Angiotensinogen Promoter Polymorphisms Predict Low Diffusing Capacity in U.S. and Spanish IPF Cohorts

Single nucleotide polymorphisms (SNPs) in angiotensinogen (AGT) at positions -20 and -6 are associated with increased severity and progression of various fibrotic diseases. Our earlier work demonstrated that the progression of idiopathic pulmonary fibrosis (IPF) was associated with the A-6 allele. This study examined the hypothesis that the homozygous CC genotype at -20 and the AA genotype at -6 would confer worse measures of pulmonary function (measured by pulmonary function tests) in IPF. Multiple logistic regression analysis was applied to a NIH Lung Tissue Research Consortium cohort and a Spanish cohort, while also adjusting for covariates to determine the effects of these SNPs on measures of pulmonary function. Analysis demonstrated that the CC genotype at -20 was strongly associated with reduced diffusing capacity in males in both cohorts (p = 0.0028 for LTRC and p = 0.017 for the Spanish cohort). In females, the AA genotype was significantly associated with lower FVC (p = 0.0082) and V (alv) (p = 0.022). In males, the haplotype CA at -20 and -6 in AGT was also strongly associated with reduced diffusing capacity in both cohorts. This study is the first to demonstrate an association of AGT polymorphisms (-20A > C and -6G > A) with lower measures of pulmonary function in IPF. It is also the first to relate the effect of gender in lung fibrosis with polymorphisms in AGT

A novel actin mRNA splice variant regulates ACTG1 expression.

Cytoplasmic actins are abundant, ubiquitous proteins in nucleated cells. However, actin expression is regulated in a tissue- and development-specific manner. We identified a novel cytoplasmic-γ-actin (Actg1) transcript that includes a previously unidentified exon (3a). Inclusion of this exon introduces an in-frame termination codon. We hypothesized this alternatively-spliced transcript down-regulates γ-actin production by targeting these transcripts for nonsense-mediated decay (NMD). To address this, we investigated conservation between mammals, tissue-specificity in mice, and developmental regulation using C2C12 cell culture. Exon 3a is 80% similar among mammals and varies in length from 41 nucleotides in humans to 45 in mice. Though the predicted amino acid sequences are not similar between all species, inclusion of exon 3a consistently results in the in the introduction of a premature termination codon within the alternative Actg1 transcript. Of twelve tissues examined, exon 3a is predominantly expressed in skeletal muscle, cardiac muscle, and diaphragm. Splicing to include exon 3a is concomitant with previously described down-regulation of Actg1 in differentiating C2C12 cells. Treatment of differentiated C2C12 cells with an inhibitor of NMD results in a 7-fold increase in exon 3a-containing transcripts. Therefore, splicing to generate exon 3a-containing transcripts may be one component of Actg1 regulation. We propose that this post-transcriptional regulation occurs via NMD, in a process previously described as "regulated unproductive splicing and translation" (RUST)

Ct values for a skeletal muscle cDNA control demonstrate no plate-to-plate variability.

<p>For each qPCR plate, a skeletal muscle cDNA control sample was included to assess plate-to-plate variability for every primer pair. For the <i>Tissue Panel</i> experiment Ct values represent three skeletal muscle cDNA biological replicates in addition to the control skeletal muscle cDNA sample. Alternatively spliced transcripts constitute approximately 30% of steady-state <i>Actg1</i> mRNA in adult skeletal muscle. Two technical replicates were averaged for every biological replicate in qPCR experiments.</p

Splicing to include exon 3a is a developmentally regulated event in skeletal muscle.

<p>(<b>A</b>) Microscopy of cell cultures before, during and after differentiation. C2C12 myoblasts were grown to 70% confluence and induced to differentiate in DMEM+10% horse serum. Partially differentiated cultures containing both myoblasts and myotubes were observed by 2 days post-differentiation. After 48 hours, medium was replaced with DMEM+2% horse serum and 10 µM Ara-C and cultured for an additional 4 days. (<b>B</b>) qPCR of RNA harvested in Trizol showed that concurrent with a decrease in normal <i>Actg1</i>, splicing to generate alternative <i>Actg1</i> increases during differentiation into myotubes. Expression of both the normal and alternative transcripts was normalized to <i>Ppia</i> and is presented as fold-difference compared to skeletal muscle. A two-tailed type 2 Student's T-test was used to compare expression differences between time points. For all time points compared, p<0.0001.</p

The sequence of primers used in this study.

