A common intronic single nucleotide variant modifies PKD1 expression level.

Autosomal dominant polycystic kidney disease (ADPKD), caused by mutations in PKD1 and PKD2 (PKD1/2), has unexplained phenotypic variability likely affected by environmental and other genetic factors. Approximately 10% of individuals with ADPKD phenotype have no causal mutation detected, possibly due to unrecognized risk variants of PKD1/2. This study was designed to identify risk variants of PKD genes through population genetic analyses. We used Wright's F-statistics (Fst) to evaluate common single nucleotide variants (SNVs) potentially favored by positive natural selection in PKD1 from 1000 Genomes Project (1KG) and genotyped 388 subjects from the Rogosin Institute ADPKD Data Repository. The variants with >90th percentile Fst scores underwent further investigation by in silico analysis and molecular genetics analyses. We identified a deep intronic SNV, rs3874648G> A, located in a conserved binding site of the splicing regulator Tra2-ß in PKD1 intron 30. Reverse-transcription PCR (RT-PCR) of peripheral blood leukocytes (PBL) from an ADPKD patient homozygous for rs3874648-A identified an atypical PKD1 splice form. Functional analyses demonstrated that rs3874648-A allele increased Tra2-ß binding affinity and activated a cryptic acceptor splice-site, causing a frameshift that introduced a premature stop codon in mRNA, thereby decreasing PKD1 full-length transcript level. PKD1 transcript levels were lower in PBL from rs3874648-G/A carriers than in rs3874648-G/G homozygotes in a small cohort of normal individuals and patients with PKD2 inactivating mutations. Our findings indicate that rs3874648G > A is a PKD1 expression modifier attenuating PKD1 expression through Tra2-ß, while the derived G allele advantageously maintains PKD1 expression and is predominant in all subpopulations.

Detection of PKD1 and PKD2 Somatic Variants in Autosomal Dominant Polycystic Kidney Cyst Epithelial Cells by Whole-Genome Sequencing.

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is a genetic disorder characterized by the development of multiple cysts in the kidneys. It is often caused by pathogenic mutations in PKD1 and PKD2 genes that encode polycystin proteins. Although the molecular mechanisms for cystogenesis are not established, concurrent inactivating germline and somatic mutations in PKD1 and PKD2 have been previously observed in renal tubular epithelium (RTE). METHODS: To further investigate the cellular recessive mechanism of cystogenesis in RTE, we conducted whole-genome DNA sequencing analysis to identify germline variants and somatic alterations in RTE of 90 unique kidney cysts obtained during nephrectomy from 24 unrelated participants. RESULTS: Kidney cysts were overall genomically stable, with low burdens of somatic short mutations or large-scale structural alterations. Pathogenic somatic "second hit" alterations disrupting PKD1 or PKD2 were identified in 93% of the cysts. Of these, 77% of cysts acquired short mutations in PKD1 or PKD2 ; specifically, 60% resulted in protein truncations (nonsense, frameshift, or splice site) and 17% caused non-truncating mutations (missense, in-frame insertions, or deletions). Another 18% of cysts acquired somatic chromosomal loss of heterozygosity (LOH) events encompassing PKD1 or PKD2 ranging from 2.6 to 81.3 Mb. 14% of these cysts harbored copy number neutral LOH events, while the other 3% had hemizygous chromosomal deletions. LOH events frequently occurred at chromosomal fragile sites, or in regions comprising chromosome microdeletion diseases/syndromes. Almost all somatic "second hit" alterations occurred at the same germline mutated PKD1/2 gene. CONCLUSIONS: These findings further support a cellular recessive mechanism for cystogenesis in ADPKD primarily caused by inactivating germline and somatic variants of PKD1 or PKD2 genes in kidney cyst epithelium.

Evaluation and Adaptation of a Laboratory-Based cDNA Library Preparation Protocol for Retrospective Sequencing of Archived MicroRNAs from up to 35-Year-Old Clinical FFPE Specimens.

