Genetics of Deafness

201256284

Gene × environment interactions for ADHD: synergistic effect of 5HTTLPR genotype and youth appraisals of inter-parental conflict

<p>Abstract</p> <p>Background</p> <p>Serotonin genes have been hypothesized to play a role in the etiology of attention-deficit hyperactivity disorder (ADHD); prior work suggests that serotonin may interact with psychosocial stressors in ADHD, perhaps via mechanisms involved in emotional dysregulation. Because the development of behavioral and emotional regulation depends heavily both on the child's experience within the family context and the child's construals of that experience, children's appraisals of inter-parental conflict are a compelling candidate potentiator of the effects of variation within the serotonin transporter gene promoter polymorphism (5HTTLPR) on liability for ADHD.</p> <p>Method</p> <p>304 youth from the local community underwent a multi-informant diagnostic assessment procedure to identify ADHD cases and non-ADHD controls. Youth also completed the Children's Perception of Inter-Parental Conflict (CPIC) scale to assess appraisals of self-blame in relation to their parents' marital disputes. The trialleic configuration of 5HTTLPR (long/short polymorphism with A> G substitution) was genotyped and participants were assigned as having high (La/La N = 78), intermediate (La/Lg, La/short, N = 137), or low (Lg/Lg, Lg/short, short/short, N = 89) serotonin transporter activity genotypes. Teacher reported behavior problems were examined as the target outcome to avoid informant overlap for moderator and outcome measures.</p> <p>Results</p> <p>Hierarchical linear regression analyses indicated significant 5HTTLPR × self-blame interactions for ADHD symptoms. Examination of the interactions indicated positive relations between reports of self-blame and ADHD symptoms for those with the high and low serotonin activity genotypes. There was no relation between self-blame and ADHD for those with intermediate activity 5HTTLPR genotypes.</p> <p>Conclusion</p> <p>Both high and low serotonergic activity may exert risk for ADHD when coupled with psychosocial distress such as children's self-blame in relation to inter-parental conflict. Results are discussed in relation to the role of serotonin in the etiology of the ADHD and related externalizing behaviors.</p

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)

Identification of Methylated Nucleosides in Messenger RNA from Novikoff Hepatoma Cells

The poly(A) tract found in eukaryotic mRNA was used to study methylation in mRNA obtained from Novikoff hepatoma cells. Methyl labeling of RNA was achieved with L-[methyl-(3)H]methionine under conditions that suppress radioactive incorporation into the purine ring. RNA that contains a poly(A) segment was obtained from polysomal RNA by chromatography on oligo(dT)-cellulose. Sucrose density gradient centrifugation of this RNA revealed a pattern expected for mRNA. The composition of the methyl-labeled nucleosides in the RNA was analyzed after complete enzymatic degradation to nucleosides. By use of DEAE-cellulose (borate) chromatography, which separates 2′-O-methylnucleosides from normal and base-methylated nucleosides, about 50% of the radioactivity was recovered in the 2′-O-methylnucleoside fraction and 50% in the base-methylnucleoside fraction. High-speed liquid chromatography (Aminex A-5) of the 2′-O-methylnucleoside fraction produced four peaks coincident with the four 2′-O-methylnucleoside standards. Analysis of the base-methylnucleoside fraction revealed a unique pattern. While ribosomal RNA and tRNA possessed complex base-methylnucleoside patterns, the distribution in mRNA was quite simple, consisting predominantly of N(6)-methyladenosine. These results demonstrate a unique distribution of methylated nucleosides in mRNA. By analogy to ribosomal RNA synthesis, the presence of methylnucleosides in mRNA may reflect a cellular mechanism for the selective processing of certain mRNA sequences

Nucleotide Methylation Patterns in Eukaryotic mRNA

This chapter reviews that the use of enzymes for selective hydrolysis, coupled with high-resolution liquid chromatography for assay of products, provides an efficient means of determining the specific patterns of methylation in eukaryotic mRNA molecules. Continuous labeling with levels of L-[methyl-3H] methionine permits normal growth of Novikoff cells to examine the methylation of specific sites of cytoplasmic mRNA as a function of time. The main site of cytoplasmic mRNA labeling is at the second position (N'') of the 5'-terminal sequence. Data obtained by comparing methylnucleoside composition of these sequences and the ratio of doubly to singly O-methylated termini as a function of labeling time is consistent with a model in which m7G, N'm and the m6A located in the mRNA molecule are all products of nuclear methylation events. Subsequently there is a cytoplasmic methylation of some singly O-methylated structures at the second (N'') position yielding the doubly O-methylated structure. The kinetics of methyl labeling and the changing composition within the caps show a distinct pattern, possibly reflecting a selection or enrichment of a stable class of mRNA molecules, many of which contain the doubly labeled structure at their 5'-terminus and are of smaller size

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