Background One of the most widely used evaluation methods in miRNA experiments is qRT-PCR. However, selecting suitable internal controls (IC) is crucial for qRT-PCR experiments. Currently, there is no consensus on the ICs for miRNA qRT-PCR experiments in breast cancer. To this end, we tried to identify the most stable (the least expression alteration) and promising miRNAs in normal and tumor breast tissues by employing TCGA miRNA-Seq data and then experimentally validated them on fresh clinical samples. Methods A multi-component scoring system was used which takes into account multiple expression stability criteria as well as correlation with clinical characteristics. Furthermore, we extended the scoring system for more than two biological sub-groups. TCGA BRCA samples were analyzed based on two grouping criteria: Tumor & Normal samples and Tumor subtypes. The top 10 most stable miRNAs were further investigated by differential expression and survival analysis. Then, we examined the expression level of the top scored miRNA (hsa-miR-361-5p) along with two commonly used ICs hsa-miR-16-5p and U48 on 34 pairs of Primary breast tumor and their adjacent normal tissues using qRT-PCR. Results According to our multi-component scoring system, hsa-miR-361-5p had the highest stability score in both grouping criteria and hsa-miR-16-5p showed significantly lower scores. Based on our qRT-PCR assay, while U48 was the most abundant IC, hsa-miR-361-5p had lower standard deviation and also was the only IC capable of detecting a significant up-regulation of hsa-miR-21-5p in breast tumor tissue. Conclusions miRNA-Seq data is a great source to discover stable ICs. Our results demonstrated that hsa-miR-361-5p is a highly stable miRNA in tumor and non-tumor breast tissue and we recommend it as a suitable reference gene for miRNA expression studies in breast cancer. Additionally, although hsa-miR-16-5p is a commonly used IC, it’s not a suitable one for breast cancer studies.
Fibrochondrogenesis 1, an autosomal recessive syndrome, is a rare disease that causes short‐limbed skeletal dysplasia. Mutations in the gene encoding the α1 chain of type XI collagen (COL11A1) are seen to be the main cause of this disease. We present an 18‐week Iranian male aborted fetus with Fibrochondrogenesis 1 from consanguineous parents. Whole‐exome sequencing revealed a novel missense variant from G to A in exon 45 of 68 in the COL11A1 gene (NM_080629.2: c.3440G > A, [p.G1147E, g.103404625]). The mutation was confirmed by Sanger sequencing and further, MutationTaster predicted this variant to be disease‐causing. Bioinformatic analysis suggests that this variant is highly conserved in both nucleotide and protein levels, suggesting that it has an important function in the proper role of COL11A1 protein. In silico analysis suggests that this mutation alters the COL11A1 protein structure through a Glycine to Glutamic acid substitution.
BACKGROUND: Identifying the molecular subtypes of breast cancer (BC) plays a crucial role in enhancing the efficacy of therapy. MiRNAs with differential expressions in different subtypes of breast tumors can be considered non-invasive biomarkers for diagnosing BC subtypes. OBJECTIVE: We aimed to investigate the efficacy of miR-190b, miR-584-5p, miR-452-5p, and miR-1306-5p as new potential diagnostic biomarkers in discriminating patients with luminal and non-luminal BCs. METHODS: A group of miRNAs significantly associated with estrogen cell receptors (ER) in breast tumors was identified using feature selection methods analysis on miRNASeq data of TCGA and GSE68085. Among them, four miRNAs were selected as novel potential biomarkers, and their expression levels were assessed within luminal tumors, non-luminal tumors, and adjacent non-tumor tissues by qRT-PCR. Their impact on diagnosis was also evaluated by ROC curve analysis. RESULTS: MiR-190b was remarkably up-regulated, while miRNA-584-5p, miRNA-452-5p, and miRNA-1306-5p were significantly down-regulated in luminal BCs. This group could discriminate luminal and non-luminal BCs at an AUC of 0.977. CONCLUSIONS: According to our findings, these four miRNAs are promising biomarkers in parallel with histologic diagnosis methods for identifying patients who are most likely responding to specific therapies based on ER status.
BackgroundFibrochondrogenesis 1, an autosomal recessive syndrome, is an infrequent and rare disease, causing short-limbed skeletal dysplasia. This syndrome is clinically characterized and distinguished by a small nose and anteverted bares, flat midface, shortened long bones, and a protuberant abdomen. Mutations in the gene encoding the α1 chain of type XI collagen (COL11A1) are seen to be the main cause of this disease.Case PresentationWe present an 18-week Iranian male aborted fetus with Fibrochondrogenesis 1 from consanguineous parents. Whole-exome sequencing (WES) revealed a novel missense variant from G to A in exon 45 of 68 in the COL11A1 gene (NM_080629.2: c.3440G>A, [p.G1147E, g.103404625]). The mutation was confirmed by Sanger sequencing and further, MutationTaster predicted this variant to be disease-causing.ConclusionBioinformatic analysis suggests that this variant is highly conserved in both nucleotide and protein levels, suggesting that it has an important function in the proper role of COL11A1 protein. In-silico analysis suggests that this mutation alters the COL11A1 protein structure through a Glycine to Glutamic acid substitution. This is a novel mutation and a rare variant as this variant is not reported in gmomAD, ExAC, or 1000 genome databases.To the best of the authors’ knowledge, this is the first study to report a novel pathogenic mutation in COL11A1 in association with Fibrochondrogenesis 1. Therefore, we suggest that WES can be used as a robust method to achieve rapid diagnosis and identification of pathogenic and novel mutations in patients.
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