Formalin-fixed paraffin-embedded (FFPE) specimens, when used in conjunction with patient clinical data history, represent an invaluable resource for molecular studies of cancer. Even though nucleic acids extracted from archived FFPE tissues are degraded, their molecular analysis has become possible. In this study, we optimized a laboratory-based next-generation sequencing barcoded cDNA library preparation protocol for analysis of small RNAs recovered from archived FFPE tissues. Using matched fresh and FFPE specimens, we evaluated the robustness and reproducibility of our optimized approach, as well as its applicability to archived clinical specimens stored for up to 35 years. We then evaluated this cDNA library preparation protocol by performing a miRNA expression analysis of archived breast ductal carcinoma in situ (DCIS) specimens, selected for their relation to the risk of subsequent breast cancer development and obtained from six different institutions. Our analyses identified six miRNAs (miR-29a, miR-221, miR-375, miR-184, miR-363, miR-455-5p) differentially expressed between DCIS lesions from women who subsequently developed an invasive breast cancer (cases) and women who did not develop invasive breast cancer within the same time interval (control). Our thorough evaluation and application of this laboratory-based miRNA sequencing analysis indicates that the preparation of small RNA cDNA libraries can reliably be performed on older, archived, clinically-classified specimens.

Improved predictions of total kidney volume growth rate in ADPKD using two-parameter least squares fitting.

Mayo Imaging Classification (MIC) for predicting future kidney growth in autosomal dominant polycystic kidney disease (ADPKD) patients is calculated from a single MRI/CT scan assuming exponential kidney volume growth and height-adjusted total kidney volume at birth to be 150 mL/m. However, when multiple scans are available, how this information should be combined to improve prediction accuracy is unclear. Herein, we studied ADPKD subjects ( n = 36 ) with 8+ years imaging follow-up (mean = 11 years) to establish ground truth kidney growth trajectory. MIC annual kidney growth rate predictions were compared to ground truth as well as 1- and 2-parameter least squares fitting. The annualized mean absolute error in MIC for predicting total kidney volume growth rate was 2.1 % ± 2 % compared to 1.1 % ± 1 % ( p = 0.002 ) for a 2-parameter fit to the same exponential growth curve used for MIC when 4 measurements were available or 1.4 % ± 1 % ( p = 0.01 ) with 3 measurements averaging together with MIC. On univariate analysis, male sex ( p = 0.05 ) and PKD2 mutation ( p = 0.04 ) were associated with poorer MIC performance. In ADPKD patients with 3 or more CT/MRI scans, 2-parameter least squares fitting predicted kidney volume growth rate better than MIC, especially in males and with PKD2 mutations where MIC was less accurate.

Low-level expression of miR-375 correlates with poor outcome and metastasis while altering the invasive properties of head and neck squamous cell carcinomas.

Small, noncoding microRNAs (miRNAs) have been shown to be abnormally expressed in every tumor type examined. We used comparisons of global miRNA expression profiles of head and neck squamous cell carcinoma (HNSCC) samples and adjacent normal tissue to rank those miRNAs that were most significantly altered in our patient population. Rank Consistency Score analysis revealed miR-375 to have the most significantly lowered miRNA levels in tumors relative to matched adjacent nonmalignant tissue from the same patient among 736 miRNAs that were evaluated. This result has been previously observed by other groups; however, we extend this finding with the unique observation that low miR-375 expression levels correlate significantly with cancer survival and distant metastasis. In a study of 123 primary HNSCC patients using multivariable Cox proportional hazard ratios (HR) and 95% confidence intervals (CI), both death from disease (HR: 12.8, 95% CI: 3 to 49) and incidence of distant metastasis (HR: 8.7, 95% CI: 2 to 31) correlated with lower expression levels of miR-375 regardless of the site or stage of the tumor. In addition, we found that oral cavity tumor cell lines (eg, UMSCC1 and UMSCC47) overexpressing miR-375 were significantly less invasive in vitro than their matched empty vector controls. We conclude that miR-375 represents a potential prognostic marker of poor outcome and metastasis in HNSCC and that it may function by suppressing the tumor's invasive properties.

Hsa-miR-375 is differentially expressed during breast lobular neoplasia and promotes loss of mammary acinar polarity.

