Multiple functional risk variants in a SMAD7 enhancer implicate a colorectal cancer risk haplotype

Genome-wide association studies (GWAS) of colorectal cancer (CRC) have led to the identification of a number of common variants associated with modest risk. Several risk variants map within the vicinity of TGFβ/BMP signaling pathway genes, including rs4939827 within an intron of SMAD7 at 18q21.1. A previous study implicated a novel SNP (novel 1 or rs58920878) as a functional variant within an enhancer element in SMAD7 intron 4. In this study, we show that four SNPs including novel 1 (rs6507874, rs6507875, rs8085824, and rs58920878) in linkage disequilibrium (LD) with the index SNP rs4939827 demonstrate allele-specific enhancer effects in a large, multi-component enhancer of SMAD7. All four SNPs demonstrate allele-specific protein binding to nuclear extracts of CRC cell lines. Furthermore, some of the risk-associated alleles correlate with increased expression of SMAD7 in normal colon tissues. Finally, we show that the enhancer is responsive to BMP4 stimulation. Taken together, we propose that the associated CRC risk at 18q21.1 is due to four functional variants that regulate SMAD7 expression and potentially perturb a BMP negative feedback loop in TGFβ/BMP signaling pathways

RNA steady-state defects in myotonic dystrophy are linked to nuclear exclusion of SHARP

We describe a new mechanism by which CTG tract expansion affects myotonic dystrophy (DM1). Changes to the levels of a panel of RNAs involved in muscle development and function that are downregulated in DM1 are due to aberrant localization of the transcription factor SHARP (SMART/HDAC1-associated repressor protein). Mislocalization of SHARP in DM1 is consistent with increased CRM1-mediated export of SHARP to the cytoplasm. A direct link between CTG repeat expression and SHARP mislocalization is demonstrated as expression of expanded CTG repeats in normal cells recapitulates cytoplasmic SHARP localization. These results demonstrate a role for the inactivation of SHARP transcription in DM1 biology

Novel Common Genetic Susceptibility Loci for Colorectal Cancer

BACKGROUND: Previous genome-wide association studies (GWAS) have identified 42 loci (P < 5 × 10-8) associated with risk of colorectal cancer (CRC). Expanded consortium efforts facilitating the discovery of additional susceptibility loci may capture unexplained familial risk. METHODS: We conducted a GWAS in European descent CRC cases and control subjects using a discovery-replication design, followed by examination of novel findings in a multiethnic sample (cumulative n = 163 315). In the discovery stage (36 948 case subjects/30 864 control subjects), we identified genetic variants with a minor allele frequency of 1% or greater associated with risk of CRC using logistic regression followed by a fixed-effects inverse variance weighted meta-analysis. All novel independent variants reaching genome-wide statistical significance (two-sided P < 5 × 10-8) were tested for replication in separate European ancestry samples (12 952 case subjects/48 383 control subjects). Next, we examined the generalizability of discovered variants in East Asians, African Americans, and Hispanics (12 085 case subjects/22 083 control subjects). Finally, we examined the contributions of novel risk variants to familial relative risk and examined the prediction capabilities of a polygenic risk score. All statistical tests were two-sided. RESULTS: The discovery GWAS identified 11 variants associated with CRC at P < 5 × 10-8, of which nine (at 4q22.2/5p15.33/5p13.1/6p21.31/6p12.1/10q11.23/12q24.21/16q24.1/20q13.13) independently replicated at a P value of less than .05. Multiethnic follow-up supported the generalizability of discovery findings. These results demonstrated a 14.7% increase in familial relative risk explained by common risk alleles from 10.3% (95% confidence interval [CI] = 7.9% to 13.7%; known variants) to 11.9% (95% CI = 9.2% to 15.5%; known and novel variants). A polygenic risk score identified 4.3% of the population at an odds ratio for developing CRC of at least 2.0. CONCLUSIONS: This study provides insight into the architecture of common genetic variation contributing to CRC etiology and improves risk prediction for individualized screenin

RNA splicing is responsive to MBNL1 dose.

Myotonic dystrophy (DM1) is a highly variable, multi-system disorder resulting from the expansion of an untranslated CTG tract in DMPK. In DM1 expanded CUG repeat RNAs form hairpin secondary structures that bind and aberrantly sequester the RNA splice regulator, MBNL1. RNA splice defects resulting as a consequence of MBNL1 depletion have been shown to play a key role in the development of DM1 pathology. In patient populations, both the number and severity of DM1 symptoms increase broadly as a function of CTG tract length. However significant variability in the DM1 phenotype is observed in patients encoding similar CTG repeat numbers. Here we demonstrate that a gradual decrease in MBNL1 levels results both in the expansion of the repertoire of splice defects and an increase in the severity of the splice alterations. Thus, MBNL1 loss does not have an all or none outcome but rather shows a graded effect on the number and severity of the ensuing splice defects. Our results suggest that once a critical threshold is reached, relatively small dose variations of free MBNL1 levels, which may reflect modest changes in the size of the CUG tract or the extent of hairpin secondary structure formation, can significantly alter the number and severity of splice abnormalities and thus contribute to the phenotype variability observed in DM1 patients

RNA half-life measurements in SkMC.

