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Absence of TGFBR1 and TGFBR2 Mutations in Patients With Bicuspid Aortic Valve and Aortic Dilation
Absence of TGFBR1 and TGFBR2 Mutations in Patients With Bicuspid Aortic Valve and Aortic Dilation Cammon B. Arrington, MD, PhDa, C. Todd Sower, BSa, Naomi Chuckwuk, BSa, Jeff Stevens, BSb, Mark F. Leppert, PhDb, Anji T. Yetman, MDa, and Neil E. Bowles, PhDa,* Mutations in the genes encoding transforming growth factor– receptor types I and II (TGFBR1 and TGFBR2, respectively) are commonly identified in patients with LoeysDietz syndrome, as well as some patients with Marfan’s syndrome or familial thoracic aortic aneurysms and dissections. This suggests that there is considerable phenotypic heterogeneity associated with mutations in these genes. Because bicuspid aortic valve (BAV) is a congenital heart defect in patients with Loeys-Dietz syndrome, this study was conducted to investigate whether variants in TGFBR1 or TGFBR2 are responsible for sporadic BAV. Analysis of these genes in 35 patients with BAVs identified only known single-nucleotide polymorphisms or novel synonymous or intronic substitutions. In conclusion, mutations in TGFBR1 and TGFBR2 rarely cause sporadic BAV. © 2008 Elsevier Inc. All rights reserved. (Am J Cardiol 2008;102:629 – 631) Bicuspid aortic valve (BAV) is the most common congenital cardiac defect, occurring in 1% to 2% of the general population.1 The defect is considered to be a heritable disorder, with a familial recurrence rate of 17% to 34%,2 and mutations in NOTCH1 have been identified in some probands.3,4 Mutations of the transforming growth factor– receptor types I and II (TGFBR1 and TGFBR2, respectively) occur in an uncommon connective tissue disorder known as Loeys-Dietz syndrome,5 which is characterized by progressive aortic dilation and the triad of hypertelorism, cleft palate or bifid uvula, and craniosynostosis. BAV is also an associated feature of the syndrome.5 Loscalzo et al6 screened the TGFBR1 and TGFBR2 genes of the probands from 13 affected families with BAVs and/or thoracic aortic aneurysms but found no mutations in either gene. We screened TGFBR1 and TGFBR2 for variants in patients presenting with sporadic BAVs (on the basis of family histories), with or without ascending aortic dilation, in the absence of other clinical features of Loeys-Dietz syndrome. Methods With University of Utah Institutional Review Board approval, children and young adults with BAVs were enrolled in the University of Utah Pediatric Cardiology GenotypePhenotype Core, and blood samples collected for deoxyribonucleic acid (DNA) isolation. Medical records were reviewed for demographic and clinical data. Subjects with Departments of aPediatrics (Division of Cardiology) and bHuman Genetics, University of Utah School of Medicine, Salt Lake City, Utah, USA. Manuscript received March 10, 2008; revised manuscript received and accepted April 4, 2008. This work was supported by funds from the Division of Cardiology, Department of Pediatrics, University of Utah, Salt Lake City, Utah, and the George S. and Dolores Doré Eccles Foundation to the Molecular Medicine and Human Genetics program at the Eccles Institute of Human Genetics, University of Utah. *Corresponding author: Tel: 801-585-7574; fax: 801-581-7404. E-mail address: [email protected] (N.E. Bowles). 0002-9149/08/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.amjcard.2008.04.044
