391
Variants in the NOTCH1 Gene in Patients with Aortic Coarctation Olga Freylikhman, PhD,* Tatyana Tatarinova, MD,* Natalia Smolina, MSc,*,† Sergey Zhuk, MSc,* Alexandra Klyushina, PhD* Artem Kiselev, MSc* Olga Moiseeva, MD, DHSci,* Gunnar Sjoberg, MD, PhD,† Anna Malashicheva, PhD,* and Anna Kostareva, MD, PhD*,† *Almazov Federal Heart, Blood and Endocrinology Center, St. Petersburg, Russia; †Department of Woman and Child Health, Center for Molecular Medicine, Karolinska Institute, Stockholm, Sweden ABSTRACT
Background and Objective. Malformations of the left ventricular outflow tract are one of the most common forms of congenital heart disorders. Recently, it has been shown that mutations in the NOTCH1 gene can lead to bicuspid aortic valve, aortic aneurysm, and hypoplastic left heart syndrome. The aim of our study was to estimate the frequency of NOTCH1 gene mutations/substitutions in patients with aortic coarctation, isolated or combined with bicuspid aortic valve. Design and Patients. The study included 51 children with coarctation. Detailed family history was obtained for every study subject, and echocardiographic data were obtained for the relatives when available. We applied a strategy of targeted mutation screening for 10 out of 34 exons of the NOTCH1 gene by direct sequencing. Control DNA was obtained from 200 healthy donors. Results. In more than half of the cases, coarctation was combined with bicuspid aortic valve, and in approximately half of the cases, it was combined with hypoplasia of the aortic arch or descending aorta. Familial history of congenital heart disease was observed in 34.3% of the cases. In total, 29 variants of the NOTCH1 gene were identified in the patient group and in the control subjects. Four of those variants led to amino acid exchange, of which only one, R1279H, was identified in both the patient group and in the controls. This variant was significantly overrepresented in the patients with aortic coarctation compared with those in the control group (P < .05). We conclude that the R1279H substitution in the NOTCH1 gene is significantly overrepresented in patients with aortic coarctation and, therefore, may represent a disease-susceptibility allele. Key Words. NOTCH1; Aortic Coarctation; Bicuspid Aortic Valve; Genetic Variant.
Introduction
M
alformations of the left ventricular outflow tract (LVOT) arise all over the world but show racial and geographic differences in prevalence, underlining the role of both genetic and environmental components in their nature. They contribute significantly to cardiovascular morbidity and mortality in the pediatric population. LVOT malformations include severe structural defects such as hypoplastic left heart syndrome (HLHS), aortic coarctation (CoA), and aortic stenosis, as well as common and benign congenital heart defects (CHDs) such as bicuspid aortic valve (BAV). In spite of major advances in prenatal diagnosis, the burden of surgical treatment and rehabilitation of children born with these heart malformations remains substantial. This is further exacerbated by the increased need for manage© 2014 Wiley Periodicals, Inc.
ment of adult CHD patients, the number of whom is progressively increasing due to the advances in quality and availability of surgical cardiac care. Thus, elucidating the basic mechanisms leading to LVOT malformations represents an important research task. The genetics of CHDs is complex. Apart from well-known chromosomal derangements and complex syndromes, single-gene mutations have recently been shown to contribute to CHDs.1 The gene list includes mostly genes coding for transcription factors, receptors, and ligands involved in cardiogenesis but also includes genes that code for structural proteins.2–5 By now, mutations in more than 30 genes have been described in connection with isolated CHDs, but application of this knowledge in genetic diagnosis and counseling is not widely accepted (for a review, see Seidman, 2013).6 Congenit Heart Dis. 2014;9:391–396
392 The genetic origin of LVOT malformations is further supported by the high incidence of intrafamilial segregation, often corresponding with autosomal dominant inheritance.7–11 It has been shown that in relatives from probands with LVOT malformations, the relative risk for various congenital heart malformations is significantly increased, underlining the substantial genetic component in the development of LVOT malformations.12,13 However, reduced penetrance and phenotypic variability make this group of disorders difficult to study with a classical Mendelian approach. Recently, it has been shown that familial and sporadic cases of bicuspid aortic valve can arise from mutations in the NOTCH1 gene.14,15 Following the identification of NOTCH1 mutations in patients with BAV, mutations were described in associated cases of aortic aneurysms and congenital LVOT malformations such as HLHS and CoA.16,17 NOTCH1 is one of the key genes in early development, regulating many important processes, such as cell lineage specification, intracellular interaction, regeneration, and apoptosis.18–21 It has an important role in cardiac development and vasculogenesis.22,23 The role of NOTCH1 signaling in development of the mature cardiac system is further supported by the fact that mutations in some other components of the NOTCH1 pathway are associated with CHDs and LVOT malformations, including CoA.24,25 Accordingly, the aim of the present study was to assess the frequency of NOTCH1 mutations in patients with CoA. Materials and Methods
Study Cohort The study protocol was approved by the local ethical committee, and written informed consent was obtained from the parents of all subjects. The study cohort included 51 children with CoA who underwent surgical treatment at the Almazov Heart, Blood and Endocrinology Center between 2008 and 2011 or in another Russian cardiosurgical center at an earlier date. The absence of aneuploidy was confirmed by standard karyotyping; structural chromosomal defects such as DiGeorge syndrome were excluded based on phenotypical examination and lack of dysmorphic features. Patients with syndromic heart defects were not enrolled in the study. All patients underwent standard echocardiography and aortography or intraoperative visualization of the coarctation zone. In some patients direct manometry was performed as a part of diagnostic or curative aortography. Congenit Heart Dis. 2014;9:391–396
Freylikhman et al. Detailed family history was obtained for every study subject, along with parents’ echocardiographic data when available. Special attention was paid to the parents’ exposure to unfavorable environmental factors, such as professional chemical exposure. DNA samples were collected from all patients with CoA and their parents where possible. Control DNA was obtained from population-matched healthy individuals from the NICA Metabolic Syndrome Study cohort who were confirmed by echocardiography to have no structural cardiac abnormalities.
