Characterization of Synonymous BRCA1:c.132C>T as a Pathogenic Variant

Breast cancer gene 1 (BRCA1) and BRCA2 are tumor suppressors involved in DNA damage response and repair. Carriers of germline pathogenic or likely pathogenic variants in BRCA1 or BRCA2 have significantly increased lifetime risks of breast cancer, ovarian cancer, and other cancer types; this phenomenon is known as hereditary breast and ovarian cancer (HBOC) syndrome. Accurate interpretation of BRCA1 and BRCA2 variants is important not only for disease management in patients, but also for determining preventative measures for their families. BRCA1:c.132C>T (p.Cys44=) is a synonymous variant recorded in the ClinVar database with “conflicting interpretations of its pathogenicity”. Here, we report our clinical tests in which we identified this variant in two unrelated patients, both of whom developed breast cancer at an early age with ovarian presentation a few years later and had a family history of relevant cancers. Minigene assay showed that this change caused a four-nucleotide loss at the end of exon 3, resulting in a truncated p.Cys44Tyrfs*5 protein. Reverse transcription-polymerase chain reaction identified two fragments (123 and 119 bp) using RNA isolated from patient blood samples, in consistency with the results of the minigene assay. Collectively, we classified BRCA1:c.132C>T (p.Cys44=) as a pathogenic variant, as evidenced by functional studies, RNA analysis, and the patients’ family histories. By analyzing variants recorded in the BRCA Exchange database, we found synonymous changes at the ends of exons could potentially influence splicing; meanwhile, current in silico tools could not predict splicing changes efficiently if the variants were in the middle of an exon, or in the deep intron region. Future studies should attempt to identify variants that influence gene expression and post-transcription modifications to improve our understanding of BRCA1 and BRCA2, as well as their related cancers.

TFEB Regulates ATP7B Expression to Promote Platinum Chemoresistance in Human Ovarian Cancer Cells

ATP7B is a hepato-specific Golgi-located ATPase, which plays a key role in the regulation of copper (Cu) homeostasis and signaling. In response to elevated Cu levels, ATP7B traffics from the Golgi to endo-lysosomal structures, where it sequesters excess copper and further promotes its excretion to the bile at the apical surface of hepatocytes. In addition to liver, high ATP7B expression has been reported in tumors with elevated resistance to platinum (Pt)-based chemotherapy. Chemoresistance to Pt drugs represents the current major obstacle for the treatment of large cohorts of cancer patients. Although the mechanisms underlying Pt-tolerance are still ambiguous, accumulating evidence suggests that lysosomal sequestration of Pt drugs by ion transporters (including ATP7B) might significantly contribute to drug resistance development. In this context, signaling mechanisms regulating the expression of transporters such as ATP7B are of great importance. Considering this notion, we investigated whether ATP7B expression in Pt-resistant cells might be driven by transcription factor EB (TFEB), a master regulator of lysosomal gene transcription. Using resistant ovarian cancer IGROV-CP20 cells, we found that TFEB directly binds to the predicted coordinated lysosomal expression and regulation (CLEAR) sites in the proximal promoter and first intron region of ATP7B upon Pt exposure. This binding accelerates transcription of luciferase reporters containing ATP7B CLEAR regions, while suppression of TFEB inhibits ATP7B expression and stimulates cisplatin toxicity in resistant cells. Thus, these data have uncovered a Pt-dependent transcriptional mechanism that contributes to cancer chemoresistance and might be further explored for therapeutic purposes.

