the full spectrum IED caused by each gene, offers novel potential for elucidating etiology, and plays a crucial role in genetic counseling and patient management.

Characterization, mRNA expression profile, subcellular distribution and association analysis with piglet diarrhea of porcine matrix metallopeptidase 7 (pMMP7)

Matrix metalloproteinase 7 (MMP7) is involved in the degradation of extracellular matrix in disease processes and therefore plays an important role in host disease resistance/susceptibility. To better understanding the effects of porcine MMP7 (pMMP7) on piglets diarrhea trait, we characterized pMMP7 gene, identified genetic variations in pMMP7 and explored the relationship between pMMP7 polymorphisms and piglets diarrhea in Min pig and Landrace populations. The complete coding sequence of pMMP7 is 804 bp encoding a protein of 267 amino acids. Sequence alignment showed that the identity between pMMP7 and human MMP7 was approximately 80%. The expression of pMMP7 in the gut of healthy piglets were weak and the distribution of the pMMP7-EGFP fusion protein was observed mainly in the cytoplasm. After the identification of 21 genetic variations in 5′ flanking region and exons, Hae III and Eco72 Ⅰ PCR-RFLP were established to genotype SNP rs327380117 and rs329429922, respectively. Statistical analysis indicated that Landrace piglets with a TT genotype at rs327380117 had a lower diarrhea score and day-14 wt than TC piglets (p < 0.05); the diarrhea score of AA Landrace animals with rs329429922 was lower than that of GG individuals (p < 0.05). The findings presented here contribute to the understanding of the biological function of pMMP7 and may provide new molecular markers for pig breeding.

Base editing-mediated perturbation of endogenous PKM1/2 splicing facilitates isoform-specific functional analysis in vitro and in vivo.

Objectives PKM1 and PKM2, which are generated from the alternative splicing of PKM gene, play important roles in tumourigenesis and embryonic development as rate‐limiting enzymes in glycolytic pathway. However, because of the lack of appropriate techniques, the specific functions of the 2 PKM splicing isoforms have not been clarified endogenously yet.Materials and methodsIn this study, we used CRISPR‐based base editors to perturbate the endogenous alternative splicing of PKM by introducing mutations into the splicing junction sites in HCT116 cells and zebrafish embryos. Sanger sequencing, agarose gel electrophoresis and targeted deep sequencing assays were utilized for identifying mutation efficiencies and detecting PKM1/2 splicing isoforms. Cell proliferation assays and RNA‐seq analysis were performed to describe the effects of perturbation of PKM1/2 splicing in tumour cell growth and zebrafish embryo development.ResultsThe splicing sites of PKM, a 5’ donor site of GT and a 3’ acceptor site of AG, were efficiently mutated by cytosine base editor (CBE; BE4max) and adenine base editor (ABE; ABEmax‐NG) with guide RNAs (gRNAs) targeting the splicing sites flanking exons 9 and 10 in HCT116 cells and/or zebrafish embryos. The mutations of the 5’ donor sites of GT flanking exons 9 or 10 into GC resulted in specific loss of PKM1 or PKM2 expression as well as the increase in PKM2 or PKM1 respectively. Specific loss of PKM1 promoted cell proliferation of HCT116 cells and upregulated the expression of cell cycle regulators related to DNA replication and cell cycle phase transition. In contrast, specific loss of PKM2 suppressed cell growth of HCT116 cells and resulted in growth retardation of zebrafish. Meanwhile, we found that mutation of PKM1/2 splicing sites also perturbated the expression of non‐canonical PKM isoforms and produced some novel splicing isoforms.ConclusionsThis work proved that CRISPR‐based base editing strategy can be used to disrupt the endogenous alternative splicing of genes of interest to study the function of specific splicing isoforms in vitro and in vivo. It also reminded us to notice some novel or undesirable splicing isoforms by targeting the splicing junction sites using base editors. In sum, we establish a platform to perturbate endogenous RNA splicing for functional investigation or genetic correction of abnormal splicing events in human diseases.