DNA Amplification and Mutation Screening We applied a strategy of targeted mutation screening for 10 out of 34 exons of the NOTCH1 gene. Genomic DNA was extracted from peripheral blood using a FlexiGene DNA purification Kit (Qiagen, GmbH, Hilden, Germany). Amplification of exons 10, 11, 12, 13, 20, 23, 24, 29, 30, and 34 was performed using primers (available upon request). The choice of these specific exons was based on previously published reports on the implication of NOTCH1 gene mutations in cardiac malformations, including BAV, aortic aneurysm, and LVOT malformations. Mutation screening in patient and control groups was performed by direct sequencing of amplified fragments with an ABI capillary sequencer (Applied Biosystems, Foster City, CA, USA) using BigDye Terminator v3.1 mix (Applied Biosystems). Obtained sequences were analyzed and aligned using BioEdit and Geneious software; new and rare variants were checked against the 1000 Genomes and EVS databases. Mutation Nomenclature Nucleotide numbering and mutation nomenclature were based on a reference NOTCH1 cDNA sequence (GeneBank Accession Number NG_007458.1, from NCBI). Statistics The difference in the distribution of genotypes between the patient group and controls was statistically analyzed by means of Fisher’s exact test to obtain a P value. A P value of less than .05 was considered significant. Odds ratios and 95% confidence intervals were calculated to express the strength of the association between a genetic variant and the disease phenotype. Correction for multiple testing was performed by false discovery rate analysis.
393
NOTCH1 Variants in Aortic Coarctation Results
Patient Characteristics We included in the study a cohort of 51 patients with CoA not associated with structural chromosomal defects or aneuploidy. Endovascular balloon aortoplasty was performed in 25 children (49%); the remaining 26 (51%) underwent surgical correction with aortic resection. In 11 patients (22%), the intervention was repeated due to re-coarctation. The mean pressure gradient in the ascending aorta was 45.7 ± 12 mm Hg. Clinical characteristics of the patient group are summarized in Table 1. CoA was twice as frequent in males compared with females. In more than half of the cases, CoA was combined with BAV, and in approximately half of the cases it was combined with hypoplasia of the aortic arch or descending aorta, including 4 cases (7.8%) with hypoplasia of both the aortic arch and the descending aorta. In the group of patients with BAV, 2 patients had complete interruption of the aortic arch and 2 had subaortic stenosis. Mitral valve pathology was observed in 9 cases, being significantly more common in the BAV subgroup than in patients without BAV. Combinations of CoA with other
Table 1.
forms of CHD were observed in 27% of the cases, with no significant difference between BAV and BAV-free subgroups. “Familial history of CHD” was defined as a documented or anamnestically reported CHD in a first- or second-degree relative and was observed in 34.3% of the cases, with no statistical difference between BAV and BAV-free subgroups (Supporting Information Table S1). There was no difference between the subgroups in the history of CHDs on either the maternal or the paternal side (Supporting Information Table S1), either for males or for females (data not shown). Echocardiographic data were available for 78.4% of the patients’ mothers but only 37.2% of their fathers. Types of CHD seen in parents included BAV, atrial septal defect, and pulmonary stenosis. Notably, persisting or corrected CoA was not observed in any of the parents. The most common CHD in parents was BAV, and it was also the predominant type of CHD among fathers, whereas in mothers the most common CHD was atrial septal defect. There was no difference in the frequency of CHD, either in fathers or in mothers, between BAV and BAVfree subgroups.
Clinical Characteristics of the Patients with Aortic Coarctation
Gender, n Male Female Age at clinical investigation (years), mean ± SD Age at surgical correction (years), mean ± SD Localization of aortic coarctation, n (%) Typical Atypical Hypoplasia, n (%) Aortic arch Descending aorta Both aortic arch and descending aorta Left subclavian artery Interrupted aortic arch Re-coarctation Repeated re-coarctation Subaortic stenosis Incomplete Shone complex Mitral valve abnormalities Mitral stenosis Mitral insufficiency Complete Shone complex Other congenital heart defects Patent ductus arteriosus Ventricular septal defect Atrial septal defect Transposition of the great arteries
Total (n = 51)
Without Bicuspid Aortic Valve (n = 170)
With Bicuspid Aortic Valve (n = 34)
34 17 11.2 ± 1.2 5.5 ± 4.9
8 9 9.1 ± 1.6 6.4 ± 5.5
26** 8 12.9 ± 1.8 4.2 ± 3.8
50 (98.0) 1 (2.0)
16 (94.1) 1 (5.9)
34 (100) 0 (0.0)
13 (25.5) 8 (15.7) 4 (7.8) 6 (11.8) 2 (3.9) 11 (21.7) 1 (2.0) 2 (3.9) 2 (3.9) 8 (15.7) 2 (3.9) 7 (13.7) 1 (2.0) 13 (25.5) 8 (15.7) 6 (11.8) 2 (3.9) 1 (2.0)
5 (29.4) 3 (17.6) 2 (11.8) 0 (0.0) 0 (0.0) 5 (29.4) 1 (5.9) 0 (0.0) 0 (0.0) 1 (5.9) 0 (0.0) 2 (11.8) 0 (0.0) 4 (23.5) 3 (17.6) 2 (11.8) 0 (0.0) 0 (0.0)
8 (23.5) 5 (14.7) 2 (5.9) 6 (17.6) 2 (5.9) 6 (17.6) 0 (0.0) 2 (5.9) 2 (5.9) 7 (20.6) 2 (5.9) 5 (14.7) 1 (2.9) 9 (26.5) 5 (14.7) 4 (11.8) 2 (5.9) 1 (2.9)
