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Journal of Anatomy logoLink to Journal of Anatomy
. 2011 Oct 30;220(1):92–101. doi: 10.1111/j.1469-7580.2011.01440.x

Genetically alike Syrian hamsters display both bifoliate and trifoliate aortic valves

Valentín Sans-Coma 1, M Carmen Fernández 1, Borja Fernández 1, Ana C Durán 1, Robert H Anderson 2, Josep M Arqué 3
PMCID: PMC3248667  PMID: 22034929

Abstract

The bifoliate, or bicuspid, aortic valve (BAV) is the most frequent congenital cardiac anomaly in man. It is a heritable defect, but its mode of inheritance remains unclear. Previous studies in Syrian hamsters showed that BAVs with fusion of the right and left coronary leaflets are expressions of a trait, the variation of which takes the form of a phenotypic continuum. It ranges from a trifoliate valve with no fusion of the coronary leaflets to a bifoliate root devoid of any raphe. The intermediate stages are represented by trifoliate valves with fusion of the coronary aortic leaflets, and bifoliate valves with raphes. The aim of this study was to elucidate whether the distinct morphological variants rely on a common genotype, or on different genotypes. We examined the aortic valves from 1 849 Syrian hamsters belonging to a family subjected to systematic inbreeding by full-sib mating. The incidence of the different trifoliate aortic valve (TAV) and bifoliate aortic valve (BAV) morphological variants widely varied in the successive inbred generations. TAVs with extensive fusion of the leaflets, and BAVs, accounted for five-sixths of the patterns found in Syrian hamsters considered to be genetically alike or virtually isogenic, with the probability of homozygosity being 0.999 or higher. The remaining one-sixth hamsters had aortic valves with a tricuspid design, but in most cases the right and left coronary leaflets were slightly fused. Results of crosses between genetically alike hamsters, with the probability of homozygosity being 0.989 or higher, revealed no significant association between the valvar phenotypes in the parents and their offspring. Our findings are consistent with the notion that the BAVs of the Syrian hamster are expressions of a quantitative trait subject to polygenic inheritance. They suggest that the genotype of the virtually isogenic animals produced by systematic inbreeding greatly predisposes to the development of anomalous valves, be they bifoliate, or trifoliate with extensive fusion of the leaflets. We infer that the same underlying genotype may account for the whole range of valvar morphological variants, suggesting that factors other than genetic ones are acting during embryonic life, creating the so-called intangible variation or developmental noise, and playing an important role in the definitive anatomic configuration of the valve. The clinical implication from our study is that congenital aortic valves with a trifoliate design, but with fusion of coronary aortic leaflets, may harbour the same inherent risks as those already recognised for BAVs with fusion of right and left coronary leaflets.

Keywords: aetiology, anatomy, bicuspid aortic valve, genetics, Syrian hamster

Introduction

It has been reported repeatedly that, in man, the aortic valve with two leaflets, known as the bifoliate or bicuspid aortic valve (BAV), is the most common congenital cardiac malformation, carrying with it the risk over a lifetime of multiple clinical complications (Sabet et al. 1999; Fedak et al. 2002; Braverman et al. 2005; De Mozzi et al. 2008; Siu & Silversides, 2010). The presence of two, as opposed to the expected three, leaflets within the aortic root is due most frequently to fusion of the leaflets guarding the right and left coronary aortic sinuses, with fusion of the right and non-coronary leaflets also being relatively common (Angelini et al. 1989; Russo et al. 2008; Calloway et al. 2011; Laforest et al. 2011). Our recent investigation, which compared the formation of BAVs in Syrian hamsters, as opposed to mice with knock-out of the gene for endothelium nitric oxide synthase (Fernández et al. 2009), showed that the morphological subtypes are distinct morphogenetical entities.