Abnormal Splicing Patterns and Novel Intron Retentions Affect Genes Regulating Splicing, NF-Kb, ERK1/2, and TP53 in Waldenstrom's Macroglobulinemia

Waldenstrom's Macroglobulinemia (WM) is characterized by activating mutations in MYD88 and CXCR4 in in 93-95% and 30-40% of patient's, respectively. Functional analysis of these mutations has revealed downstream activation of NF-kB, through BTK/IRAK as well as AKT/ERK activation downstream of HCK and CXCR4. Transcriptome analysis using next generation sequencing demonstrated strong upregulation of BCL2 and overall dysregulation of the BCL2 family of genes.Isoform based evaluation of splicing assumes that the data consists of a mixture of known isoforms, which may not be a reasonable assumption for a cancer transcriptome wherein mutational events and transcription factor dysregulation may cause unique combinations of splicing events not observed in healthy donors (HD). To explore alternative splicing in Waldenstrom's Macroglobulinemia (WM), we developed a MISO (Katz et al, Nat Meth 2010) based protocol to analyze next generation RNA sequencing data from 77 WM patients, 16 HD plasma cell samples, 9 HD CD19+CD27- peripheral blood B-cells and 9 HD CD19+CD27+ memory B-cells. Fifty-five patients harbored MYD88 mutations, 20 of whom also carried activating mutations in CXCR4. Twenty-two patients were wild-type (WT) for both genes, and was specifically enriched to insure adequate numbers for comparative study. To identify dysregulated splicing events, the memory B-cell cohort was merged and down-sampled for use as common comparator control for the WM and other HD samples. Estimating the variance in the control group is critical for statistical analysis so a jackknife permutation analysis where control samples pulled out and compared to the remaining samples was performed. This analysis was build into an automated pipeline based on two-pass STAR aligned bam files and corresponding tools to aggregate and analyze the data were developed using R. Novel intron retentions were investigated by identifying intronic regions not overlapping known exons. The number or reads mapping to these regions relative their flanking exons was calculated and compared.Filtering against events observed in the control permutation analysis and other healthy donor samples allowed for the identification of individual splicing events associated with WM. Splicing events were present in genes that regulated splicing itself (SF1, PTBP1), as well as pathways critical to WM growth and survival including NF-kB (ZFAND5, LYN, PKCD), ERK1/2 (CTBP1, VPS28, CCM2), and TP53 (PML, EIF4G2). PCR was successfully used to validate many of these splicing events (Figure 1). Preliminary intron retention analysis identified 4/77 (5%) of patients had intronic retentions on CD79B that were caused by intronic somatic mutations near the intron exon boundary (Figure 2). CD79B is a critical component of the B-cell receptor pathway and coding mutations have been observed in 7-15% of WM patients (Hunter et al, Blood 2013; Poulain et al, Am J Hematol, 2013).The retentions identified in this study represent a novel form of CD79B mutation that has not been previously reported. Additional analysis and functional characterization of these events is planned. These studies aim to identify novel biomarkers and therapeutic targets for WM while developing analytical tools applicable to other malignancies. [Display omitted] DisclosuresCastillo:Janssen: Consultancy, Research Funding; Millennium: Research Funding; Pharmacyclics: Consultancy, Research Funding; Abbvie: Research Funding. Anderson:MedImmune: Membership on an entity's Board of Directors or advisory committees; Millenium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Oncopep: Other: scientific founder; Gilead Sciences: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Other: scientific founder; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Treon:Pharmacyclics: Consultancy, Research Funding.