Association between SNCA Intron and genetic predisposition to sporadic Parkinson's disease: a meta-analysis based on 49576 individuals

Background The aetiology of Parkinson's disease (PD) is indistinct, but previous studies of different ethnicities have shown that genetic variations in synuclein alpha (SNCA) have an essential character in the risk of PD. The relation between SNCA intronic single nucleotide polymorphisms (SNPs) and the risk of PD is unclear. Based on the general population and five ethnic groups, this article managed a meta-analysis about the connection of SNCA intronic SNPs with the PD genetic predisposition. Methods This study was implemented according to the 24-step guideline, with strict criteria. The analysis was performed using Stata 16.0 software. Five genetic models were used to analyze the strength of the association, which was quantified by OR value and 95% CI. Results We included 15433 cases and 34143 controls from 31 articles. 6 SNPs in the intron region were screened, and 5 SNPs were statistically significant. Three variants augmented the PD susceptibility (rs2736990, rs3822086, and rs3857059), and two SNPs decreased the risk (rs356186 and rs7684318). Subgroup analysis showed that rs2736990 and rs3822086 carriers added the PD genetic predisposition in the East Asian group. European and Latin group carrying rs3857059 and rs2736990 is the high-risk populations of PD. Conclusions This study finally found 5 SNCA intronic SNPs related to the risk of PD. And racial factors should not be ignored.

Selection of functional EPHB2 genotypes from ENU mutated grass carp treated with GCRV

Background N-ethyl-N-nitrosourea (ENU) mutagenesis is a useful method for the genetic engineering of plants, and the production of functional mutants in animal models including mice and zebrafish. Grass carp reovirus (GCRV) is a haemorrhagic disease of grass carp which has caused noteworthy losses in fingerlings over the last few years. To overcome this problem, we used ENU mutant grass carp in an attempt to identify functional resistance genes for future hereditary rearing projects in grass carp. Results This study used ENU-mutated grass carp to identify genetic markers associated with resistance to the haemorrhagic disease caused by GCRV. Bulked segregant analysis (BSA) was performed on two homozygous gynogenetic ENU grass carp groups who were susceptible or resistant to GCRV. This analysis identified 466,162 SNPs and 197,644 InDels within the genomes of these mixed pools with a total of 170 genes annotated in the associated region, including 49 genes with non-synonymous mutations at SNP sites and 25 genes with frame shift mutations at InDel sites. Of these 170 mutated genes, 5 randomly selected immune-related genes were shown to be more strongly expressed in the resistant group as compared to the susceptible animals. In addition, we found that one immune-related gene, EPHB2, presented with two heterozygous SNP mutations which altered the animal’s responded to GCRV disease. These SNPs were found in the intron region of EPHB2 at positions 5859 (5859G > A) and 5968 (5968G > A) and were significantly (p = 0.002, 0.003) associated with resistance to GCRV. These SNP sites were also shown to correlate with the GCRV-resistant phenotype in these ENU grass carp. We also evaluated the mortality of the different ENU fish genotypes in response to GCRV and the SNPs in EPHB2. The outcomes of these evaluations will be useful in future selections of GCRV-resistant genes for genetic breeding in grass carp. Conclusion Our results provide a proof of concept for the application of BSA-sequence analysis in detecting genes responsible for specific functional genotypes and may help to develop better methods for marker-assisted selection, especially for disease resistance in response to GCRV.

Novel 61-bp Indel of RIN2 Is Associated With Fat and Hatching Weight Traits in Chickens

The Ras and Rab interactor 2 (RIN2) gene, which encodes RAS and Rab interacting protein 2, can interact with GTP-bound Rab5 and participate in early endocytosis. This study found a 61-bp insertion/deletion (indel) in the RIN2 intron region, and 3 genotypes II, ID, and DD were observed. Genotype analysis of mutation sites was performed on 665 individuals from F2 population and 8 chicken breeds. It was found that the indel existed in each breed and that yellow feathered chickens were mainly of the DD genotype. Correlation analysis of growth and carcass traits in the F2 population of Xinghua and White Recessive Rock chickens showed that the 61-bp indel was significantly correlated with abdominal fat weight, abdominal fat rate, fat width, and hatching weight (P < 0.05). RIN2 mRNA was expressed in all the tested tissues, and its expression in abdominal fat was higher than that in other tissues. In addition, the expression of the RIN2 mRNA in the abdominal fat of the DD genotype was significantly higher than that of the II genotype (P < 0.05). The transcriptional activity results showed that the luciferase activity of the pGL3-DD vector was significantly higher than that of the pGL3-II vector (P < 0.01). Moreover, the results indicate that the polymorphisms in transcription factor binding sites (TFBSs) of 61-bp indel may affect the transcriptional activity of RIN2, and thus alter fat traits in chicken. The results of this study showed that the 61-bp indel was closely related to abdominal fat-related and hatching weight traits of chickens, which may have reference value for molecular marker-assisted selection of chickens.