**P < .05 vs. female.
Congenit Heart Dis. 2014;9:391–396
394 Table 2.
Freylikhman et al. Clinical Characteristics of Patients with and without the R1279H Variant of the NOTCH1 Gene Not Carrying R1279H
Carrying R1279H
Abnormality, n (%)
Total (n = 44)
Male (n = 29)
Female (n = 14)
Total (n = 7)
Male (n = 5)
Female (n = 2)
Bicuspid aortic valve Hypoplasia of aortic arch Hypoplasia of descending aorta Hypoplasia of both aortic arch and descending aorta Hypoplasia of left subclavian artery Interrupted aortic arch Mitral valve abnormalities Other congenital heart defects
29 (66) 10 (23) 6 (13.6) 2 (4.5)
21 (72) 6 (20.7) 4 (13.8) 1 (3.4)
8 (57) 4 (28.6) 2 (14.3) 1 (7.1)
5 (71) 3 (42.9) 2 (28.6) 2 (28.6)**
5 (100.0) 2 (40.0) 1 (20.0) 1 (20.0)
0 (0.0) 1 (50.0) 1 (50.0) 1 (50.0)
5 (11.4) 2 (4.5) 7 (16) 11 (25)
4 (13.8) 1 (3.4) 2 (6.9) 6 (20.7)
1 (7.1) 1 (7.1) 5 (35.7) 5 (35.7)
1 (14.3) 0 (0.0) 1 (14.3) 2 (28.6)
1 (20.0) 0 (0.0) 1 (20.0) 1 (20.0)
0 (0.0) 0 (0.0) 0 (0.0) 1 (50.0)
**P < .05.
Sequencing of the NOTCH1 Gene in Patients with CoA Ten out of 34 exons of the NOTCH1 gene and adjacent intronic fragments were sequenced in the subjects with CoA and in the control group. We applied the strategy of targeting mutation screening to the exons previously shown to be associated with structural cardiovascular malformations (congenital heart defects and aortic aneurysms). Most of the sequenced exons correspond to multiple extracellular epidermal growth factor (EGF)-like repeats of the protein and code for missense variants previously associated with LVOT malformations. Exons 29, 30, and 34, corresponding to the intracellular domain of the protein, were chosen due to their previously reported association with CoA and BAV.15,17 In total, 29 variants were identified in the patient group and in the control subjects; of those, 13 were in the coding regions (Supporting Information Table S2). Nine out of 29 variants had not previously been reported, either in databases or in the literature, including 5 located within exons. Seven variants, including 6 exonic ones, were found only in the control group, 3 of which had not been reported previously. The P2377L variant was found only once, in the control group. Out of 29 identified variants, 3 led to amino acid exchange, of which only one, R1279H, was identified in both the patient group and in the controls. However, this variant was significantly overrepresented in the patients with CoA compared with the control group without structural heart defects (7 out of 51 and 4 out of 200, respectively; P < .05). This association remained after correction for multiple testing; therefore, this variant may represent a diseasesusceptibility allele. The clinical data of the patients with CoA are presented in Table 2, according to whether or not they carried the R1279H substitution. Patients Congenit Heart Dis. 2014;9:391–396
with CoA who carried the R1279H variant were significantly more likely to have hypoplasia of both the aortic arch and descending aorta compared with noncarriers. However, the low number of cases in the study means further investigation is needed to verify this association. There was no significant difference in any other clinical characteristic between these two groups. The other two variants coding for amino acid substitutions were identified only in the control group. The F1259I variant was observed in 8 control subjects and is therefore considered a polymorphism, and the P2377L variant is a newly reported gene variant of unknown significance. Among the intronic variants, three were significantly overrepresented in the patient group compared with the controls. The genetic variant in intron 12 (g.30667C/T) had a frequency of 10/51 in cases compared with 2/200 in controls in our study, which in combination with data reported by Mohamed et al.15 gives the summative frequency of 11/99 vs. 2/200 (Fisher’s exact test, P < .005). The other two variants, in introns 22 (g.38735C/ T) and 24 (g.39335G/A), have not previously been reported in patients with LVOT malformations and were also significantly overrepresented in the patient group (13/51 vs. 8/200, P < .05, and 10/51 vs. 5/200, P < .05). Discussion
In this study we searched for NOTCH1 gene mutations in patients with CoA. It has been shown previously that NOTCH1 mutations are associated with various cardiac abnormalities, such as familial and sporadic cases of BAV, aortic aneurysms associated with BAV, and more severe structural malformations of the LVOT, such as HLHS. McBride et al.17 previously reported NOTCH1 mutations in two patients with CoA in combination with BAV,
395
NOTCH1 Variants in Aortic Coarctation with a frequency of approximately 22%, but not in patients with CoA alone. We aimed the mutation search at patients with CoA with or without BAV or any other CHD. We compared the frequency of the identified gene variants between the patient group and the control group with no structural heart defects. This is the first study to report data obtained from the direct sequencing of 10 exons of the NOTCH1 gene in 200 control subjects. We identified several new and previously reported gene variants, both in the patients with CoA and in the control subjects. In line with data reported by other authors, our study confirms the high variability of the NOTCH1 gene, finding several rare genetic variants at a frequency of below 1%. For example, the P2377L substitution was found only once, in a control subject. This substitution is very well conserved among 6 species and lies in a functional PEST domain. Taking into account the data reported