A generic assay for the identification of splicing variants that induce nonsense-mediated decay in Pompe disease

DNA variants affecting mRNA expression and processing in genetic diseases are often missed or poorly characterized. We previously reported a generic assay to identify variants that affect mRNA expression and splicing in Pompe disease, a monogenic disorder caused by deficiency of acid α-glucosidase (GAA). However, this assay could miss mRNA that is subjected to degradation. Here, we inhibited mRNA degradation using cycloheximide and performed unbiased splicing analysis of all GAA exons using exon flanking RT-PCR and exon internal RT-qPCR. In four patients that were suspected of harboring splicing variants but for which aberrant splicing could not be detected in normally growing cells, we detected a total of 10 novel splicing events in cells treated with cycloheximide. In addition, we found that sequences of GAA introns 6 and 12 were naturally included in a subset of transcripts from patients and healthy controls, indicating inefficient canonical splicing. Identification of aberrant splicing caused by the common Asian variant c.546G>T allowed the development of an antisense oligonucleotide that promoted canonical GAA pre-mRNA splicing and elevated GAA enzymatic activity. Our results indicate that this extended generic splicing assay allows the detection of aberrant splicing in cases of mRNA degradation to enable functional analysis of unknown splicing variants and the development of targeted treatment options.

Conjugation as a Highly Sensitive Assay to Study Group II Intron Splicing In Vivo.

Group II introns are noncoding sequences that interrupt genes, and that must be removed or spliced-out at the RNA level during gene expression. Following the transcription of interrupted genes, group II introns self-splice while concurrently ligating their flanking exons to generate mature mRNAs ready for translation. Ll.LtrB, the model group II intron from the gram-positive bacterium Lactococcus lactis, interrupts the gene coding for a relaxase enzyme that initiates the transfer of mobile elements by conjugation. This functional link between group II intron splicing and conjugative transfer enabled us to engineer highly sensitive splicing assays using the native biological context of Ll.LtrB. The splicing efficiency/conjugation assay was developed to determine the splicing competence of various Ll.LtrB mutants, whereas the splicing selection/conjugation assay was established to isolate splicing-proficient variants from a randomly generated bank of mutated introns.

Spliceosomopathies: Diseases and mechanisms.

The spliceosome is a complex of RNA and proteins that function together to identify intron-exon junctions in precursor messenger-RNAs, splice out the introns, and join the flanking exons. Mutations in any one of the genes encoding the proteins that make up the spliceosome may result in diseases known as spliceosomopathies. While the spliceosome is active in all cell types, with the majority of the proteins presumably expressed ubiquitously, spliceosomopathies tend to be tissue-specific as a result of germ line or somatic mutations, with phenotypes affecting primarily the retina in retinitis pigmentosa, hematopoietic lineages in myelodysplastic syndromes, or the craniofacial skeleton in mandibulofacial dysostosis. Here we describe the major spliceosomopathies, review the proposed mechanisms underlying retinitis pigmentosa and myelodysplastic syndromes, and discuss how this knowledge may inform our understanding of craniofacial spliceosomopathies.

Epigenetic Regulation of Alternative mRNA Splicing in Dilated Cardiomyopathy.

In recent years, the genetic architecture of dilated cardiomyopathy (DCM) has been more thoroughly elucidated. However, there is still insufficient knowledge on the modifiers and regulatory principles that lead to the failure of myocardial function. The current study investigates the association of epigenome-wide DNA methylation and alternative splicing, both of which are important regulatory principles in DCM. We analyzed screening and replication cohorts of cases and controls and identified distinct transcriptomic patterns in the myocardium that differ significantly, and we identified a strong association of intronic DNA methylation and flanking exons usage (p < 2 × 10−16). By combining differential exon usage (DEU) and differential methylation regions (DMR), we found a significant change of regulation in important sarcomeric and other DCM-associated pathways. Interestingly, inverse regulation of Titin antisense non-coding RNA transcript splicing and DNA methylation of a locus reciprocal to TTN substantiate these findings and indicate an additional role for non-protein-coding transcripts. In summary, this study highlights for the first time the close interrelationship between genetic imprinting by DNA methylation and the transport of this epigenetic information towards the dynamic mRNA splicing landscape. This expands our knowledge of the genome–environment interaction in DCM besides simple gene expression regulation.

