Wieben, Eric D.

Abstract Long non-coding RNAs (lncRNA) are key regulatory RNAs that do not code for protein. Recently lncRNAs have been recognized as key regulators of gene expression and chromatin organization. Although, deregulation of lncRNAs such as HOTAIR, MALAT1 and H19 has been reported in solid cancers, role of lncRNAs in lymphoma or other hematologic malignancies is not known. The aim of this study is to identify lymphoma-associated lncRNAs and elucidate their role in disease pathogenesis. Next generation RNA-sequencing and validation studies were carried out on mantle cell lymphoma (MCL) patient samples along with normal controls. As a result, hundreds of lncRNAs were found overexpressed in the MCL samples as compared with normal B cells, including RP11-24J23.2 (50 fold), RP11-12A2.3 (36 fold) and DLEU7-AS1 (19 fold). Quantitative RT-PCR (QRT-PCR) confirmed higher expression of RP11-24J23.2, RP11-12A2.3, and DLEU7-AS1 in MCL patient samples as compared to normal B cells. To investigate the biological significance of lncRNAs in MCL cells, we overexpressed RP11-24J23.2, RP11-12A2.3, and DLEU7-AS1 lncRNAs and analyzed effects on cell proliferation and apoptosis. To achieve overexpression lncRNAs, RP11-24J23.2, RP11-12A2.3, and DLEU7-AS1 were cloned individually in expression pcDNA3.1 generating constructs: pcDNA3RP11-24J23.2, pcDNA3RP11-12A2.3 and pcDNA3DLEU7-AS1and then transfected into MCL cell lines along with pcDNA3.1. QRT-PCR revealed effective overexpression (~100 fold increase over baseline) for each transfected lncRNAs construct in Granta and Mino cells. Analysis of proliferation and survival showed that overexpressing RP11-24J23.2, RP11-12A2.3 or DLEU7-AS1 promotes cell proliferation and survival in these cells as compared to the vector controls. Most of the lncRNAs found so far have been linked to the transcriptional regulation of the target genes through their association with chromatin remodeling factors such as Polycomb Repressive Complex 2 (PRC2). To identify the functional relevance of RP11-24J23.2, RP11-12A2.3, and DLEU7-AS1 lncRNAs, we examined the binding of the lymphoma specific lncRNAs with PRC2 complex, by performing RNA immunoprecipitation (RNA-IP) using specific antibodies to EZH2, EED and SUZ12. The RNA-IP results demonstrate high affinity binding of all three lncRNAs with SUZ12 and EED but a weak binding with EZH2. Furthermore, RNA-IP using antibody to histone 3-lysine 27 trimethylation (H3K27me3), a suppressive histone mark associated with PRC2 complex, also showed efficient binding with RP11-24J23.2, RP11-12A2.3, and DLEU7-AS1. These results suggest that RP11-24J23.2, RP11-12A2.3, and DLEU7-AS1 lncRNAs can directly associated with PRC2 complex at chromatin. To understand the mechanism of increased cell growth after overexpression of lncRNAs, RP11-24J23.2, RP11-12A2.3 or DLEU7-AS1, we analyzed expression level of important genes signature of MCL pathogenesis that include cyclin D1, cyclin D3, c-Myc, P14, and SOX11, a member of the group C SOX (SRY-related HMG-box) transcription factor family. Our results demonstrate that overexpression of lncRNAs RP11-24J23.2, RP11-12A2.3 or DLEU7-AS1 enhanced the levels of cyclin D1 (>2 fold) mRNA without any impact on cyclin D3 or c-Myc transcript levels. In contrast overexpression of RP11-24J23.2, RP11-12A2.3 or DLEU7-AS1 lncRNAs decreased SOX11 (0.08, 0.1 and 0.06 respectively) and P14 (0.06, 0.7 and 0.40 respectively) mRNA expression in Mino cells. To gain understanding of the mechanism involved in the suppression of SOX11 by the lncRNAs, we performed Chromatin immunoprecipitation assays with EZH2, EED and SUZ12 antibodies on Mino cells transfected with pcDNA3RP11-24J23.2, pcDNA3RP11-12A2.3 and pcDNA3DLEU7-AS1 or vector control. The data showed that overexpression of RP11-12A2.3 and DLEU7-AS1 significantly increased the binding of EED and to a lesser extent of EZH2 to SOX11 promoter while the binding of SUZ12 to SOX11 promoter was not changed as compared to vector alone. In conclusion, our results imply that lncRNAs are overexpressed in the MCL. Moreover, lymphoma associated lncRNAs (RP11-24J23.2, RP11-12A2.3 and DLEU7-AS1) regulate SOX11 expression via PRC2 complex contributing towards growth of MCL cells. Specific nature of lncRNAs found in lymphoma samples suggests that lncRNAs can be used as potential biomarkers for mantle cell lymphoma. Disclosures No relevant conflicts of interest to declare.