by McBride et al.,17 the total frequency of this substitution is 1/416, which makes it unlikely to be a rare polymorphism. However, whether this mutation causes disease is not clear, as it was found in a healthy adult subject with no structural heart defect. All the data presented here underline the particular importance of proving the pathogenic status of an identified gene variant/mutation by investigating a sufficient number of population-matched control subjects accompanied by functional studies. The most important finding of our study is the association of the R1279H gene variant with the development of CoA. Earlier, this gene variant was described by McBride et al.17 both in a group of patients with LVOT malformations (3/91) and in a control group (4/207). The frequency of R1279H substitution in the control group without structural heart defects in our study is similar to that reported by McBride et al.17 (4/200 and 4/207). This substitution lies in the functionally important EGF-like domain and is well conserved among species. Functional studies performed on this substitution show a clear tendency toward reduced activation of ligand-induced NOTCH1 activity in NIH3T3 cells, though not a statistically significant one as is the case with known disease-causing mutations.17 The NOTCH1 signaling pathway plays an important role in regulating the endothelial-to-mesenchymal transition, which is an early and critical event in LVOT formation and valvulogenesis.26–28 Thus, a slightly impaired functional variant of the protein can alter the outcome of early cardiogenic events underlying LVOT formation and, later, the effectiveness of aortic func-
tion in adult individuals. Until now, no regulatory sites or sequences encoding or binding miRNA have been reported covering this region; however, further work is needed to uncover the functional consequences of this DNA variant. Taking all the data presented together, we hypothesize that the R1279H substitution may represent a diseaseassociated allele that may promote the development of CoA. Acknowledgement This work was supported by the Russian Federal “Scientific and Educational Resources in Russian Innovation” program, grant agreements N1062, N8120, N11.512.11 .029, and the Swedish Heart–Lung Foundation.
Author Contributions Olga Freylikhman: Data analysis and interpretation. Tatyana Tatarinova: Data analysis and interpretation, data collection. Natalia Smolina: Data analysis and interpretation. Sergey Zhuk: Data analysis and interpretation. Alexandra Klyushina: Data analysis and interpretation, data collection. Artem Kiselev: Data analysis, working with databases. Olga Moiseeva: Concept/design, data collection, critical revision of article, approval of article. Gunnar Sjoberg: Concept/design, drafting of article, critical revision of article, approval of article. Anna Malashicheva: Concept/design, critical revision of article, approval of article. Anna Kostareva: Concept/design, drafting of article, approval of article.
Corresponding Author: Anna Kostareva, MD, PhD, Department Molecular Biology and Genetics Almazov Federal Heart, Blood and Endocrinology Centre, Akkuratova 2, St. Petersburg, 197341, Russian Federation. Tel: (+7) 812-7023777; Fax: (+7) 812-7023701; E-mail: [email protected] Conflict of interest: None. Accepted in final form: November 24, 2013.
References
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396 2 Ching YH, Ghosh TK, Cross SJ, et al. Mutation in myosin heavy chain 6 causes atrial septal defect. Nat Genet. 2005;37:423–428. 3 Garg V, Kathiriya IS, Barnes R, et al. GATA4 mutations cause human congenital heart defects and reveal an interaction with TBX5. Nature. 2003; 424:443–447. 4 Reamon-Buettner SM, Borlak J. TBX5 mutations in non-Holt–Oram syndrome (HOS) malformed hearts. Hum Mutat. 2004;24:104. 5 Wessels MW, Willems PJ. Mutations in sarcomeric protein genes not only lead to cardiomyopathy but also to congenital cardiovascular malformations. Clin Genet. 2008;74:16–19. 6 Fahed AC, Gelb BD, Seidman JG, Seidman CE. Genetics of congenital heart disease: the glass half empty. Circ Res. 2013;112:707–720. 7 Clementi M, Notari L, Borghi A, Tenconi R. Familial congenital bicuspid aortic valve: a disorder of uncertain inheritance. Am J Med Genet. 1996;62:336–338. 8 Cripe L, Andelfinger G, Martin LJ, Shooner K, Benson DW. Bicuspid aortic valve is heritable. J Am Coll Cardiol. 2004;44:138–143. 9 Huntington KA, Hunter G, Chan KL. A prospective study to assess the frequency of familial clustering of congenital bicuspid aortic valve. J Am Coll Cardiol. 1997;30:1809–1812. 10 Pradat P, Francannet C, Harris J, Robert E. The epidemiology of cardiovascular defects, part I: a study based on data from three large registries of congenital malformations. Pediatr Cardiol. 2003; 24:195–221. 11 Roberts WC. The congenitally bicuspid aortic valve: a study of 85 autopsy cases. Am J Cardiol. 1970;26:72–83. 12 Lewin MB, McBride KL, Pignatelli R, et al. Echocardiographic evaluation of asymptomatic parental and sibling cardiovascular anomalies associated with congenital left ventricular outflow tract lesions. Pediatrics. 2004;114:691–696. 13 McBride KL, Pignatelli R, Lewin M, et al. Inheritance analysis of congenital left ventricular outflow tract obstruction malformations: segregation, multiplex relative risk, and heritability. Am J Med Genet. 2005;134A:180–186. 14 Garg V, Muth AN, Ransom JF, et al. Mutations in NOTCH1 cause aortic valve disease. Nature. 2005; 437:270–274. 15 Mohamed SA, Aherrahrou Z, Liptau H, et al. Novel missense mutations (p.T596M and p.P1797H) in NOTCH1 in patients with bicuspid aortic valve. Biochem Biophys Res Commun. 2006;345: 1460–1465. 16 McKellar SH, Tester DJ, Yagubyan M, Majumdar R, Ackerman MJ, Sundt TM. Novel NOTCH1 mutations in patients with bicuspid aortic valve