Metal ions and sugar puckering balance single-molecule kinetic heterogeneity in RNA and DNA tertiary contacts

The fidelity of group II intron self-splicing and retrohoming relies on long-range tertiary interactions between the intron and its flanking exons. By single-molecule FRET, we explore the binding kinetics of the most important, structurally conserved contact, the exon and intron binding site 1 (EBS1/IBS1). A comparison of RNA-RNA and RNA-DNA hybrid contacts identifies transient metal ion binding as a major source of kinetic heterogeneity which typically appears in the form of degenerate FRET states. Molecular dynamics simulations suggest a structural link between heterogeneity and the sugar conformation at the exon-intron binding interface. While Mg2+ ions lock the exon in place and give rise to long dwell times in the exon bound FRET state, sugar puckering alleviates this structural rigidity and likely promotes exon release. The interplay of sugar puckering and metal ion coordination may be an important mechanism to balance binding affinities of RNA and DNA interactions in general.

A series of 10 Polish patients with thromboembolic events and antithrombin deficiency: two new c.1154-1 G>C and c.1219-534 A>G SERPINC1 gene splicing mutations.

: Inherited antithrombin (AT) deficiency, with prevalence in the general population ranging 0.02-0.17%, is an autosomal dominant disorder associated with a high risk of venous thromboembolism. In most cases, deficiency is caused by mutations in the AT-coding gene (SERPINC1). Only 24 splicing defects have been described causing AT deficiency, all affecting exon flanking regions. The aim of the current study was to characterize the mutations underlying AT deficiency in 10 venous thromboembolism Polish patients aged 42.9 (14-63) years. Whole SERPINC1 gene sequencing was done by next generation sequencing methods. Eight cases had mutations previously described. However, we identified two new intronic mutations that might affect the correct splicing of exon 6 according to in-silico predictions: c.1154-1 G>C, which strongly disturbs the acceptor sequence and c.1219-534 A>G, a deep intronic mutation that might generate a cryptic donor sequence; both might compete with the wild-type donor sequence and explain the associated moderate AT deficiency of carriers. In conclusion, we show the molecular base of AT deficiency in 10 new Polish patients, including two novel SERPINC1 gene mutations potentially affecting splicing.

Targeting the Tumour-Specific Spliceosome Through In Silico Virtual Screening for Discovery of New SF3B1 Small Molecule Inhibitors

Background: The coding (exon) and noncoding (intron) eukaryotic genes are expressed as precursor messenger RNA (premRNA). mRNA splicing defines the process by which introns are excised from premRNA and flanking exons are ligated together. This process is catalyzed by the spliceosome in which combination of small nuclear ribonucleoproteins (U1, U2, U4, U5, U6) forms a spliceosome. The SF3B1 protein is a core component of the U2 snRNP that binds to the branch site and facilitate RNA splicing. Recent studies have identified mutations and dysregulation of in SF3B1 activities in subsets of human cancers including chronic lymphocytic leukemias (CLLs), uveal melanomas, pancreatic cancers and breast cancers. Despite promising in vivo results indicating the potential of spliceosome modulators in targeting the refractory breast cancers, the preclinical and clinical development of such modulators will take several years to complete. Aim: In the current study, we sought to identify new SF3B1 modulators using massive virtual screening of FDA-approved drugs or novel agents for drug repurposing. Methods: A total of 3000 compounds were screened and the hits were identified based on the binding free energy (kcal/mol) of the molecules to the predicted binding sites. Of the 90 hits, vitamin D3 and its analogs (calcipotriol and calcitriol) were identified as a putative SF3B1 modulators. Results: Further in vitro testing revealed that vitamin D3 and its analogs induced significant mRNA misplicing and tumor-specific cell death in MCF7 and MDA-MB-468. Further analyses revealed that vitamin D3 and its analogs significantly reduce SF3B1 protein expression with no changes in its mRNA expression. Conclusion: These results suggest that vitamin D3 and its analogs might interact with SF3B1 to induce protein degradation rather than transcriptional activation.

Variants in the CYP19A1 gene can affect in vitro embryo production traits in cattle.