known genetic abnormalities or other significant syndromes (e.g., Turner’s, Loeys-Dietz, Marfan’s) were excluded. Aortic root and ascending aortic dimensions were obtained from echocardiograms performed within 6 months of study enrollment. Parasternal long-axis images were used to make these measurements. DNA was isolated from peripheral blood samples using a Gentra Autopure LS (Qiagen, Valencia, California). Polymerase chain reaction (PCR) primers were designed to amplify the coding exons of TGFBR1 and TGFBR2 using the Exon Primer utility (http://ihg.gsf.de/ihg/ExonPrimer.html), avoiding known single-nucleotide polymorphisms and repeat elements. All patient DNA samples were amplified by PCR in duplicate, along with negative (water) controls, using Platinum Taq DNA polymerase (Invitrogen, Carlsbad, California); primer sequences and amplification conditions are available on request. After amplification, 0.5 l of LC Green I (Idaho Technologies, Salt Lake City, Utah) was added to each 10-l reaction. The mix was heated at 95°C for 5 minutes and then cooled to 4°C. The PCR products were analyzed on a Lightscanner (Idaho Technologies), according to the manufacturer’s instructions. This instrument analyzes PCR products by high-resolution melting analysis.7 This technique is highly sensitive for the detection of point mutations and approaches 100% for PCR products ⬍ 400 base pairs.8 All heterozygotes and most homozygotes can be detected without mixing.9,10 The plates were heated at 0.1°C/s, and fluorescence was collected from 70°C to 98°C. Melting curves were analyzed using Call-IT software (Idaho Technologies). Briefly, after exponential background subtraction, melting curves were normalized between 0% and 100%. Normalized and temperature-overlaid curves were viewed on difference plots to magnify variations in melting curve shape. Difference plots were generated by subtracting each curve from the mean wild-type curve, defined as the most common genotype. Samples giving abnormal curves were treated with 4 l of Exo-SAP-IT (USB, Cleveland, Ohio) at 37°C for 2 hours and 80°C for www.AJConline.org
630
The American Journal of Cardiology (www.AJConline.org)
Table 1 Demographics of the patient cohort
Table 2 Gene variants detected in TGFBR1 and TGFBR2
Variable
Value
Gender Male Female Age at echocardiographic examination (yrs) Other presentations Aortic stenosis Aortic coarctation Ventricular septal defect Subaortic stenosis Atrial septal defect Thoracic aortic aneurysm Interventions Ross procedure z score Aortic valve annulus Aortic sinus Aortic sinotubular junction Ascending aorta
Gene TGFBR1
26 (74%) 9 (26%) 14.3 (1 to 30) TGFBR2 19 (54%) 17 (49%) 4 (11%) 2 (6%) 1 (3%) 0 2 (6%) 0.64 (⫺1.5 to 6.3) 0.58 (⫺2.1 to 5.7) 0.77 (⫺1.9 to 8.1) 2.13 (⫺1.9 to 5.6)
Data are expressed as number (percentage) or as median (range).
0
5
10
15
20
25
30
35
Age (years)
Figure 1. Normalized ascending aortic dimension (z score) as a function of age in patients with BAVs. Dilation of the ascending aorta (z score ⬎ 2) was common and present in ⬎1/2 of the patients in this study. No obvious systematic variation of z score with age was seen.
15 minutes. An aliquot of DNA was analyzed by agarose gel electrophoresis, and then samples were submitted to the University of Utah DNA sequencing core laboratory for analysis. DNA sequences were compared with published sequences using Basic Local Alignment and Search Tool analysis (http://www.ncbi.nlm.nih.gov/blast/ Blast.cgi) and with genomic sequences downloaded from Ensembl (http://www.ensembl.org/Homo_sapiens/index.html). Results Thirty-five patients with BAVs were included in this study and ranged in age from 1 to 30 years (median 14.3, 26% female; Table 1). Fifty-four percent of study participants had some degree of aortic valve dysfunction, and 6% required surgical valve replacement. Many study participants had additional congenital heart defects, including coarctation of the aorta (49%), ventricular septal defects (11%), subaortic membranes (6%), and atrial septal defects (3%). Aortic root (aortic valve annulus, sinuses of Valsalva, and sinotubular junction) and ascending aortic measurements were normalized to body surface area using the pro-
Exon
Variant
1 3 7 8 1 4
rs11466445: ⫺/GGCGGCGGC c.396A⬎G rs334354:G⬎A c.1462⫹70T⬎A rs2306856:C⬎G rs11466512:T⬎A
