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Endogenous Reference Genes for Gene Expression Studies on Bicuspid Aortic Valve Associated Aortopathy in Humans.

ABSTRACT: Bicuspid aortic valve (BAV) disease is the most common congenital cardiac abnormality and predisposes patients to life-threatening aortic complications including aortic aneurysm. Quantitative real-time reverse transcription PCR (qRT-PCR) is one of the most commonly used methods to investigate underlying molecular mechanisms involved in aortopathy. The accuracy of the gene expression data is dependent on normalization by appropriate housekeeping (HK) genes, whose expression should remain constant regardless of aortic valve morphology, aortic diameter and other factors associated with aortopathy. Here, we identified an appropriate set of HK genes to be used as endogenous reference for quantifying gene expression in ascending aortic tissue using a spin column-based RNA extraction method. Ascending aortic biopsies were collected intra-operatively from patients undergoing aortic valve and/or ascending aortic surgery. These patients had BAV or tricuspid aortic valve (TAV), and the aortas were either dilated (?4.5cm) or undilated. The cohort had an even distribution of gender, valve disease and hypertension. The expression stability of 12 reference genes were investigated (ATP5B, ACTB, B2M, CYC1, EIF4A2, GAPDH, SDHA, RPL13A, TOP1, UBC, YWHAZ, and 18S) using geNorm software. The most stable HK genes were found to be GAPDH, UBC and ACTB. Both GAPDH and UBC demonstrated relative stability regardless of valve morphology, aortic diameter, gender and age. The expression of B2M and SDHA were found to be the least stable HK genes. We propose the use of GAPDH, UBC and ACTB as reference genes for gene expression studies of BAV aortopathy using ascending aortic tissue.

SUBMITTER: Harrison OJ

PROVIDER: S-EPMC5058495 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Endogenous Reference Genes for Gene Expression Studies on Bicuspid Aortic Valve Associated Aortopathy in Humans.

Harrison Oliver J OJ Moorjani Narain N Torrens Christopher C Ohri Sunil K SK Cagampang Felino R FR

PloS one 20161011 10

Bicuspid aortic valve (BAV) disease is the most common congenital cardiac abnormality and predisposes patients to life-threatening aortic complications including aortic aneurysm. Quantitative real-time reverse transcription PCR (qRT-PCR) is one of the most commonly used methods to investigate underlying molecular mechanisms involved in aortopathy. The accuracy of the gene expression data is dependent on normalization by appropriate housekeeping (HK) genes, whose expression should remain constant ...[more]

PMID: 27727313

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Project description:ImportanceThe prevalence and characteristics of bicuspid aortic valve (BAV) are mainly reported from selected cohorts. BAV is associated with aortopathy, but it is unclear if it represents a fetal developmental defect or is secondary to abnormal valve dynamics.ObjectiveTo determine the prevalence of BAV and BAV subtypes and to describe the associated aortopathy in a large, population-based cohort of newborns.Design, setting, and participantsThe Copenhagen Baby Heart Study was a cross-sectional, population-based study open to all newborns born in Copenhagen between April 1, 2016, and October 31, 2018. Newborns with BAV were matched 1:2 to newborns with a tricuspid aortic valve (non-BAV group) on sex, singleton/twin pregnancy, gestational age, weight, and age at time of examination.ExposuresTransthoracic echocardiography within 60 days after birth.Main outcomes and measuresPrimary outcome was BAV prevalence and types, ie, number of raphes and spatial orientation of raphes or cusps (no raphes), according to the classification system of Sievers and Schmidtke (classified as type 0, 1, or 2, with numbers indicating the number of raphes). Secondary outcome was valve function and BAV-associated aortopathy, defined as aortic diameter z score of 3 or greater or coarctation.ResultsIn total, 25 556 newborns (51.7% male; mean age, 12 [SD, 8] days) underwent echocardiography. BAV was diagnosed in 196 newborns (prevalence, 0.77% [95% CI, 0.67%-0.88%]), with male-female ratio 2.1:1. BAV was classified as type 0 in 17 newborns (8.7% [95% CI, 5.5%-13.5%]), type 1 in 178 (90.8% [95% CI, 86.0%-94.1%]) (147 [75.0% {95% CI, 68.5%-80.5%}] right-left coronary raphe, 27 [13.8% {95% CI, 9.6%-19.3%}] right coronary-noncoronary raphe, 4 [2.0% {95% CI, 0.8%-5.1%}] left coronary-noncoronary raphe), and type 2 in 1 (0.5% [95% CI, 0.1%-2.8%]). Aortic regurgitation was more prevalent in newborns with BAV (n = 29 [14.7%]) than in those without BAV (1.3%) (absolute % difference, 13.4% [95% CI, 7.8%-18.9%]; P < .001). Newborns with BAV had higher flow velocities across the valve (0.67 [95% CI, 0.65-0.69] m/s vs 0.61 [95% CI, 0.60-0.62] m/s; mean difference, 0.06 m/s [95% CI, 0-0.1]) and larger aortic root and tubular ascending aortic diameters than those without BAV (10.7 [95% CI, 10.7-10.9] mm vs 10.3 [95% CI, 10.2-10.4] mm; mean difference, 0.43 mm [95% CI, 0.2-0.6 mm] and 9.8 [95% CI, 9.6-10.0] mm vs 9.4 [95% CI, 9.3-9.5] mm; mean difference, 0.46 mm [95% CI, 0.30-0.70], respectively) (P < .001 for all). Aortopathy was seen in 65 newborns (33.2%) with BAV (62 with aortic z score ≥3; 3 with coarctation).Conclusions and relevanceAmong newborns in Copenhagen, the prevalence of BAV was 0.77%. Aortopathy was common in newborns with BAV, suggesting that it also represents a fetal malformation.