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disease and thoracic aortic aneurysms. J Thorac Cardiovasc Surg. 2007;134:290–296. McBride KL, Riley MF, Zender GA, et al. NOTCH1 mutations in individuals with left ventricular outflow tract malformations reduce ligand-induced signaling. Hum Mol Genet. 2008;17: 2886–2893. Grego-Bessa J, Luna-Zurita L, del Monte G, et al. Notch signaling is essential for ventricular chamber development. Dev Cell. 2007;12:415–429. High FA, Epstein JA. The multifaceted role of Notch in cardiac development and disease. Nat Rev Genet. 2008;9:49–61. High FA, Zhang M, Proweller A, et al. An essential role for Notch in neural crest during cardiovascular development and smooth muscle differentiation. J Clin Invest. 2007;117:353–363. Niessen K, Karsan A. Notch signaling in cardiac development. Circ Res. 2008;102:1169–1181. Iso T, Hamamori Y, Kedes L. Notch signaling in vascular development. Arterioscler Thromb Vasc Biol. 2003;23:543–553. Itoh F, Itoh S, Goumans MJ, et al. Synergy and antagonism between Notch and BMP receptor signaling pathways in endothelial cells. EMBO J. 2004;23:541–551. Eldadah ZA, Hamosh A, Biery NJ, et al. Familial tetralogy of Fallot caused by mutation in the jagged1 gene. Hum Mol Genet. 2001;10:163–169. Tan HL, Glen E, Topf A, et al. Nonsynonymous variants in the SMAD6 gene predispose to congenital cardiovascular malformation. Hum Mutat. 2012;33:720–727. Noseda M, McLean G, Niessen K, et al. Notch activation results in phenotypic and functional changes consistent with endothelial-tomesenchymal transformation. Circ Res. 2004;94: 910–917. Riley MF, McBride KL, Cole S. NOTCH1 missense alleles associated with left ventricular outflow tract defects exhibit impaired receptor processing and defective EMT. Biochim Biophys Acta. 2011; 1812:121–129. Timmerman LA, Grego-Bessa J, Raya A, et al. Notch promotes epithelial–mesenchymal transition during cardiac development and oncogenic transformation. Genes Dev. 2004;18:99–115.
Supporting Information Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: Table S1. Familial history of the patients with aortic coarctation. Table S2. Genetic variants of the human NOTCH1 gene in patients with aortic coarctation and controls.
Variants in the NOTCH1 Gene in Patients with Aortic Coarctation Olga Freylikhman, PhD,* Tatyana Tatarinova, MD,* Natalia Smolina, MSc,*,† Sergey Zhuk, MSc,* Alexandra Klyushina, PhD* Artem Kiselev, MSc* Olga Moiseeva, MD, DHSci,* Gunnar Sjoberg, MD, PhD,† Anna Malashicheva, PhD,* and Anna Kostareva, MD, PhD*,† *Almazov Federal Heart, Blood and Endocrinology Center, St. Petersburg, Russia; †Department of Woman and Child Health, Center for Molecular Medicine, Karolinska Institute, Stockholm, Sweden ABSTRACT
Background and Objective. Malformations of the left ventricular outflow tract are one of the most common forms of congenital heart disorders. Recently, it has been shown that mutations in the NOTCH1 gene can lead to bicuspid aortic valve, aortic aneurysm, and hypoplastic left heart syndrome. The aim of our study was to estimate the frequency of NOTCH1 gene mutations/substitutions in patients with aortic coarctation, isolated or combined with bicuspid aortic valve. Design and Patients. The study included 51 children with coarctation. Detailed family history was obtained for every study subject, and echocardiographic data were obtained for the relatives when available. We applied a strategy of targeted mutation screening for 10 out of 34 exons of the NOTCH1 gene by direct sequencing. Control DNA was obtained from 200 healthy donors. Results. In more than half of the cases, coarctation was combined with bicuspid aortic valve, and in approximately half of the cases, it was combined with hypoplasia of the aortic arch or descending aorta. Familial history of congenital heart disease was observed in 34.3% of the cases. In total, 29 variants of the NOTCH1 gene were identified in the patient group and in the control subjects. Four of those variants led to amino acid exchange, of which only one, R1279H, was identified in both the patient group and in the controls. This variant was significantly overrepresented in the patients with aortic coarctation compared with those in the control group (P < .05). We conclude that the R1279H substitution in the NOTCH1 gene is significantly overrepresented in patients with aortic coarctation and, therefore, may represent a disease-susceptibility allele. Key Words. NOTCH1; Aortic Coarctation; Bicuspid Aortic Valve; Genetic Variant.
Introduction
M
alformations of the left ventricular outflow tract (LVOT) arise all over the world but show racial and geographic differences in prevalence, underlining the role of both genetic and environmental components in their nature. They contribute significantly to cardiovascular morbidity and mortality in the pediatric population. LVOT malformations include severe structural defects such as hypoplastic left heart syndrome (HLHS), aortic coarctation (CoA), and aortic stenosis, as well as common and benign congenital heart defects (CHDs) such as bicuspid aortic valve (BAV). In spite of major advances in prenatal diagnosis, the burden of surgical treatment and rehabilitation of children born with these heart malformations remains substantial. This is further exacerbated by the increased need for manage© 2014 Wiley Periodicals, Inc.