gram z-score (Boston, Massachusetts). Z scores ⬎2 and ⬍⫺2 represent aortic dimensions that are greater than the 95th percentile and less than the 5th percentile for body surface area, respectively.11,12 Average z scores for the aortic root fell within the normal range and were found to be 0.96 at the aortic valve annulus, 0.91 at the sinuses of Valsalva, and 0.8 at the sinotubular junction. In contrast, many study participants had dilation of the ascending aorta, with an average z score of 2.1 (range ⫺1.9 to 5.6, median 2.1, SD 2.2). Dilation of the ascending aorta was noted across all ages (Figure 1). For exon 3 of TGFBR1 and exon 1 of TGFBR2, abnormal melting profiles were obtained on the Lightscanner for 1 patient sample, while exons 1, 7, and 8 of TGFBR1 and exon 4 of TGFBR1 showed multiple subjects with abnormal but similar profiles (Figure 2). DNA sequence analysis revealed the presence of known single-nucleotide polymorphisms in 4 of these exons (1 subject was homozygous for rs11466512), a substitution in intron 8 of TGFBR1, and a novel synonymous substitution in exon 3 of TGFBR1 (Table 2). Analysis of the synonymous substitution using splice prediction algorithms (http://www.fruitfly.org/seq_tools/splice.html) or exonic splice enhancer prediction software (http://genes.mit.edu/burgelab/ rescue-ese/) did not indicate functional changes. There was no obvious correlation between the presence of single-nucleotide polymorphisms and aortic dilation or complex anatomy in these patients (data not shown). Discussion These data confirm that Lightscanner analysis of PCR products provides a rapid, sensitive, and cost-effective approach to screening patients for genetic variants. In addition, these data support the conclusion of Loscalzo et al6 that mutations in TGFBR1 or TGFBR2 genes are rarely identified in patients with BAVs but extend their study to a larger patient cohort, focusing on patients without family histories and without aortic dissection or rupture or surgical interventions for thoracic aortic aneurysms. Furthermore, the data suggest that BAV with or without ascending aortic dilation is not part of the phenotypic spectrum of Loeys-Dietz syndrome from a genetic standpoint. However, in animal models, transforming growth factor– does exert control over endothelial to mesenchymal transdifferentiation,13 a key process in the development of the aortic valve. Therefore, defects in other molecular pathways involving transforming growth factor–, including Cx45/NFATc1, Snail, and BMP, may be involved in the pathogenesis of BAV.
Congenital Heart Disease/BAV and TGFBR Mutations
631
Figure 2. Detection of DNA variants using the Lightscanner. Difference curves are shown for the TGFBR1 396A⬎G variant (A) in a single patient and the rs334354 single-nucleotide polymorphism (B) in 9 patients (red). 1. National Center for Biotechnology Information. Online Mendelian Inheritance in Man #109730: aortic valve disease. Available at http:// www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?id⫽109730. Accessed February 21, 2008. 2. Roberts WC. The congenitally bicuspid aortic valve. A study of 85 autopsy cases. Am J Cardiol 1970;26:72– 83. 3. Garg V, Muth AN, Ransom JF, Schluterman MK, Barnes R, King IN, Grossfeld PD, Srivastava D. Mutations in NOTCH1 cause aortic valve disease. Nature 2005;437:270 –274. 4. McKellar SH, Tester DJ, Yagubyan M, Majumdar R, Ackerman MJ, Sundt TM III. Novel NOTCH1 mutations in patients with bicuspid aortic valve disease and thoracic aortic aneurysms. J Thorac Cardiovasc Surg 2007;134:290 –296. 5. Loeys BL, Chen J, Neptune ER, Judge DP, Podowski M, Holm T, Meyers J, Leitch CC, Katsanis N, Sharifi N, et al. A syndrome of altered cardiovascular, craniofacial, neurocognitive and skeletal development caused by mutations in TGFBR1 or TGFBR2. Nat Genet 2005;37:275–281. 6. Loscalzo ML, Goh DL, Loeys B, Kent KC, Spevak PJ, Dietz HC. Familial thoracic aortic dilation and bicommissural aortic valve: a prospective analysis of natural history and inheritance. Am J Med Genet A 2007;143:1960 –1967.