This study aimed to associate DNA variants in promoter and exon flanking regions of the CYP19A1 gene with in vitro embryo production traits in cattle. The role of transcription factor binding sites created or lost due to DNA sequence variation and their possible effect on gene expression was also evaluated. We collected date from Gyr dairy oocyte donor cows (Bos taurus indicus) at a commercial in vitro embryo production farm and analyzed the genotype-phenotype association with in vitro production traits. Using Sanger sequencing and web-based software, we assessed important CYP19A1 gene regions in oocyte donor cows and analyzed the effects of variants on the transcription factor binding sites. Two SNP mutations significantly associated with oocyte production, oocyte viability, embryo development, and pregnancies were found (T > C in the untranslated exon 1 flanking region ([GenBank: AJ250379.1]: rs718446508 T > C), and a T > C in the 5'-upstream region (1.1 promoter) ([GenBank: AC_000167.1]: rs41651668 T > C). Six new transcription factor binding sites were created. A binding site for transcription factors associated with the development of the placenta and embryo implantation was eliminated due to variations in the DNA sequence identified. The CYP19A1 gene contributes to genetic variation of in vitro embryo production traits in cattle. The complexity of the physiological phenomena related to estrogen pathways and their influence on reproduction in cattle allow indication of the mutations evaluated here as possible genetic markers for embryo production traits, which should be validated in the next steps of marker-assisted selection.

Abstract 419: Reproducibility assessment of mutations calls in exome- and whole-genome sequencing using consensus calling from TCGA and ICGC

Abstract Two large cancer genomic consortia recently published the largest and highest-quality consensus mutations calls for both whole-exome sequencing (WES) and whole-genome sequencing (WGS) in cancer: The Cancer Genome Atlas (TCGA), and the International Cancer Genetics Consortia (ICGC), respectively. Together these datasets encompass more than 60M mutations from ~13,000 samples (~10,000 WES and ~3,000 WGS). An intersecting set of 742 samples, from 22 cancer types, was sequenced using both platforms and mutations were identified using a combined 13 variant calling tools (7 WES and 5 WGS). These samples represent an ideal dataset to compare and contrast WES with WGS performance, reliability, and reproducibility of mutation calling in exons, and provide the community with key regions flanking exons that play a role in carcinogenesis. MAF files were collected using strict filtering criteria for initial file release, including the elimination of germline contaminants, 8-oxo-guanine artifacts, depth filtering and repeat masking. Additional filtering included minimum coverage requirements and restriction of both WES and WGS to variants detected within targeted exons. Finally, we restricted our data to known cancer genes. This final step suggests that these 742 samples have anywhere between 11.5K to 12.3K mutations from covered exons in potential cancer driver genes—WES and WGS, respectively. Preliminary results found that ~70% of samples had had &amp;gt;80% congruent mutations between both platforms; ~25% of samples had had &amp;gt;80% congruent mutations calls in one or the other platform; and the remaining samples had poor performance in replicating identical mutations. We observed that a majority of the variants unique to a sequencing platform were primarily from mutations with low VAF. We also sought to explore regions of the genome that are captured by both technologies despite the knowledge that WES did not target these regions. This is made possible by obtaining access to the primary data resources, and relaxing filtering criteria to include other regions such as 3' and 5' UTR, exon flanking regions, and intronic regions. We identified many recurrent mutations from non-exonic regions that were corroborated using both platforms that have not been previously reported in pan-cancer efforts. At this historic junction in time, as preliminary results from whole-genome sequencing efforts emerge and large exome sequencing efforts taper, 742 samples spanning both efforts can provide insights into the lessons learned from exome sequencing, and provide a solid foundation stepping forward into whole-genome analysis. We will continue to glean insights into the etiology of human disease by using both technologies; however, these mutation calls highlight the challenges that still exist in somatic variant calling, and provide grounds for more critical evaluation of genomic findings in cancer. Citation Format: Matthew H. Bailey, Liang-Bo Wang, Wen-Wei Liang, Steven Foltz, Guanlan Dong, Michael C. Wendl, Michael McLellan, Angela C. Hirbe, Jared Simpson, Mark Gerstein, Li Ding. Reproducibility assessment of mutations calls in exome- and whole-genome sequencing using consensus calling from TCGA and ICGC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 419.