Invasive lobular carcinoma (ILC) of the breast, characterized by loss of E-cadherin expression, accounts for 5-15% of invasive breast cancers and it is believed to arise via a linear histological progression. Genomic studies have identified a clonal relationship between ILC and concurrent lobular carcinoma in situ (LCIS) lesions, suggesting that LCIS may be a precursor lesion. It has been shown that an LCIS diagnosis confers a 15-20% risk of progression to ILC over a lifetime. Currently no molecular test or markers can identify LCIS lesions likely to progress to ILC. Since microRNA (miRNA) expression changes have been detected in a number of other cancer types, we explored whether their dysregulation might be detected during progression from LCIS to ILC. Using the Illumina miRNA profiling platform, designed for simultaneous analysis of 470 mature miRNAs, we analysed the profiles of archived normal breast epithelium, LCIS lesions found alone, LCIS lesions concurrent with ILC, and the concurrent ILCs as a model of linear histological progression towards ILC. We identified two sets of differentially expressed miRNAs, the first set highly expressed in normal epithelium, including hsa-miR-224, -139, -10b, -450, 140, and -365, and the second set up-regulated during lobular neoplasia progression, including hsa-miR-375, -203, -425-5p, -183, -565, and -182. Using quantitative RT-PCR, we validated a trend of increasing expression for hsa-miR-375, hsa-miR-182, and hsa-miR-183 correlating with ILC progression. As we detected increased expression of hsa-miR-375 in LCIS lesions synchronous with ILC, we sought to determine whether hsa-miR-375 might induce phenotypes reminiscent of lobular neoplasia by expressing it in the MCF-10A 3D culture model of mammary acinar morphogenesis. Increased expression of hsa-miR-375 resulted in loss of cellular organization and acquisition of a hyperplastic phenotype. These data suggest that dysregulated miRNA expression contributes to lobular neoplastic progression.

Extracellular Vesicle Capture by AnTibody of CHoice and Enzymatic Release (EV-CATCHER): A customizable purification assay designed for small-RNA biomarker identification and evaluation of circulating small-EVs.

Circulating nucleic acids, encapsulated within small extracellular vesicles (EVs), provide a remote cellular snapshot of biomarkers derived from diseased tissues, however selective isolation is critical. Current laboratory-based purification techniques rely on the physical properties of small-EVs rather than their inherited cellular fingerprints. We established a highly-selective purification assay, termed EV-CATCHER, initially designed for high-throughput analysis of low-abundance small-RNA cargos by next-generation sequencing. We demonstrated its selectivity by specifically isolating and sequencing small-RNAs from mouse small-EVs spiked into human plasma. Western blotting, nanoparticle tracking, and transmission electron microscopy were used to validate and quantify the capture and release of intact small-EVs. As proof-of-principle for sensitive detection of circulating miRNAs, we compared small-RNA sequencing data from a subset of small-EVs serum-purified with EV-CATCHER to data from whole serum, using samples from a small cohort of recently hospitalized Covid-19 patients. We identified and validated, only in small-EVs, hsa-miR-146a and hsa-miR-126-3p to be significantly downregulated with disease severity. Separately, using convalescent sera from recovered Covid-19 patients with high anti-spike IgG titers, we confirmed the neutralizing properties, against SARS-CoV-2 in vitro, of a subset of small-EVs serum-purified by EV-CATCHER, as initially observed with ultracentrifuged small-EVs. Altogether our data highlight the sensitivity and versatility of EV-CATCHER.

Effective DNA/RNA co-extraction for analysis of microRNAs, mRNAs, and genomic DNA from formalin-fixed paraffin-embedded specimens.

BACKGROUND: Retrospective studies of archived human specimens, with known clinical follow-up, are used to identify predictive and prognostic molecular markers of disease. Due to biochemical differences, however, formalin-fixed paraffin-embedded (FFPE) DNA and RNA have generally been extracted separately from either different tissue sections or from the same section by dividing the digested tissue. The former limits accurate correlation whilst the latter is impractical when utilizing rare or limited archived specimens. PRINCIPAL FINDINGS: For effective recovery of genomic DNA and total RNA from a single FFPE specimen, without splitting the proteinase-K digested tissue solution, we optimized a co-extraction method by using TRIzol and purifying DNA from the lower aqueous and RNA from the upper organic phases. Using a series of seven different archived specimens, we evaluated the total amounts of genomic DNA and total RNA recovered by our TRIzol-based co-extraction method and compared our results with those from two commercial kits, the Qiagen AllPrep DNA/RNA FFPE kit, for co-extraction, and the Ambion RecoverAll™ Total Nucleic Acid Isolation kit, for separate extraction of FFPE-DNA and -RNA. Then, to accurately assess the quality of DNA and RNA co-extracted from a single FFPE specimen, we used qRT-PCR, gene expression profiling and methylation assays to analyze microRNAs, mRNAs, and genomic DNA recovered from matched fresh and FFPE MCF10A cells. These experiments show that the TRIzol-based co-extraction method provides larger amounts of FFPE-DNA and -RNA than the two other methods, and particularly provides higher quality microRNAs and genomic DNA for subsequent molecular analyses. SIGNIFICANCE: We determined that co-extraction of genomic DNA and total RNA from a single FFPE specimen is an effective recovery approach to obtain high-quality material for parallel molecular and high-throughput analyses. Our optimized approach provides the option of collecting DNA, which would otherwise be discarded or degraded, for additional or subsequent studies.