7. Wittwer CT, Reed GH, Gundry CN, Vandersteen JG, Pryor RJ. Highresolution genotyping by amplicon melting analysis using LCGreen. Clin Chem 2003;49:853– 860. 8. Reed GH, Wittwer CT. Sensitivity and specificity of single-nucleotide polymorphism scanning by high-resolution melting analysis. Clin Chem 2004;50:1748 –1754. 9. Liew M, Pryor R, Palais R, Meadows C, Erali M, Lyon E, Wittwer C. Genotyping of single-nucleotide polymorphisms by high-resolution melting of small amplicons. Clin Chem 2004;50:1156 –1164. 10. Palais RA, Liew MA, Wittwer CT. Quantitative heteroduplex analysis for single nucleotide polymorphism genotyping. Anal Biochem 2005; 346:167–175. 11. Daubeney PE, Blackstone EH, Weintraub RG, Slavik Z, Scanlon J, Webber SA. Relationship of the dimension of cardiac structures to body size: an echocardiographic study in normal infants and children. Cardiol Young 1999;9:402– 410. 12. Hanseus K, Bjorkhem G, Lundstrom NR. Dimensions of cardiac chambers and great vessels by cross-sectional echocardiography in infants and children. Pediatr Cardiol 1988;9:7–15. 13. Armstrong EJ, Bischoff J. Heart valve development: endothelial cell signaling and differentiation. Circ Res 2004;95:459 – 470.
known genetic abnormalities or other significant syndromes (e.g., Turner’s, Loeys-Dietz, Marfan’s) were excluded. Aortic root and ascending aortic dimensions were obtained from echocardiograms performed within 6 months of study enrollment. Parasternal long-axis images were used to make these measurements. DNA was isolated from peripheral blood samples using a Gentra Autopure LS (Qiagen, Valencia, California). Polymerase chain reaction (PCR) primers were designed to amplify the coding exons of TGFBR1 and TGFBR2 using the Exon Primer utility (http://ihg.gsf.de/ihg/ExonPrimer.html), avoiding known single-nucleotide polymorphisms and repeat elements. All patient DNA samples were amplified by PCR in duplicate, along with negative (water) controls, using Platinum Taq DNA polymerase (Invitrogen, Carlsbad, California); primer sequences and amplification conditions are available on request. After amplification, 0.5 l of LC Green I (Idaho Technologies, Salt Lake City, Utah) was added to each 10-l reaction. The mix was heated at 95°C for 5 minutes and then cooled to 4°C. The PCR products were analyzed on a Lightscanner (Idaho Technologies), according to the manufacturer’s instructions. This instrument analyzes PCR products by high-resolution melting analysis.7 This technique is highly sensitive for the detection of point mutations and approaches 100% for PCR products ⬍ 400 base pairs.8 All heterozygotes and most homozygotes can be detected without mixing.9,10 The plates were heated at 0.1°C/s, and fluorescence was collected from 70°C to 98°C. Melting curves were analyzed using Call-IT software (Idaho Technologies). Briefly, after exponential background subtraction, melting curves were normalized between 0% and 100%. Normalized and temperature-overlaid curves were viewed on difference plots to magnify variations in melting curve shape. Difference plots were generated by subtracting each curve from the mean wild-type curve, defined as the most common genotype. Samples giving abnormal curves were treated with 4 l of Exo-SAP-IT (USB, Cleveland, Ohio) at 37°C for 2 hours and 80°C for www.AJConline.org
630
The American Journal of Cardiology (www.AJConline.org)
Table 1 Demographics of the patient cohort
Table 2 Gene variants detected in TGFBR1 and TGFBR2
Variable
Value
Gender Male Female Age at echocardiographic examination (yrs) Other presentations Aortic stenosis Aortic coarctation Ventricular septal defect Subaortic stenosis Atrial septal defect Thoracic aortic aneurysm Interventions Ross procedure z score Aortic valve annulus Aortic sinus Aortic sinotubular junction Ascending aorta
Gene TGFBR1
26 (74%) 9 (26%) 14.3 (1 to 30) TGFBR2 19 (54%) 17 (49%) 4 (11%) 2 (6%) 1 (3%) 0 2 (6%) 0.64 (⫺1.5 to 6.3) 0.58 (⫺2.1 to 5.7) 0.77 (⫺1.9 to 8.1) 2.13 (⫺1.9 to 5.6)
Data are expressed as number (percentage) or as median (range).