Investigation of the 5′ flanking region and exon 3 polymorphisms of IGF-1 gene showed moderate association with semen quality in Sanjabi breed rams

In this study, semen samples were collected from 96 Sanjabi rams in order to investigate the IGF-1 gene polymorphisms and their relationship with the characteristics of semen quality and testicular size. The dimensions of scrotal length, width and circumference were measured during autumn and spring over two years. Blood samples were simultaneously collected from jugular vein to extract DNA. PCR was performed using specific primers to amplify 294 and 272bp fragments including 5′ regulatory region and exon 3 of IGF-1 gene, respectively. PCR products were digested by BFOI and Eco88l restriction enzymes, respectively. Two genotypes including AA (194 and 100bp), AB (294, 194 and 100bp) and all possible genotypes including CC (182 and 90bp), CT (272, 182, and 90bp) and TT (272bp) were observed for 5′ flanking region and exon 3 of IGF-1 gene, respectively. The significant differences among IGF-1 genotypes for testicular dimensions were not observed. However, the polymorphism of 5′ flanking region in the studied population had significant effect on individual motility and percent morphology traits. Animals with AB genotype had significantly higher individual motility compared with AA genotype (P < 0.05). Also, animals with AA genotype had significantly the highest percent morphology compared with AB genotype (P < 0.1). The exon 3 of IGF-1 gene had significant effect on individual motility, concentration, morphology and water test traits. Animals with CT genotype had the highest sperm concentration (P < 0.1) and water test (P < 0.05) compared to CC and TT genotypes. Moreover, animals with TT genotype had significantly the highest percent morphology compared with other genotypes (P < 0.05). Briefly, the results indicated that individual motility, concentration, percent morphology and water test traits could be in association with IGF-1 genotypes. It might be concluded that polymorphisms in IGF-1gene can be considered to develop male fertility in future and for using in selection process of better animals under masker assisted selection programs.

Molecular characterization, expression analysis of the myostatin gene and its association with growth traits in sea cucumber (Apostichopus japonicus).

Myostatin (MSTN), also referred to as growth and differentiation factor-8 (GDF-8), is a member of the transforming growth factor-β superfamily (TGF-β) and an important negative regulator for skeletal muscle development and growth in vertebrates. However, its function is not clear in invertebrates. In this study, we cloned and analyzed the MSTN gene (Aj-MSTN) from sea cucumber (Apostichopus japonicus). The full-length cDNA sequence of Aj-MSTN gene was composed of 2912bp, which contained a 5' UTR of 487bp, an ORF of 1356bp encoding 452 amino acids and a 3' UTR of 1069bp. The structure of Aj-MSTN included a putative signal peptide, a TGF-β propeptide domain and a conserved TGF-β domain. Phylogenetic analysis showed that the Aj-MSTN gene was clustered in the same subgroup with the MSTN-like gene found in Strongylocentrotus purpuratus. Quantitative real-time PCR detection results indicated that the Aj-MSTN gene expressed widely in adult tissues and the highest expression level was observed in the body wall. At different developmental stages, the expression levels were increased significantly at early auricularia and doliolaria stages, and reached the peak at juvenile stage. Six SNPs were identified in 5' flanking region and exons of the Aj-MSTN gene. Association analysis showed that SNP-1, SNP-2 and SNP-4 had significant effects on dry body weight. The results suggested that Aj-MSTN gene could be used as a candidate gene for the selective breeding of A. japonicus.

Gene expression of vascular endothelial growth factor A and hypoxic adaptation in Tibetan pig.