Purpose: To investigate single nucleotide polymorphisms (SNPs) and trinucleotide repeat (TNR) expansion in the transcription factor 4 (TCF4) gene in a large cohort of German patients with Fuchs endothelial corneal dystrophy (FECD). Methods: Genomic DNA was obtained from 398 patients with FECD and from 58 non-FECD controls. Thirty-seven previously reported SNPs were evaluated by genotyping. The 398 FECD samples were analyzed for TNR expansions by short tandem repeat assays and Southern blotting. The possible associations between the TNR length and clinical parameters (age, sex, visual acuity, and central corneal thickness) were analyzed in 132 patients. Results: The SNPs in COL8A2, TCF8, LOXHD1, and AGBL1 showed no heterogeneity in 36 cases, although SLCA411 showed 3 nonsense mutations. SNPs were detected for TCF4 (rs613872, rs2123392, rs17089887, rs1452787, and rs1348047), but only rs613872 showed a significant association with FECD (P = 9.93 × 10−12). Overall, 315/398 (79%) patients harbored TNR lengths >50, whereas no non-FECD controls harbored TNR lengths >50. The TCF4 SNP rs613872 genotype was TT: 39 (67%), TG: 18 (31%), and GG: 1 (2%) in non-FECD controls; TT: 39 (47%), TG: 38 (46%), and GG: 6 (7%) in FECD cases harboring TNR <50; and TT: 23 (8%), TG: 224 (79%), and GG: 38 (13%) in FECD cases harboring TNR >50 (P = 2.93 × 10−25). No significant association was detected between the TNR length and clinical parameters. Conclusions: Our large German cohort demonstrated a significant association between the risk allele G in rs613872 and FECD, irrespective of TNR expansion, although this risk allele was more frequent in FECD cases with TNR expansion than without.

AbstractObjectiveWe identified a pedigree over five generations with 49 members, some of whom had chronic megacolon presenting in adolescence or adulthood. We aimed to assess the genetic cause of chronic megacolon through clinical and DNA studies.DesignAfter ethical approval and informed consent, family members provided answers to standard bowel disease questionnaires, radiological or surgical records, and DNA (buccal mucosal scraping). Exome DNA sequencing of colon tissue or blood DNA from seven family members with colon or duodenal dilatation, or no megacolon (n = 1) was carried out. Sanger sequencing was performed in 22 additional family members to further evaluate candidate variants. The study focused on genes of potential relevance to enteric nerve (ENS) maturation and Hirschsprung's disease or megacolon, based on the literature (GFRA1, NKX2‐1, KIF26A, TPM3, ACTG2, SCN10A, and C17orf107 [CHRNE]) and other genetic variants that co‐segregated with megacolon in the six affected family members.ResultsInformation was available in all except five members alive at time of study; among 30 members who provided DNA, six had definite megacolon, one megaduodenum, seven significant constipation without bowel dilatation, and 16 normal bowel function by questionnaire. Among genes studied, SEMA3F (g.3:50225360A>G; c1873A>G) was found in 6/6 family members with megacolon. The SEMA3F gene variant was assessed as potentially pathogenic, based on M‐CAP in silico prediction. SEMA3F function is associated with genes (KIT and PDGFRB) that impact intestinal pacemaker function.ConclusionFamilial chronic megacolon appears to be associated with SEMA3F, which is associated with genes impacting enteric nerve or pacemaker function.

AbstractBackground: Pathogenic germline mutations in the CDKN2A tumor suppressor gene are rare and associated with highly penetrant familial melanoma and pancreatic cancer in non-Hispanic whites (NHW). To date, the prevalence and impact of CDKN2A rare coding variants (RCV) in racial minority groups remain poorly characterized. We examined the role of CDKN2A RCVs on the risk of pancreatic cancer among minority subjects.Methods: We sequenced CDKN2A in 220 African American (AA) pancreatic cancer cases, 900 noncancer AA controls, and 183 Nigerian controls. RCV frequencies were determined for each group and compared with that of 1,537 NHW patients with pancreatic cancer. Odds ratios (OR) and 95% confidence intervals (CI) were calculated for both a case–case comparison of RCV frequencies in AAs versus NHWs, and case–control comparison between AA cases versus noncancer AA controls plus Nigerian controls. Smaller sets of Hispanic and Native American cases and controls also were sequenced.Results: One novel missense RCV and one novel frameshift RCV were found among AA patients: 400G&gt;A and 258_278del. RCV carrier status was associated with increased risk of pancreatic cancer among AA cases (11/220; OR, 3.3; 95% CI, 1.5–7.1; P = 0.004) compared with AA and Nigerian controls (17/1,083). Further, AA cases had higher frequency of RCVs: 5.0% (OR, 13.4; 95% CI, 4.9–36.7; P &lt; 0.001) compared with NHW cases (0.4%).Conclusions: CDKN2A RCVs are more common in AA than in NHW patients with pancreatic cancer and associated with moderately increased pancreatic cancer risk among AAs.Impact: RCVs in CDKN2A are frequent in AAs and are associated with risk for pancreatic cancer. Cancer Epidemiol Biomarkers Prev; 27(11); 1364–70. ©2018 AACR.

AbstractBackgroundCongenital myopathies due to ryanodine receptor (RYR1) mutations are increasingly identified and correlate with a wide range of phenotypes, most commonly that of malignant hyperthermia susceptibility and central cores on muscle biopsy with rare reports of distal muscle weakness, but in the setting of early onset global weakness.MethodsWe report a case of a patient presenting with childhood onset hand stiffness and adult onset progressive hand weakness and jaw contractures discovered to have two variants in the RYR1 gene.ResultsThe patient manifested with distal upper limb weakness which progressed to involve the distal lower limb, proximal upper limb, as well as the face in addition to limited jaw opening. Creatine kinase was mildly elevated with EMG findings supporting a myopathy. Muscle biopsy showed features consistent with centronuclear myopathy. Whole exome sequencing revealed a novel heterozygous pathogenic variant in RYR1 (c.12315_12328delAGAAATCCAGTTCC, p.Glu4106Alafs*8), and a heterozygous missense variant (c.10648C>T, p.Arg3550Trp) of unknown significance in compound heterozygous state.ConclusionWe expand the spectrum of RYR1‐related myopathy with the description of a novel phenotype in an adult patient presenting with hand weakness and suggest considering RYR1 analysis in the diagnosis of distal myopathies.