BACKGROUND Fragile X Syndrome (FXS) is a trinucleotide repeat disease that is caused by the expansion of CGG sequences in the 5’ untranslated region of the FMR1 gene. Molecular diagnoses of FXS and other emerging FMR1 disorders typically rely on two tests, PCR and Southern blotting. However, performance or throughput limitations in these methods currently constrain routine testing. METHODS We evaluated a novel FMR1 gene-specific PCR technology with 20 cell line DNA templates and 146 blinded clinical specimens. The CGG repeat number was determined by fragment sizing of PCR amplicons using capillary electrophoresis and compared with the results of FMR1 Southern blotting performed with the same samples. RESULTS The FMR1 PCR accurately detected full mutation alleles up to at least 1300 CGG repeats and comprising >99% GC character. All categories of alleles detected by Southern blot, including 66 specimens with full mutations, were also identified by FMR1 PCR for each of 146 clinical specimens. Since all full mutation alleles in heterozygous female samples were detected by PCR, allele zygosity was reconciled in every case. The PCR reagents also detected a 1% mass fraction of a 940 CGG allele in a background of 99% 23 CGG allele—roughly 5-fold greater sensitivity than Southern blotting. CONCLUSIONS The novel PCR technology can accurately categorize the spectrum of FMR1 alleles, including alleles previously considered too large to amplify, reproducibly detect low abundance full mutation alleles, and correctly infer homozygosity in female specimens, thus greatly reducing the need for sample reflexing to Southern blot.

(CGG) n repeat expansion in the FMR1 gene is associated with fragile X syndrome and other disorders. Current methods for FMR1 molecular testing rely on Southern blot analysis to detect expanded alleles too large to be PCR-amplified and to identify female homozygous alleles that often confound interpretations of PCR data. A novel , single-tube CGG repeat primed FMR1 PCR technology was designed with two genespecific primers that flank the triplet repeat region, as well as a third primer that is complementary to the (CGG) n repeat. This PCR was evaluated with 171 unique DNA samples , including a blinded set of 146 clinical specimens. The method detected all alleles reported by Southern blot analysis , including full mutations in 66 clinical samples and comprised up to 1300 CGG. Furthermore , a blinded cohort of 42 female homozygous and heterozygous specimens, including 21 with full mutation alleles , was resolved with 100% accuracy. Last , AGG interrupter sequences, which may influence the risk of (CGG) n expansion in the children of some carriers , were each correctly identified in 14 male and female clinical samples as referenced to DNA sequencing. As a result , this PCR provides robust detection of expanded alleles and resolves allele zygosity , thus minimizing the number of samples that require Southern blot analysis and producing more comprehensive FMR1 genotyping data than other methods. Expansion of cytosine-guanine-guanine (CGG) triplet repeats in the 5Ј-untranslated region of the fragile X mental retardation 1 (FMR1, NM_002024.4) gene is associated with several disorders, including fragile X syndrome, fragile X-associated tremor/ataxia syndrome, and fragile X-associated primary ovarian insufficiency. [1][2][3][4] Patients with the FMR1 full mutation (Ͼ200 CGG repeats) may be affected by a range of neurological, psychiatric, or emotional challenges, including mental retardation and/or autism.5 Deficits in development and particularly in attention and social communication have also been noted for many children with the FMR1 premutation. Moreover, premutation carriers (55 to 200 CGG repeats) are known to be at risk for fragile X-associated primary ovarian insufficiency and fragile X-associated tremor/ataxia syndrome, and some of these individuals may present additional complications, such as hypothyroidism and fibromyalgia.6 As a result, FMR1 disorders are linked to a range of clinical conditions, necessitating testing patients at different times during their life span. 7Fragile X syndrome molecular diagnosis is usually based on quantification of the (CGG) n repeat elements and the assessment of the methylation state of expanded alleles.5 Although PCR is the preferred approach to determine the (CGG) n repeat length of FMR1 alleles, typically only alleles with less than 100 to 150 CGG have

Implementation of highly sophisticated technologies, such as next-generation sequencing (NGS), into routine clinical practice requires compatibility with common tumor biopsy types, such as formalin-fixed, paraffin-embedded (FFPE) and fine-needle aspiration specimens, and validation metrics for platforms, controls, and data analysis pipelines. In this study, a two-step PCR enrichment workflow was used to assess 540 known cancer-relevant variants in 16 oncogenes for high-depth sequencing in tumor samples on either mature (Illumina GAIIx) or emerging (Ion Torrent PGM) NGS platforms. The results revealed that the background noise of variant detection was elevated approximately twofold in FFPE compared with cell line DNA. Bioinformatic algorithms were optimized to accommodate this background. Variant calls from 38 residual clinical colorectal cancer FFPE specimens and 10 thyroid fine-needle aspiration specimens were compared across multiple cancer genes, resulting in an accuracy of 96.1% (95% CI, 96.1% to 99.3%) compared with Sanger sequencing, and 99.6% (95% CI, 97.9% to 99.9%) compared with an alternative method with an analytical sensitivity of 1% mutation detection. A total of 45 of 48 samples were concordant between NGS platforms across all matched regions, with the three discordant calls each represented at <10% of reads. Consequently, NGS of targeted oncogenes in real-life tumor specimens using distinct platforms addresses unmet needs for unbiased and highly sensitive mutation detection and can accelerate both basic and clinical cancer research.