ment of adult CHD patients, the number of whom is progressively increasing due to the advances in quality and availability of surgical cardiac care. Thus, elucidating the basic mechanisms leading to LVOT malformations represents an important research task. The genetics of CHDs is complex. Apart from well-known chromosomal derangements and complex syndromes, single-gene mutations have recently been shown to contribute to CHDs.1 The gene list includes mostly genes coding for transcription factors, receptors, and ligands involved in cardiogenesis but also includes genes that code for structural proteins.2–5 By now, mutations in more than 30 genes have been described in connection with isolated CHDs, but application of this knowledge in genetic diagnosis and counseling is not widely accepted (for a review, see Seidman, 2013).6 Congenit Heart Dis. 2014;9:391–396
392 The genetic origin of LVOT malformations is further supported by the high incidence of intrafamilial segregation, often corresponding with autosomal dominant inheritance.7–11 It has been shown that in relatives from probands with LVOT malformations, the relative risk for various congenital heart malformations is significantly increased, underlining the substantial genetic component in the development of LVOT malformations.12,13 However, reduced penetrance and phenotypic variability make this group of disorders difficult to study with a classical Mendelian approach. Recently, it has been shown that familial and sporadic cases of bicuspid aortic valve can arise from mutations in the NOTCH1 gene.14,15 Following the identification of NOTCH1 mutations in patients with BAV, mutations were described in associated cases of aortic aneurysms and congenital LVOT malformations such as HLHS and CoA.16,17 NOTCH1 is one of the key genes in early development, regulating many important processes, such as cell lineage specification, intracellular interaction, regeneration, and apoptosis.18–21 It has an important role in cardiac development and vasculogenesis.22,23 The role of NOTCH1 signaling in development of the mature cardiac system is further supported by the fact that mutations in some other components of the NOTCH1 pathway are associated with CHDs and LVOT malformations, including CoA.24,25 Accordingly, the aim of the present study was to assess the frequency of NOTCH1 mutations in patients with CoA. Materials and Methods
Study Cohort The study protocol was approved by the local ethical committee, and written informed consent was obtained from the parents of all subjects. The study cohort included 51 children with CoA who underwent surgical treatment at the Almazov Heart, Blood and Endocrinology Center between 2008 and 2011 or in another Russian cardiosurgical center at an earlier date. The absence of aneuploidy was confirmed by standard karyotyping; structural chromosomal defects such as DiGeorge syndrome were excluded based on phenotypical examination and lack of dysmorphic features. Patients with syndromic heart defects were not enrolled in the study. All patients underwent standard echocardiography and aortography or intraoperative visualization of the coarctation zone. In some patients direct manometry was performed as a part of diagnostic or curative aortography. Congenit Heart Dis. 2014;9:391–396
Freylikhman et al. Detailed family history was obtained for every study subject, along with parents’ echocardiographic data when available. Special attention was paid to the parents’ exposure to unfavorable environmental factors, such as professional chemical exposure. DNA samples were collected from all patients with CoA and their parents where possible. Control DNA was obtained from population-matched healthy individuals from the NICA Metabolic Syndrome Study cohort who were confirmed by echocardiography to have no structural cardiac abnormalities.
DNA Amplification and Mutation Screening We applied a strategy of targeted mutation screening for 10 out of 34 exons of the NOTCH1 gene. Genomic DNA was extracted from peripheral blood using a FlexiGene DNA purification Kit (Qiagen, GmbH, Hilden, Germany). Amplification of exons 10, 11, 12, 13, 20, 23, 24, 29, 30, and 34 was performed using primers (available upon request). The choice of these specific exons was based on previously published reports on the implication of NOTCH1 gene mutations in cardiac malformations, including BAV, aortic aneurysm, and LVOT malformations. Mutation screening in patient and control groups was performed by direct sequencing of amplified fragments with an ABI capillary sequencer (Applied Biosystems, Foster City, CA, USA) using BigDye Terminator v3.1 mix (Applied Biosystems). Obtained sequences were analyzed and aligned using BioEdit and Geneious software; new and rare variants were checked against the 1000 Genomes and EVS databases. Mutation Nomenclature Nucleotide numbering and mutation nomenclature were based on a reference NOTCH1 cDNA sequence (GeneBank Accession Number NG_007458.1, from NCBI). Statistics The difference in the distribution of genotypes between the patient group and controls was statistically analyzed by means of Fisher’s exact test to obtain a P value. A P value of less than .05 was considered significant. Odds ratios and 95% confidence intervals were calculated to express the strength of the association between a genetic variant and the disease phenotype. Correction for multiple testing was performed by false discovery rate analysis.
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NOTCH1 Variants in Aortic Coarctation Results
Patient Characteristics We included in the study a cohort of 51 patients with CoA not associated with structural chromosomal defects or aneuploidy. Endovascular balloon aortoplasty was performed in 25 children (49%); the remaining 26 (51%) underwent surgical correction with aortic resection. In 11 patients (22%), the intervention was repeated due to re-coarctation. The mean pressure gradient in the ascending aorta was 45.7 ± 12 mm Hg. Clinical characteristics of the patient group are summarized in Table 1. CoA was twice as frequent in males compared with females. In more than half of the cases, CoA was combined with BAV, and in approximately half of the cases it was combined with hypoplasia of the aortic arch or descending aorta, including 4 cases (7.8%) with hypoplasia of both the aortic arch and the descending aorta. In the group of patients with BAV, 2 patients had complete interruption of the aortic arch and 2 had subaortic stenosis. Mitral valve pathology was observed in 9 cases, being significantly more common in the BAV subgroup than in patients without BAV. Combinations of CoA with other
Table 1.
forms of CHD were observed in 27% of the cases, with no significant difference between BAV and BAV-free subgroups. “Familial history of CHD” was defined as a documented or anamnestically reported CHD in a first- or second-degree relative and was observed in 34.3% of the cases, with no statistical difference between BAV and BAV-free subgroups (Supporting Information Table S1). There was no difference between the subgroups in the history of CHDs on either the maternal or the paternal side (Supporting Information Table S1), either for males or for females (data not shown). Echocardiographic data were available for 78.4% of the patients’ mothers but only 37.2% of their fathers. Types of CHD seen in parents included BAV, atrial septal defect, and pulmonary stenosis. Notably, persisting or corrected CoA was not observed in any of the parents. The most common CHD in parents was BAV, and it was also the predominant type of CHD among fathers, whereas in mothers the most common CHD was atrial septal defect. There was no difference in the frequency of CHD, either in fathers or in mothers, between BAV and BAVfree subgroups.