BackgroundVascular endothelial growth factor A (VEGFA) can induce endothelial cell proliferation, promote cell migration, and inhibit apoptosis. These processes play key roles in physiological blood vessel formation and pathological angiogenesis.MethodsIn this study, we examined VEGFA gene expression in the heart, liver, and kidney of Tibetan pigs (TP), Yorkshire pigs that migrated to high altitudes (YH), and Yorkshire pigs that lived at low altitudes (YL). We used PCR and Sanger sequencing to screen for single nucleotide polymorphisms (SNPs) in 5ʹ-flanking DNA and exons of the VEGFA gene. Quantitative real-time PCR and western blots were used to measure expression levels and PCR products were sequenced.ResultsResults showed that the VEGFA mRNA and protein expression in heart, liver and kidney of TP was higher than that in YH and YL. In addition, the mRNA sequence of the pig VEGFA gene was conserved among pig breeds, and only five SNPs were found in the 5ʹ-flanking region of the VEGFA gene, the allele frequency distributions of the 5 SNPs were not significantly different between the TP, Yorkshire (YL), and Diannan small-ear (DN) pig populations.ConclusionIn conclusion, the Tibetan pig showed high levels of VEGFA gene expression in several hypoxic tissues, which suggests that the VEGFA gene may play a major functional role in hypoxic adaptation.

Abstract P2-09-10: Double heterozygosity for BRCA1 and BRCA2 pathogenic variants in a French metastatic breast cancer patient

Abstract Double heterozygosity is an extremely rare occurrence in hereditary breast and ovarian cancer syndrome (HBOC [MIM 604370; MIM 612555]) where two pathogenic variants, one in BRCA1 and one in BRCA2, are found in an individual. To date, only a few case reports and case series have been reported in the literature (1-3). Furthermore, little is known about the clinical characteristics, family history, and tumor histology in these patients. In this study, we utilized targeted gene testing with next-generation sequencing (NGS) technology in an early-onset metastatic breast cancer patient from France. We evaluated germline variants using Pathway Genomics' BRCATrueTM NGS test, which analyzes variants covering all exons and exon flanking regions in both the BRCA1 and BRCA2 genes. All variant calls were determined after alignment and mapping to the GRCh37/hg19 reference genome. Variant calls were confirmed by Sanger sequencing. In this patient, a c.1016dupA (p.V340GfsX6) frameshift variant was found in BRCA1 along with a c.6814delA (p.R2272EfsX8) frameshift variant in BRCA2. Both frameshift variants are predicted to truncate the BRCA proteins. The BRCA1 c.1016dupA variant is considered a Norwegian founder mutation but has also been observed in individuals who are of French-Canadian, French, Italian or Dutch ancestry (4-7). The BRCA2 c.6814delA (p.R2272Efs*8) pathogenic variant, also known as 7042delA, is predicted to truncate the BRCA2 protein and has been identified in individuals with a personal or family history of breast and/or ovarian cancer (8,9). To the best of our knowledge, the combination of these two pathogenic variants in an individual has not been previously reported. In a clinical diagnostic setting, the possibility of double heterozygosity of pathogenic variants in more than one susceptibility gene should be considered, especially in patients with early-onset metastatic cancers. Furthermore, genetic testing and genetic counseling should also be indicated for high-risk family members. 1. Heidemann, S. et al. (2012) Breast cancer research and treatment 134, 1229-1239 2. Lavie, O., et al. (2011) Annals of oncology : official journal of the European Society for Medical Oncology / ESMO 22, 964-966 3. Nomizu, T., et al. (2012). Breast cancer 4. Andersen, T. I., Borresen, A. L., and Moller, P. (1996) American journal of human genetics 59, 486-487 5. Caputo, S., et al. (2012) Nucleic acids research 40, D992-1002 6. Dorum, A., et al. (1999). American journal of human genetics 65, 671-679 7. Simard, J., et al. (1994). Nature genetics 8, 392-398 8. Novakovic, S., et al. (2012) International journal of oncology 41, 1619-1627 9. Tea, M. K., et al. (2014) Maturitas 77, 68-72. Citation Format: Curtit E, Meynard G, Villanueva C, Mansi L, Chaix M, Vilalta A, Kuo JZ, Villa M, Neidich J, Tomar A, Arianpour A, Lebahar P, Pivot X. Double heterozygosity for BRCA1 and BRCA2 pathogenic variants in a French metastatic breast cancer patient. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-09-10.

PCK1 is negatively regulated by bta-miR-26a, and a single-nucleotide polymorphism in the 3' untranslated region is involved in semen quality and longevity of Holstein bulls.