Chemical inhibition of the mitochondrial electron transport chain (mtETC) by antimycin A (AA) or the TCA cycle by monofluoroacetate (MFA) causes increased expression of nucleus-encoded alternative oxidase (AOX) genes in plants. In order to better understand the mechanisms of this mitochondrial retrograde regulation (MRR) of gene expression, constructs containing deleted and mutated versions of a promoter region of the Arabidopsis thaliana AOX1a gene (AtAOX1a) controlling expression of the coding region of the enhanced firefly luciferase gene were employed to identify regions of the AtAOX1a promoter important for induction in response to mtETC or TCA cycle inhibition. Transient transformation coupled with in vitro and in vivo assays as well as plants containing transgenes with truncated promoter regions were used to identify a 93 base pair portion of the promoter, termed the MRR region, that was necessary for induction. Further mutational analyses showed that most of the 93 bp MRR region is important for both AA and MFA induction. Sub-regions within the MRR region that are especially important for strong induction by both AA or MFA were identified. Specific mutations in a W-box and Dof motifs in the MRR region indicate that these specific motifs are not important for induction. Recent evidence suggests that MRR of AOX genes following inhibition of the mtETC is via a separate signaling pathway from MRR resulting from metabolic shifts, such as those that result from MFA treatment. Our data suggest that these signaling pathways share regulatory regions in the AtAOX1a promoter. Arabidopsis proteins interacted specifically with a probe containing the MRR region, as shown by electrophoretic mobility shift assays and Southwestern blotting. These interactions were eliminated under reducing conditions.

Purpose: MicroRNA-21 (miRNA-21) has proto-oncogenic properties, though no miRNA-21 specific targets have been found in head and neck squamous cell carcinoma (HNSCC). Further study of miRNA-21 and its specific targets is essential to understanding HNSCC biology. Experimental Design: miRNA expression profiles of 10 HNSCC and 10 normal mucosa samples were investigated using a custom miRNA microarray. 13 HNSCC and 5 normal mucosa primary tissue specimens underwent mRNA expression microarray analysis. To identify miRNA-21 downstream targets, oral keratinocyte cells were subjected to microarray analysis after miRNA-21 transient transfection. miRNA and mRNA expression were validated by RT-qPCR in a separate cohort of 16 HNSCC and 15 normal mucosal samples. Microarray and bioinformatics analyses were integrated to identify potential gene targets. In vitro assays looked at the function and interaction of miRNA-21 and its specific gene targets. Results: miRNA-21 was upregulated in HNSCC and stimulated cell growth. Integrated analyses identified Clusterin (CLU) as a potential miRNA-21 gene target. CLU was downregulated after forced expression of miRNA-21 in normal and HNSCC cell lines. The activity of a luciferase construct containing the 3’UTR of CLU was repressed by the ectopic expression of miRNA-21. CLU was also downregulated in primary HNSCC and correlated with miRNA-21 over-expression. CLU variant 1 (CLU-1) was the predominant splice variant in HNSCC, and showed growth suppression function that was reversed by miRNA-21 over-expression. Conclusions: CLU is a specific, functional target of oncogenic miRNA-21 in HNSCC. CLU-1 isoform is the predominant growth suppressive variant targeted by miRNA-21.

<p>PDF file - 133K, Supplementary Figure 1. miRNA-21 is upregulated in HNSCC tumors. Supplementary Figure 2. RT-qPCR validation of miRNA-21 overexpression by transient transfection. (A) pre-miRNA-21 and (B) miRNA-21 expression level. Supplementary Figure 3. miRNA-21 overexpression stimulates cell proliferation. All measurements were obtained with the Cell Counting Kit -8 cell proliferation assay. ∗p<0.01. Supplementary Figure 4. miRNA-21 loses specificity for CLU transcript when miRNA-21 binding seed sequence is deleted from the CLU 3'UTR sequence. ∗p<0.03. Supplementary Figure 5. Relative expression of CLU-1 in normal and HNSCC cell lines. Supplementary Figure 6. RT-qPCR validation of CLU-1 overexpression by transient transfection. Supplementary Figure 7. CLU-1 has growth inhibitory effects in JHU-O28, FaDu and SCC9 HNSCC cell lines. Overexpression of CLU-1 inhibits cell proliferation. All measurements were obtained by taking direct cell counts at each time point. ∗p<0.05.</p>

<p>PDF file - 67K</p>

<p>XLSX file - 841K, Supplementary Table 4. miRNA array analysis of discovery cohort. Supplementary Table 5. miRNA array significant call signals. Supplementary Table 6. RT-qPCR validation of miRNA array.</p>