Effects of genetic variation in the 20th intron of Sansui duck ATP2A2 gene on eggshell quality

In order to explore the influence of the polymorphism in the 20 intron region of the Sansui duck ATP2A2 gene on the eggshell quality, this study used Primer Premier 5 software to design and synthesize a pair of primers in the 20 intron region, two-way direct sequencing and sequence alignment to mine SNPs Sites, SPSS 18.0 software was used to analyze the relationship between SNP sites and eggshell quality of Sansui duck. Results Three SNP sites were found in the 20 intron region of the ATP2A2 gene: g.40874 T>C, g.40920 G>A and g.40990 T=C, all of which were moderately polymorphic, at the site g.40874 T ＞C and g.40920 G＞A both deviated significantly from Hardy-Weinberg equilibrium (P>0.05), position g.40990 T=C accords with Hardy-Weinberg equilibrium (P<0.05), and position g.40874 T＞C There is a strong linkage disequilibrium between g.40990 T=C; a total of 4 haplotypes and 9 double types were detected at 3 SNP loci; the results of association analysis show that g.40874 T＞C mutation has an effect on eggshell strength The eggshell strength of CC genotype was significantly higher than that of TC and TT genotypes (P<0.05), and the eggshell weight of CC genotype was significantly higher than that of TC genotype (P<0.05), g. The 40990 T=C mutation has a significant effect on the eggshell strength, and the eggshell strength of the TC genotype is significantly higher than that of the TT genotype (P<0.05). In summary, the g.40874 T>C and g.40990 T=C found in the 20th intron region of the Sansui duck ATP2A2 gene may be the marker sites that affect the quality of the eggshell.

Selection of functional EPHB2 genotype in ENU mutated grass carp response to GCRV by the method of BSA Sequence Analysis

Background N-ethyl-N-nitrosourea (ENU) mutagenesis is a useful method for genetic development of plants, as well as for inducing functional mutants in animal models including mice and zebrafish. GCRV (Grass carp Reovirus) is the hemorrhage disease of grass carp and causes noteworthy loss of fingerlings in the last few years. To overcome this problem, we have used ENU mutant grass carp might be a helpful to find out some resistant functional gene for future hereditary rearing projects in grass carp.Results In the present study, ENU-mutated grass carp were used to identify genetic markers correlated with anti hemorrhagic disease caused by grass carp reovirus (GCRV). The BSA (Bulked segregant analysis) technology were performed on two homozygous gynogenetic ENU grass carp groups with susceptible or resistant in response of GCRV. Our results showed that 466,162 SNPs and 197644 InDel were found out in the mixed pool. A total of 170 genes were annotated in the associated region, including 49 genes with non-synonymous mutations at SNP sites and 25 genes with frame shift mutations at InDel sites. Among these 170 mutated genes, 5 randomly selected immune-related genes expression was higher in resistant group as compared to susceptible. Furthermore, we found one immune-related gene EPHB2 has confirmed two heterozygous SNP mutations which responded to GCRV disease. The SNPs within gene EPHB2 were found out in the intron region with position 5859 (5859G>A) and 5968 (5968G>A) were significantly (p = 0.002, 0.003) associated with the resistance against GCRV. These SNP sites were confirmed to correlate with the GCRV-resistant phenotype of ENU grass carp. The SNPs associated with GCRV resistance are useful in selecting GCRV-resistant genes for genetic breeding in grass carp.Conclusion Our results proved the possibility of BSA-sequence analysis in detecting genes responsible for the exciting phenotypes and will help in carrying out the marker-assisted selection, especially for disease resistance in the response of GCRV.

