The gene family of serine protease inhibitors of the Kazal type (SPINK) are functional and positional candidate genes for celiac disease (CD). Our aim was to assess the gut mucosal gene expression and genetic association of SPINK1, -2, -4, and -5 in the Dutch CD population. Gene expression was determined for all four SPINK genes by quantitative reverse-transcription polymerase chain reaction in duodenal biopsy samples from untreated (n=15) and diet-treated patients (n=31) and controls (n=16). Genetic association of the four SPINK genes was tested within a total of 18 haplotype tagging SNPs, one coding SNP, 310 patients, and 180 controls. The SPINK4 study cohort was further expanded to include 479 CD cases and 540 controls. SPINK4 DNA sequence analysis was performed on six members of a multigeneration CD family to detect possible point mutations or deletions. SPINK4 showed differential gene expression, which was at its highest in untreated patients and dropped sharply upon commencement of a gluten-free diet. Genetic association tests for all four SPINK genes were negative, including SPINK4 in the extended case/control cohort. No SPINK4 mutations or deletions were observed in the multigeneration CD family with linkage to chromosome 9p21-13 nor was the coding SNP disease-specific. SPINK4 exhibits CD pathology-related differential gene expression, likely derived from altered goblet cell activity. All of the four SPINK genes tested do not contribute to the genetic risk for CD in the Dutch population.

Oral leukoplakia is the most common oral potentially malignant disorder with a malignant transformation rate into oral squamous cell carcinoma of 1-3% annually. The presence and grade of World Health Organization defined dysplasia is an important histological marker to assess the risk for malignant transformation, but is not sufficiently accurate to personalize treatment and surveillance. Differentiated dysplasia, known from differentiated vulvar intraepithelial neoplasia, is hitherto not used in oral dysplasia grading. We hypothesized that assessing differentiated dysplasia besides World Health Organization defined (classic) dysplasia will improve risk assessment of malignant transformation of oral leukoplakia. We investigated a retrospective cohort consisting of 84 oral leukoplakia patients. Biopsies were assessed for dysplasia presence and grade, and the expression of keratins 13 (CK13) and 17, known to be dysregulated in dysplastic vulvar mucosa. In dysplastic oral lesions, differentiated dysplasia is as common as classic dysplasia. In 25 out of 84 (30%) patients, squamous cell carcinoma of the upper aerodigestive tract developed during follow-up. Considering only classic dysplasia, 11 out of 56 (20%) patients with nondysplastic lesions progressed. With the incorporation of differentiated dysplasia, only 2 out of 30 (7%) patients with nondysplastic lesions progressed. The risk of progression increased from 3.26 (Hazard ratio, p = 0.002) when only classic dysplasia is considered to 7.43 (Hazard ratio, p = 0.001) when classic and differentiated dysplasia are combined. Loss of CK13, combined with presence of dysplasia, is associated with greater risk of malignant progression (p = 0.006). This study demonstrates that differentiated dysplasia should be recognized as a separate type of dysplasia in the oral mucosa and that its distinction from classic dysplasia is of pathological and clinical significance since it is a strong (co) prognostic histopathological marker for oral malignant transformation. In oral lesions without dysplasia and retained CK13 staining the risk for progression is very low.

Summary Chromosomal copy number aberrations can be efficiently detected and quantified using low-coverage whole-genome sequencing, but analysis is hampered by the lack of knowledge on absolute DNA copy numbers and tumor purity. Here, we describe an analytical tool for Absolute Copy number Estimation, ACE, which scales relative copy number signals from chromosomal segments to optimally fit absolute copy numbers, without the need for additional genetic information, such as SNP data. In doing so, ACE derives an estimate of tumor purity as well. ACE facilitates analysis of large numbers of samples, while maintaining the flexibility to customize models and generate output of single samples. Availability and implementation ACE is freely available via www.bioconductor.org and at www.github.com/tgac-vumc/ACE. Supplementary information Supplementary data are available at Bioinformatics online.

Malignant melanoma is an aggressive form of skin cancer with poor prognosis. Despite improvements in awareness and prevention of this disease, its incidence is rapidly increasing. MicroRNAs (miRNAs) are a class of small RNA molecules that regulate cellular processes by repressing messenger RNAs (mRNAs) with partially complementary target sites. Several miRNAs have already been shown to attenuate cancer phenotypes, by limiting proliferation, invasiveness, tumor angiogenesis, and stemness. Here, we employed a genome-scale lentiviral human miRNA expression library to systematically survey which miRNAs are able to decrease A375 melanoma cell viability. We highlight the strongest inhibitors of melanoma cell proliferation, including the miR-15/16, miR-141/200a and miR-96/182 families of miRNAs and miR-203. Ectopic expression of these miRNAs resulted in long-term inhibition of melanoma cell expansion, both in vitro and in vivo. We show specifically miR-16, miR-497, miR-96 and miR-182 are efficient effectors when introduced as synthetic miRNAs in several melanoma cell lines. Our study provides a comprehensive interrogation of miRNAs that interfere with melanoma cell proliferation and viability, and offers a selection of miRNAs that are especially promising candidates for application in melanoma therapy.

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BackgroundMicroRNAs (miRNAs) are a class of small regulatory RNAs that target sequences in messenger RNAs (mRNAs) to inhibit their protein output. Dissecting the complexities of miRNA function continues to prove challenging as miRNAs are predicted to have thousands of targets, and mRNAs can be targeted by dozens of miRNAs.ResultsTo systematically address biological function of miRNAs, we constructed and validated a lentiviral miRNA expression library containing 660 currently annotated and 422 candidate human miRNA precursors. The miRNAs are expressed from their native genomic backbone, ensuring physiological processing. The arrayed layout of the library renders it ideal for high-throughput screens, but also allows pooled screening and hit picking. We demonstrate its functionality in both short- and long-term assays, and are able to corroborate previously described results of well-studied miRNAs.ConclusionsWith the miRNA expression library we provide a versatile tool for the systematic elucidation of miRNA function.

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