0
5
10
15
20
25
30
35
Age (years)
Figure 1. Normalized ascending aortic dimension (z score) as a function of age in patients with BAVs. Dilation of the ascending aorta (z score ⬎ 2) was common and present in ⬎1/2 of the patients in this study. No obvious systematic variation of z score with age was seen.
15 minutes. An aliquot of DNA was analyzed by agarose gel electrophoresis, and then samples were submitted to the University of Utah DNA sequencing core laboratory for analysis. DNA sequences were compared with published sequences using Basic Local Alignment and Search Tool analysis (http://www.ncbi.nlm.nih.gov/blast/ Blast.cgi) and with genomic sequences downloaded from Ensembl (http://www.ensembl.org/Homo_sapiens/index.html). Results Thirty-five patients with BAVs were included in this study and ranged in age from 1 to 30 years (median 14.3, 26% female; Table 1). Fifty-four percent of study participants had some degree of aortic valve dysfunction, and 6% required surgical valve replacement. Many study participants had additional congenital heart defects, including coarctation of the aorta (49%), ventricular septal defects (11%), subaortic membranes (6%), and atrial septal defects (3%). Aortic root (aortic valve annulus, sinuses of Valsalva, and sinotubular junction) and ascending aortic measurements were normalized to body surface area using the pro-
Exon
Variant
1 3 7 8 1 4
rs11466445: ⫺/GGCGGCGGC c.396A⬎G rs334354:G⬎A c.1462⫹70T⬎A rs2306856:C⬎G rs11466512:T⬎A
gram z-score (Boston, Massachusetts). Z scores ⬎2 and ⬍⫺2 represent aortic dimensions that are greater than the 95th percentile and less than the 5th percentile for body surface area, respectively.11,12 Average z scores for the aortic root fell within the normal range and were found to be 0.96 at the aortic valve annulus, 0.91 at the sinuses of Valsalva, and 0.8 at the sinotubular junction. In contrast, many study participants had dilation of the ascending aorta, with an average z score of 2.1 (range ⫺1.9 to 5.6, median 2.1, SD 2.2). Dilation of the ascending aorta was noted across all ages (Figure 1). For exon 3 of TGFBR1 and exon 1 of TGFBR2, abnormal melting profiles were obtained on the Lightscanner for 1 patient sample, while exons 1, 7, and 8 of TGFBR1 and exon 4 of TGFBR1 showed multiple subjects with abnormal but similar profiles (Figure 2). DNA sequence analysis revealed the presence of known single-nucleotide polymorphisms in 4 of these exons (1 subject was homozygous for rs11466512), a substitution in intron 8 of TGFBR1, and a novel synonymous substitution in exon 3 of TGFBR1 (Table 2). Analysis of the synonymous substitution using splice prediction algorithms (http://www.fruitfly.org/seq_tools/splice.html) or exonic splice enhancer prediction software (http://genes.mit.edu/burgelab/ rescue-ese/) did not indicate functional changes. There was no obvious correlation between the presence of single-nucleotide polymorphisms and aortic dilation or complex anatomy in these patients (data not shown). Discussion These data confirm that Lightscanner analysis of PCR products provides a rapid, sensitive, and cost-effective approach to screening patients for genetic variants. In addition, these data support the conclusion of Loscalzo et al6 that mutations in TGFBR1 or TGFBR2 genes are rarely identified in patients with BAVs but extend their study to a larger patient cohort, focusing on patients without family histories and without aortic dissection or rupture or surgical interventions for thoracic aortic aneurysms. Furthermore, the data suggest that BAV with or without ascending aortic dilation is not part of the phenotypic spectrum of Loeys-Dietz syndrome from a genetic standpoint. However, in animal models, transforming growth factor– does exert control over endothelial to mesenchymal transdifferentiation,13 a key process in the development of the aortic valve. Therefore, defects in other molecular pathways involving transforming growth factor–, including Cx45/NFATc1, Snail, and BMP, may be involved in the pathogenesis of BAV.