Clinical Characteristics of the Patients with Aortic Coarctation
Gender, n Male Female Age at clinical investigation (years), mean ± SD Age at surgical correction (years), mean ± SD Localization of aortic coarctation, n (%) Typical Atypical Hypoplasia, n (%) Aortic arch Descending aorta Both aortic arch and descending aorta Left subclavian artery Interrupted aortic arch Re-coarctation Repeated re-coarctation Subaortic stenosis Incomplete Shone complex Mitral valve abnormalities Mitral stenosis Mitral insufficiency Complete Shone complex Other congenital heart defects Patent ductus arteriosus Ventricular septal defect Atrial septal defect Transposition of the great arteries
Total (n = 51)
Without Bicuspid Aortic Valve (n = 170)
With Bicuspid Aortic Valve (n = 34)
34 17 11.2 ± 1.2 5.5 ± 4.9
8 9 9.1 ± 1.6 6.4 ± 5.5
26** 8 12.9 ± 1.8 4.2 ± 3.8
50 (98.0) 1 (2.0)
16 (94.1) 1 (5.9)
34 (100) 0 (0.0)
13 (25.5) 8 (15.7) 4 (7.8) 6 (11.8) 2 (3.9) 11 (21.7) 1 (2.0) 2 (3.9) 2 (3.9) 8 (15.7) 2 (3.9) 7 (13.7) 1 (2.0) 13 (25.5) 8 (15.7) 6 (11.8) 2 (3.9) 1 (2.0)
5 (29.4) 3 (17.6) 2 (11.8) 0 (0.0) 0 (0.0) 5 (29.4) 1 (5.9) 0 (0.0) 0 (0.0) 1 (5.9) 0 (0.0) 2 (11.8) 0 (0.0) 4 (23.5) 3 (17.6) 2 (11.8) 0 (0.0) 0 (0.0)
8 (23.5) 5 (14.7) 2 (5.9) 6 (17.6) 2 (5.9) 6 (17.6) 0 (0.0) 2 (5.9) 2 (5.9) 7 (20.6) 2 (5.9) 5 (14.7) 1 (2.9) 9 (26.5) 5 (14.7) 4 (11.8) 2 (5.9) 1 (2.9)
**P < .05 vs. female.
Congenit Heart Dis. 2014;9:391–396
394 Table 2.
Freylikhman et al. Clinical Characteristics of Patients with and without the R1279H Variant of the NOTCH1 Gene Not Carrying R1279H
Carrying R1279H
Abnormality, n (%)
Total (n = 44)
Male (n = 29)
Female (n = 14)
Total (n = 7)
Male (n = 5)
Female (n = 2)
Bicuspid aortic valve Hypoplasia of aortic arch Hypoplasia of descending aorta Hypoplasia of both aortic arch and descending aorta Hypoplasia of left subclavian artery Interrupted aortic arch Mitral valve abnormalities Other congenital heart defects
29 (66) 10 (23) 6 (13.6) 2 (4.5)
21 (72) 6 (20.7) 4 (13.8) 1 (3.4)
8 (57) 4 (28.6) 2 (14.3) 1 (7.1)
5 (71) 3 (42.9) 2 (28.6) 2 (28.6)**
5 (100.0) 2 (40.0) 1 (20.0) 1 (20.0)
0 (0.0) 1 (50.0) 1 (50.0) 1 (50.0)
5 (11.4) 2 (4.5) 7 (16) 11 (25)
4 (13.8) 1 (3.4) 2 (6.9) 6 (20.7)
1 (7.1) 1 (7.1) 5 (35.7) 5 (35.7)
1 (14.3) 0 (0.0) 1 (14.3) 2 (28.6)
1 (20.0) 0 (0.0) 1 (20.0) 1 (20.0)
0 (0.0) 0 (0.0) 0 (0.0) 1 (50.0)