<p>Primers were designed using Primer3 to amplify cDNA from either human (Hs), mouse (Mm), or dog (Cf) and cat (Fc).</p

Splicing to include exon 3a into the <i>Actg1</i> transcript is tissue specific.

<p>(<b>A</b>) Two PCR-based assays were employed to screen for the presence of <i>Actg1</i> – competitive end-point PCR and splice-specific qPCR. <i>Actg1</i>-specific primers were designed to amplify either both the normal and alternative transcripts in a single reaction (competitive end-point PCR), or specifically the normal or alternative transcripts in separate reactions (splice-specific qPCR). (<b>B</b>) The competitive end-point PCR assay was used to amplify all <i>Actg1</i> transcripts in various tissues from adult mouse. The larger PCR products at ∼390 bp and ∼290 bp are intermediate spliceforms of <i>Actg1</i>. The 147 bp product represents alternatively spliced, exon 3a-containing <i>Actg1</i> transcripts, while the 102 bp product represents normally spliced <i>Actg1</i> transcripts. (<b>C</b>) Expression data from qPCR to amplify normal <i>Actg1</i> (exon 3 – exon 4) shows a high degree of variability of γ-actin expression between tissues. (<b>D</b>) Splicing to include exon 3a, as measured by qPCR is primarily limited to skeletal and cardiac muscle, with very low levels in the brain, eye, and intestine. All qPCR data is normalized to <i>Rplp0</i> and <i>Rrn18s</i> expression and presented as a fold-difference to skeletal muscle.</p

Exon 3a containing alternatively spliced <i>Actg1</i> is exported to the cytoplasm, but does not produce a stable protein.

<p>Nuclear (<b>A</b>) and cytoplasmic (<b>B</b>) RNA fractions were harvested from C2C12 myotubes and evaluated for the presence of alternative <i>Actg1</i> using competitive, end-point PCR. All <i>Actg1</i> spliceforms, including partially spliced transcripts, were present in the nuclear fraction, however, only the normal and exon 3a transcripts were observed in the cytoplasmic fraction. (<b>C</b>) Western blot using an anti-γ-actin specific antibody was used to probe mouse skeletal muscle lysate for the presence of a protein product corresponding to the inclusion of exon 3a. Usage of the in-frame stop codon in exon 3a would generate a 15 kDa protein. Alternatively, read-through of the stop codon would increase the size of the ACTG1 protein by 2 kDa, resulting in a 44 kDa protein. Neither of these protein products is present. A γ-actin protein at 52 kDa is observed and likely represents a post-translational modification. Molecular weight marker is prestained Low Range (BioRad).</p

Cycloheximide (CHX) treatment of myotubes results in a 7-fold increase in alternative <i>Actg1</i>.

<p>(<b>A,B</b>) Competitive end-point PCR and (<b>C</b>) qPCR were used to evaluate relative abundance in untreated (<b>A,C</b>) and CHX treated cells (<b>B,C</b>). Expression of both the normal and alternative transcripts were normalized to <i>Ppia</i> and are presented as fold-difference compared to skeletal muscle. A two-tailed type 2 Student's T-test was used to compare expression differences between samples. * p<0.05, ** p<0.001.</p

Expression of GJB2 and GJB6 Is Reduced in a Novel DFNB1 Allele

In a large kindred of German descent, we found a novel allele that segregates with deafness when present in trans with the 35delG allele of GJB2. Qualitative polymerase chain reaction–based allele-specific expression assays showed that expression of both GJB2 and GJB6 from the novel allele is dramatically reduced. This is the first evidence of a deafness-associated regulatory mutation of GJB2 and of potential coregulation of GJB2 and GJB6