Congenital Heart Disease/BAV and TGFBR Mutations
631
Figure 2. Detection of DNA variants using the Lightscanner. Difference curves are shown for the TGFBR1 396A⬎G variant (A) in a single patient and the rs334354 single-nucleotide polymorphism (B) in 9 patients (red). 1. National Center for Biotechnology Information. Online Mendelian Inheritance in Man #109730: aortic valve disease. Available at http:// www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?id⫽109730. Accessed February 21, 2008. 2. Roberts WC. The congenitally bicuspid aortic valve. A study of 85 autopsy cases. Am J Cardiol 1970;26:72– 83. 3. Garg V, Muth AN, Ransom JF, Schluterman MK, Barnes R, King IN, Grossfeld PD, Srivastava D. Mutations in NOTCH1 cause aortic valve disease. Nature 2005;437:270 –274. 4. McKellar SH, Tester DJ, Yagubyan M, Majumdar R, Ackerman MJ, Sundt TM III. Novel NOTCH1 mutations in patients with bicuspid aortic valve disease and thoracic aortic aneurysms. J Thorac Cardiovasc Surg 2007;134:290 –296. 5. Loeys BL, Chen J, Neptune ER, Judge DP, Podowski M, Holm T, Meyers J, Leitch CC, Katsanis N, Sharifi N, et al. A syndrome of altered cardiovascular, craniofacial, neurocognitive and skeletal development caused by mutations in TGFBR1 or TGFBR2. Nat Genet 2005;37:275–281. 6. Loscalzo ML, Goh DL, Loeys B, Kent KC, Spevak PJ, Dietz HC. Familial thoracic aortic dilation and bicommissural aortic valve: a prospective analysis of natural history and inheritance. Am J Med Genet A 2007;143:1960 –1967.
7. Wittwer CT, Reed GH, Gundry CN, Vandersteen JG, Pryor RJ. Highresolution genotyping by amplicon melting analysis using LCGreen. Clin Chem 2003;49:853– 860. 8. Reed GH, Wittwer CT. Sensitivity and specificity of single-nucleotide polymorphism scanning by high-resolution melting analysis. Clin Chem 2004;50:1748 –1754. 9. Liew M, Pryor R, Palais R, Meadows C, Erali M, Lyon E, Wittwer C. Genotyping of single-nucleotide polymorphisms by high-resolution melting of small amplicons. Clin Chem 2004;50:1156 –1164. 10. Palais RA, Liew MA, Wittwer CT. Quantitative heteroduplex analysis for single nucleotide polymorphism genotyping. Anal Biochem 2005; 346:167–175. 11. Daubeney PE, Blackstone EH, Weintraub RG, Slavik Z, Scanlon J, Webber SA. Relationship of the dimension of cardiac structures to body size: an echocardiographic study in normal infants and children. Cardiol Young 1999;9:402– 410. 12. Hanseus K, Bjorkhem G, Lundstrom NR. Dimensions of cardiac chambers and great vessels by cross-sectional echocardiography in infants and children. Pediatr Cardiol 1988;9:7–15. 13. Armstrong EJ, Bischoff J. Heart valve development: endothelial cell signaling and differentiation. Circ Res 2004;95:459 – 470.