**P < .05.
Sequencing of the NOTCH1 Gene in Patients with CoA Ten out of 34 exons of the NOTCH1 gene and adjacent intronic fragments were sequenced in the subjects with CoA and in the control group. We applied the strategy of targeting mutation screening to the exons previously shown to be associated with structural cardiovascular malformations (congenital heart defects and aortic aneurysms). Most of the sequenced exons correspond to multiple extracellular epidermal growth factor (EGF)-like repeats of the protein and code for missense variants previously associated with LVOT malformations. Exons 29, 30, and 34, corresponding to the intracellular domain of the protein, were chosen due to their previously reported association with CoA and BAV.15,17 In total, 29 variants were identified in the patient group and in the control subjects; of those, 13 were in the coding regions (Supporting Information Table S2). Nine out of 29 variants had not previously been reported, either in databases or in the literature, including 5 located within exons. Seven variants, including 6 exonic ones, were found only in the control group, 3 of which had not been reported previously. The P2377L variant was found only once, in the control group. Out of 29 identified variants, 3 led to amino acid exchange, of which only one, R1279H, was identified in both the patient group and in the controls. However, this variant was significantly overrepresented in the patients with CoA compared with the control group without structural heart defects (7 out of 51 and 4 out of 200, respectively; P < .05). This association remained after correction for multiple testing; therefore, this variant may represent a diseasesusceptibility allele. The clinical data of the patients with CoA are presented in Table 2, according to whether or not they carried the R1279H substitution. Patients Congenit Heart Dis. 2014;9:391–396
with CoA who carried the R1279H variant were significantly more likely to have hypoplasia of both the aortic arch and descending aorta compared with noncarriers. However, the low number of cases in the study means further investigation is needed to verify this association. There was no significant difference in any other clinical characteristic between these two groups. The other two variants coding for amino acid substitutions were identified only in the control group. The F1259I variant was observed in 8 control subjects and is therefore considered a polymorphism, and the P2377L variant is a newly reported gene variant of unknown significance. Among the intronic variants, three were significantly overrepresented in the patient group compared with the controls. The genetic variant in intron 12 (g.30667C/T) had a frequency of 10/51 in cases compared with 2/200 in controls in our study, which in combination with data reported by Mohamed et al.15 gives the summative frequency of 11/99 vs. 2/200 (Fisher’s exact test, P < .005). The other two variants, in introns 22 (g.38735C/ T) and 24 (g.39335G/A), have not previously been reported in patients with LVOT malformations and were also significantly overrepresented in the patient group (13/51 vs. 8/200, P < .05, and 10/51 vs. 5/200, P < .05). Discussion
In this study we searched for NOTCH1 gene mutations in patients with CoA. It has been shown previously that NOTCH1 mutations are associated with various cardiac abnormalities, such as familial and sporadic cases of BAV, aortic aneurysms associated with BAV, and more severe structural malformations of the LVOT, such as HLHS. McBride et al.17 previously reported NOTCH1 mutations in two patients with CoA in combination with BAV,
395
NOTCH1 Variants in Aortic Coarctation with a frequency of approximately 22%, but not in patients with CoA alone. We aimed the mutation search at patients with CoA with or without BAV or any other CHD. We compared the frequency of the identified gene variants between the patient group and the control group with no structural heart defects. This is the first study to report data obtained from the direct sequencing of 10 exons of the NOTCH1 gene in 200 control subjects. We identified several new and previously reported gene variants, both in the patients with CoA and in the control subjects. In line with data reported by other authors, our study confirms the high variability of the NOTCH1 gene, finding several rare genetic variants at a frequency of below 1%. For example, the P2377L substitution was found only once, in a control subject. This substitution is very well conserved among 6 species and lies in a functional PEST domain. Taking into account the data reported by McBride et al.,17 the total frequency of this substitution is 1/416, which makes it unlikely to be a rare polymorphism. However, whether this mutation causes disease is not clear, as it was found in a healthy adult subject with no structural heart defect. All the data presented here underline the particular importance of proving the pathogenic status of an identified gene variant/mutation by investigating a sufficient number of population-matched control subjects accompanied by functional studies. The most important finding of our study is the association of the R1279H gene variant with the development of CoA. Earlier, this gene variant was described by McBride et al.17 both in a group of patients with LVOT malformations (3/91) and in a control group (4/207). The frequency of R1279H substitution in the control group without structural heart defects in our study is similar to that reported by McBride et al.17 (4/200 and 4/207). This substitution lies in the functionally important EGF-like domain and is well conserved among species. Functional studies performed on this substitution show a clear tendency toward reduced activation of ligand-induced NOTCH1 activity in NIH3T3 cells, though not a statistically significant one as is the case with known disease-causing mutations.17 The NOTCH1 signaling pathway plays an important role in regulating the endothelial-to-mesenchymal transition, which is an early and critical event in LVOT formation and valvulogenesis.26–28 Thus, a slightly impaired functional variant of the protein can alter the outcome of early cardiogenic events underlying LVOT formation and, later, the effectiveness of aortic func-
tion in adult individuals. Until now, no regulatory sites or sequences encoding or binding miRNA have been reported covering this region; however, further work is needed to uncover the functional consequences of this DNA variant. Taking all the data presented together, we hypothesize that the R1279H substitution may represent a diseaseassociated allele that may promote the development of CoA. Acknowledgement This work was supported by the Russian Federal “Scientific and Educational Resources in Russian Innovation” program, grant agreements N1062, N8120, N11.512.11 .029, and the Swedish Heart–Lung Foundation.
Author Contributions Olga Freylikhman: Data analysis and interpretation. Tatyana Tatarinova: Data analysis and interpretation, data collection. Natalia Smolina: Data analysis and interpretation. Sergey Zhuk: Data analysis and interpretation. Alexandra Klyushina: Data analysis and interpretation, data collection. Artem Kiselev: Data analysis, working with databases. Olga Moiseeva: Concept/design, data collection, critical revision of article, approval of article. Gunnar Sjoberg: Concept/design, drafting of article, critical revision of article, approval of article. Anna Malashicheva: Concept/design, critical revision of article, approval of article. Anna Kostareva: Concept/design, drafting of article, approval of article.
Corresponding Author: Anna Kostareva, MD, PhD, Department Molecular Biology and Genetics Almazov Federal Heart, Blood and Endocrinology Centre, Akkuratova 2, St. Petersburg, 197341, Russian Federation. Tel: (+7) 812-7023777; Fax: (+7) 812-7023701; E-mail: [email protected] Conflict of interest: None. Accepted in final form: November 24, 2013.
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Supporting Information Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: Table S1. Familial history of the patients with aortic coarctation. Table S2. Genetic variants of the human NOTCH1 gene in patients with aortic coarctation and controls.