<p>Normal myoblasts were treated with a combination of actinomycin-D and α-aminitin to inhibit transcription. Myoblasts were harvested at different time-points after treatment (0, 0.5, 4, 8, 16 and 24 h) and RNA was extracted. Synthesized cDNAs were subjected to RT-PCR analysis to measure RNA half-lives as previously described (17,18). <i>MYC</i>, a short-lived RNA and the long-lived 18S RNA were used as controls. Graphical representation of the average percent of RNA plotted against time from two independent experiments is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048825#pone.0048825.s002" target="_blank">Figure S2</a>.</p

Number and severity of splice defects increase when MBNL1 is silenced incrementally from ∼79% to ∼98%.

<p>SkMC were transfected with siRNAs directed against MBNL1 and cell samples on each subsequent day post-siRNA transfection for a period of 5 days, were divided into 4 aliquots where one aliquot was used to measure MBNL1 levels and total RNA was extracted from each of the three other aliquots. Scrambled siRNA transfected samples were harvested on Day 5, the last time point of the experiment. <b>(A)</b> Total protein (10 µg) was analyzed by western blot to measure the silencing achieved for MBNL1 at 24 h intervals for 5 days. Blots were probed for GAPDH as an internal control. <b>(B)</b> Synthesized cDNAs were subjected to PCR analysis to study RNA splicing as indicated with <i>GAPDH</i> RNA as an internal control. In each case the levels of exon inclusion obtained in the experiment shown are indicated. <b>(C)</b> The results of RNA splicing as a function of MBNL1 levels in SkMC are tabulated.</p

Splice defects in <i>Mbnl1^+/ΔE3</i> and <i>Mbnl1^ΔE3/ΔE3</i> skeletal muscle.

<p>Lower limb skeletal muscles from adult wild-type, <i>Mbnl1^+/ΔE3</i> and <i>Mbnl1^ΔE3/ΔE3</i> mice were harvested and divided into 2 aliquots. One aliquot was used to measure Mbnl1 levels and the other aliquot was used study RNA splicing. <b>(A)</b> Western blot analysis of steady-state Mbnl1 levels in skeletal muscle of wild-type, <i>Mbnl1^+/ΔE3</i> and <i>Mbnl1^ΔE3/ΔE3</i> mice are shown with Gapdh as an internal loading control. <b>(B)</b> cDNAs synthesized from skeletal muscle of wild-type, <i>Mbnl1^+/ΔE3</i> and <i>Mbnl1^ΔE3/ΔE3</i> mice were subjected to PCR analysis as indicated with <i>Gapdh</i> RNA as an internal control. In each case the levels of exon inclusion obtained in the experiment shown are indicated. <b>(C)</b> The results of RNA splicing examined as a function of Mbnl1 levels are tabulated.</p

Incremental depletion of MBNL1 results in an increase of both the number and severity of RNA splice defects.

<p>RNA splice defects that manifest with the depletion of MBNL1 in SkMC and in <i>Mbnl1^+/ΔE3</i> and <i>Mbnl1^ΔE3/ΔE3</i> skeletal muscle are shown. Line thickness represents the severity of the splice defect.</p

Multiple functional risk variants in a SMAD7 enhancer implicate a colorectal cancer risk haplotype

Genome-wide association studies (GWAS) of colorectal cancer (CRC) have led to the identification of a number of common variants associated with modest risk. Several risk variants map within the vicinity of TGFβ/BMP signaling pathway genes, including rs4939827 within an intron of SMAD7 at 18q21.1. A previous study implicated a novel SNP (novel 1 or rs58920878) as a functional variant within an enhancer element in SMAD7 intron 4. In this study, we show that four SNPs including novel 1 (rs6507874, rs6507875, rs8085824, and rs58920878) in linkage disequilibrium (LD) with the index SNP rs4939827 demonstrate allele-specific enhancer effects in a large, multi-component enhancer of SMAD7. All four SNPs demonstrate allele-specific protein binding to nuclear extracts of CRC cell lines. Furthermore, some of the risk-associated alleles correlate with increased expression of SMAD7 in normal colon tissues. Finally, we show that the enhancer is responsive to BMP4 stimulation. Taken together, we propose that the associated CRC risk at 18q21.1 is due to four functional variants that regulate SMAD7 expression and potentially perturb a BMP negative feedback loop in TGFβ/BMP signaling pathways

Fragment A Enhancer is responsive to BMP4 stimulation but not TGFβ1.

<p>(<b>A</b>) Enhancer activity was measured by luciferase assay for the fragment A containing the major (CGTC) and minor (TCCG) haplotypes in serum starved HCT-116 and SW480 cells incubated with TGFβ1 for 6 hours. Samples are plotted as a fold change in activity relative to non-treated cells on the same plate. (<b>B</b>) Enhancer activity was stimulated by 6 hours of BMP4 treatment for the CGTC haplotype in HCT-116 (<i>p</i> = 4.14×10⁻³) and SW480 cells (<i>p</i> = 1.22×10⁻¹⁰). The TCCG haplotype in fragment A shows lower levels of stimulation, and only in SW480 (<i>p</i> = 1.61×10⁻⁶). (HCT-116 <i>p</i> = 0.38). Effect of BMP is plotted as fold change over untreated samples for each haplotype on the same plate. (<b>C</b>) Enhancer activity comparison between the CGTC and TCCG haplotypes following BMP4 treatment. Relative luciferase activity was plotted for each haplotype in HCT-116 and SW480 using the same experimental dataset as (B).</p