Phosphoenolpyruvate carboxykinase 1 (PCK1) is a multi-functional enzyme that plays important roles in physiological processes, including reproduction. We previously reported that the PCK1 transcript has five splice variants; PCK1-AS4, which lacks exon 5, is enriched in the testis of Holstein bulls. In the present study, we profiled select PCK1 transcript variants in the testis, epididymus, and semen of high- and low-performance bulls, and examined the possibility that microRNAs may be involved in single nucleotide polymorphism (SNP)-mediated modulation of PCK1 expression. PCK1-AS4 abundance is not significantly different between high- and low-performance bulls. Luciferase reporter assays, however, showed that bovine PCK1 expression is repressed by bta-miR-26a in HepG2 hepatocyte cells. One SNP (c. + 2183 G > T) at the miRNA-binding site of PCK1 does not influence PCK1 expression, but is associated with elevated ejaculation volume, fresh sperm motility, and genomic estimated breeding value of longevity, as well as with reduced values of composite index and calving ease. Collectively, the identified 3'-untranslated-region SNP variant highlights the importance of PCK1 in the fecundity of Holstein bulls, and implicates a role for bta-miR-26a in regulating PCK1 abundance. Further study is needed to assess the effects of other genetic variants in 5'-flanking region and exons of PCK1 on enzyme levels in the testis and sperm. Mol. Reprod. Dev. 83: 217-225, 2016. © 2016 Wiley Periodicals, Inc.

A gene feature enumeration approach for describing HLA allele polymorphism

HLA genotyping via next generation sequencing (NGS) poses challenges for the use of HLA allele names to analyze and discuss sequence polymorphism. NGS will identify many new synonymous and non-coding HLA sequence variants. Allele names identify the types of nucleotide polymorphism that define an allele (non-synonymous, synonymous and non-coding changes), but do not describe how polymorphism is distributed among the individual features (the flanking untranslated regions, exons and introns) of a gene. Further, HLA alleles cannot be named in the absence of antigen-recognition domain (ARD) encoding exons. Here, a system for describing HLA polymorphism in terms of HLA gene features (GFs) is proposed. This system enumerates the unique nucleotide sequences for each GF in an HLA gene, and records these in a GF enumeration notation that allows both more granular dissection of allele-level HLA polymorphism and the discussion and analysis of GFs in the absence of ARD-encoding exon sequences.

Polymorphisms in the Perilipin Gene May Affect Carcass Traits of Chinese Meat-type Chickens.

Improved meat quality and greater muscle yield are highly sought after in high-quality chicken breeding programs. Past studies indicated that polymorphisms of the Perilipin gene (PLIN1) are highly associated with adiposity in mammals and are potential molecular markers for improving meat quality and carcass traits in chickens. In the present study, we screened single nucleotide polymorphisms (SNPs) in all exons of the PLIN1 gene with a direct sequencing method in six populations with different genetic backgrounds (total 240 individuals). We evaluated the association between the polymorphisms and carcass and meat quality traits. We identified three SNPs, located on the 5′ flanking region and exon 1 of PLIN1 on chromosome 10 (rs315831750, rs313726543, and rs80724063, respectively). Eight main haplotypes were constructed based on these SNPs. We calculated the allelic and genotypic frequencies, and genetic diversity parameters of the three SNPs. The polymorphism information content (PIC) ranged from 0.2768 to 0.3750, which reflected an intermediate genetic diversity for all chickens. The CC, CT, and TT genotypes influenced the percentage of breast muscle (PBM), percentage of leg muscle (PLM) and percentage of abdominal fat at rs315831750 (p<0.05). Diplotypes (haplotype pairs) affected the percentage of eviscerated weight (PEW) and PBM (p<0.05). Compared with chickens carrying other diplotypes, H3H7 had the greatest PEW and H2H2 had the greatest PBM, and those with diplotype H7H7 had the smallest PEW and PBM. We conclude that PLIN1 gene polymorphisms may affect broiler carcass and breast muscle yields, and diplotypes H3H7 and H2H2 could be positive molecular markers to enhance PEW and PBM in chickens.