Project description:BackgroundAortic dissection is a life-threatening complication of bicuspid aortic valve (BAV)-associated aortopathy. In these populations, whilst prophylactic replacement of proximal thoracic aortic aneurysms (TAAs) is generally recommended at threshold diameters ≥5.5 cm, dissection may occur in smaller aortas. An alternative size-based parameter, the cross-sectional aortic area/patient height ratio (indexed aortic area, IAA), correlates with increased dissection risk at abnormal values > 10 cm2/m. We sought to assess the utility of the IAA in identifying at-risk BAV-associated TAAs with abnormal IAA, albeit with sub-threshold aortic diameter.Materials and methodsWe retrospectively identified 69 patients with BAV-associated TAAs who underwent surgical repair between 2010 and 2016. Aortic diameter was measured on pre-operative imaging, and IAA calculated, at the mid-sinus of Valsalva, sino-tubular junction and mid-ascending aorta for each patient. We determined proportions of aneurysms with IAA >10 cm2/m, median IAAs corresponding to aortic diameters <4.0 cm, 4.0-4.5 cm, 4.5-5.0 cm, 5.0-5.5 cm and >5.5 cm, and median aortic diameters corresponding to an abnormal IAA.Results50.9%, 12.5% and 64.6% of aneurysms at the sinus of Valsalva, sino-tubular junction and mid-ascending aorta, respectively, had an abnormal IAA. 51.9% and 88.9% of patients with aortic diameter 4.5-5.0 cm and 5.0-5.5 cm, respectively, had an abnormal IAA. In aneurysms with abnormal IAA involving the sinus of Valsalva, sino-tubular junction, and mid-ascending aorta, median aortic diameters were 4.98 cm, 5.04 cm and 5.11 cm, respectively. Overall, 57/72 (79.2%) at-risk aneurysms with IAA >10 cm2/m had diameters smaller than the 5.5 cm guideline cut-off for surgical intervention.ConclusionSignificant proportions of BAV-associated TAAs are at increased risk of aortic dissection attending an IAA >10 cm2/m, whilst not fulfilling the size criteria indicating aortic surgery in contemporary guidelines. Further analysis of IAA in larger BAV cohorts is necessary to clarify its role in patient selection and optimal timing for prophylactic aortic replacement.

Project description:OBJECTIVES:The objectives of this study were to investigate an association between the magnitude of flow-mediated aortic wall shear stress (WSS) and medial wall histopathology in patients with bicuspid aortic valve (BAV) with aortopathy. METHODS:Patients with BAV (n = 27; 52 ± 15 years; 3 women; proximal thoracic aorta diameter = 4.4 ± 0.7 and 4.6 ± 0.5 cm) who underwent prophylactic aortic resection received preoperative 3-dimensional time-resolved phase-contrast magnetic resonance imaging with 3-dimensional velocity encoding to quantify WSS relative to a population of healthy age- and sex-matched tricuspid aortic valve control participants (n = 20). Quantitative histopathology was conducted on BAV aorta tissue samples resected at surgery (n = 93), and correlation was performed between elastic fiber thickness and in vivo aortic WSS as continuous variables. Validation of elastic fiber thickness was achieved by correlation relative to tissue stiffness determined using biaxial biomechanical testing (n = 22 samples). RESULTS:Elastic fibers were thinner and WSS was higher along the greater curvature compared with other circumferential regions (vs anterior wall: P = .003 and P = .0001, respectively; lesser curvature: both P = .001). Increased regional WSS was associated with decreased elastic fiber thickness (r = -0.25; P = .02). Patient stratification with subanalysis showed an increase in the correlation between WSS and histopathology with aortic valve stenosis (r = -0.36; P = .002) and smaller aortic diameters (<4.5 cm: r = -0.39; P = .03). Elastic fiber thinning was associated with circumferential stiffness (r = -0.41; P = .06). CONCLUSIONS:For patients with BAV, increased aortic valve-mediated WSS is significantly associated with elastic fiber thinning, particularly with aortic valve stenosis and in earlier stages of aortopathy. Elastic fiber thinning correlates with impaired tissue biomechanics. These novel findings further implicate valve-mediated hemodynamics in the progression of BAV aortopathy.

Dataset Information

Endogenous Reference Genes for Gene Expression Studies on Bicuspid Aortic Valve Associated Aortopathy in Humans.

Publications

Endogenous Reference Genes for Gene Expression Studies on Bicuspid Aortic Valve Associated Aortopathy in Humans.

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