The complete plastomes of red fleshed pitaya (Selenicereus monacanthus) and three related Selenicereus species: insights into gene losses, IR expansions and phylogenomic implications

Background: Selenicereus is a genus of perennial shrub from the family Cactaceae, and some of them play an important role in the food industry, pharmaceuticals, cosmetics and medicine. To date, there are few reports on Selenicereus plastomes, which limits our understanding of this genus. Here, we reported the complete plastomes of four Selenicereus species (S. monacanthus, S. annthonyanus, S. grandiflorus and S. validus, and carried out a comprehensive comparative analysis.Results: The four Selenicereus plastomes all have a typical quartile structure. The plastome size ranged from 133,146 bp to 134,450 bp, and contained 104 unique genes, including 30 tRNA genes, 4 rRNA genes and 70 protein-coding genes. Comparative analysis showed that there were massive losses of ndh genes in Selenicereus. Besides, we observed the IR regions had undergone a dramatic expansion and formed a previously unreported SC/IR border in the intron region of the atpF gene. Furthermore, we identified 6 hypervariable regions (trnF-GAA-rbcL, ycf1, accD, clpP-trnS-GCU, clpP-trnT-CGU and rpl22-rps19) that could be used as potential DNA barcodes for the identification of Selenicereus species. Phylogenetic analysis indicated that Hylocereus was nested in Selenicereus.Conclusion: Our study enriches the plastomic resources in the family Cactaceae, and provides the basis for the reconstruction of phylogenetic relationships.

A Second Hit Somatic (P.R905W) and a Novel Germline Intron-Mutation of TSC2 Gene is Found in Intestinal Lymphangioleiomyomatosis: A Case Report with Literature Review

Background: Tuberous sclerosis complex (TSC) is an autosomal dominant disorder associated with germline mutations in TSC1 and TSC2, including exonic, intronic, or mosaic mutations. Gastrointestinal (GI) tract Lymphangioleiomyomatosis (LAM) is an extremely rare manifestation of TSC, with few reported cases. Herein, we aimed to determine the driver mutation, pathogenesis, and relationship of germline and somatic mutations of LAM through whole-genome sequencing (WGS) of the tumor and blood samples and whole transcriptome sequencing (WTS) analysis. Case presentation: A nine-year-old girl with a full-blown TSC presented with abdominal masses detected during a routine check-up. Resected intestinal masses were diagnosed as LAM by thorough pathological examination. Interestingly, the LAM presented a somatic TSC2 gene mutation in exon 24 (p.R905W, c.C2713T), and the patient had intron retention by a novel germline mutation in the intron region of TSC2 (chr16:2126489, C>G). Conclusion: Our case suggests that intron retention by a single nucleotide intronic mutation of TSC2 is sufficient to develop severe manifestations of TSC, but the development of LAM requires an additional somatic oncogenic mutation.

QKI-5 regulates the alternative splicing of cytoskeletal gene ADD3 in lung cancer

Accumulating evidence indicates that the alternative splicing program undergoes extensive changes during cancer development and progression. The RNA-binding protein QKI-5 is frequently down-regulated and exhibits anti-tumor activity in lung cancer. However, little is known about the functional targets and regulatory mechanism of QKI-5. Here, we report that up-regulation of exon 14 inclusion of cytoskeletal gene Adducin 3 (ADD3) significantly correlates with a poor prognosis in lung cancer. QKI-5 inhibits cell proliferation and migration in part through suppressing the splicing of ADD3 exon 14. Through genome-wide mapping of QKI-5 binding sites in vivo at nucleotide resolution by iCLIP-seq analysis, we found that QKI-5 regulates alternative splicing of its target mRNAs in a binding position-dependent manner. By binding to multiple sites in an upstream intron region, QKI-5 represses the splicing of ADD3 exon 14. We also identified several QKI mutations in tumors, which cause dysregulation of the splicing of QKI targets ADD3 and NUMB. Taken together, our results reveal that QKI-mediated alternative splicing of ADD3 is a key lung cancer-associated splicing event, which underlies in part the tumor suppressor function of QKI.