The Syrian hamsters we used belonged to a single inbred family having a high incidence of BAVs characterized by fusion of the right and left coronary aortic leaflets. In preceding papers, we showed that such valves are expressions of a trait, the variation of which takes the form of a phenotypic continuum (Sans-Coma et al. 1993b, 1996; Fernández et al. 2000). The continuum ranges from a trifoliate, or tricuspid, aortic valve (TAV) with no fusion of the coronary arterial leaflets, to a BAV devoid of any raphe in the conjoined valvar leaflet. The intermediate stages are represented by TAVs with greater or less extensive fusion of the coronary aortic leaflets, and BAVs with greater or less extensive development of the valvar raphe. Embryological findings revealed that each valvar primordium acquires its morphological condition soon after its initial development from the mesenchymal outflow cushions, and that the entirety of the phenotypic spectrum seen in adult hamsters can be well recognised prior to the completion of valvogenesis (Sans-Coma et al. 1996).

In the clinical setting, evidence points to genetic factors being the primary cause of formation of two as opposed to three leaflets in the aortic root in man (Emanuel et al. 1978; McDonald & Maurer, 1989; Glick & Roberts, 1994; Clementi et al. 1996; Huntington et al. 1997; Cripe et al. 2004; Mohamed et al. 2005, 2006; Garg et al. 2005; Garg, 2006; Martin et al. 2007; Hinton et al. 2009; Calloway et al. 2011; Laforest et al. 2011). Several genomic regions have been identified that may account for the inheritance of such valves (Garg et al. 2005; Mohamed et al. 2005, 2006; Garg, 2006; Martin et al. 2007; Hinton et al. 2009). With this background in mind, we have investigated whether the distinct morphological variants found in our hamsters are dependent on a common genotype, or on different genotypes. As our initial approach to this issue, we analysed data from full-sib matings performed until individuals were produced that could be considered genetically alike, in other words virtually isogenic. We then tested the hypothesis that genetically similar hamsters would display similar aortic valvar morphology. As we now report, our results failed to support this notion. We believe that our findings contribute to providing a better understanding of the relationship between genotype and phenotype in the formation of aortic valves with fused coronary leaflets. Moreover, they may underscore future studies on aortic valvar dysfunction, and complications of the ascending aorta, in man.

Materials and methods

Animals

We used Syrian hamsters belonging to a family subjected to systematic inbreeding in our laboratory by mating siblings. In the family, which originated from an unrelated pair with TAVs, there is a high incidence of BAVs with fused right and left coronary leaflets (Fernández et al. 2009). The characteristics of this unique inbred family have been reported previously (Sans-Coma et al. 1993b, 1996; Fernández et al. 2000). We have now produced 39 inbred generations, permitting assessment of valvar morphology in 1849 animals, aged 1–816 days, of which 919 were male and 930 female. We were able to assess the morphology of the aortic valve only after death of the animals, so we could not select hamsters with known morphology for the purposes of breeding. Our present data, therefore, represent random results, and not the results of crosses between known phenotypes. For comparative purposes we used 92 Syrian hamsters belonging to a closed colony that had been outbred since 1951 (strain code 049; Charles River, Germany). The hamsters were handled in accordance with the international guides for animal welfare. There was no known exposure of the animals to teratogenic agents. Hamsters were killed by overdosing with chloroform or diethyl ether, or by inhalation of carbon dioxide.

Techniques

In 462 of the animals, we assessed the aortic valvar morphology by making internal casts of the heart, great arterial trunks and coronary arteries, injecting vinyl resin through the apex of the ventricles, and subsequently macerating the specimens in a bath of hydrochloric acid (Durán et al. 1992; Sans-Coma et al. 1993a). In a further 1258 hamsters, we dissected the hearts, assessing the gross anatomy of the aortic valve by stereomicroscopy. Some of these specimens were processed by scanning electron microscopy following the protocol described in a preceding paper (Sans-Coma et al. 1996). We determined the morphology of the aortic valves in the remaining 129 hamsters by making serial histological sections, again as described previously (Sans-Coma et al. 1993b, 1996). Until the fifth inbred generation, we assessed the condition of the aortic valve exclusively by means of internal casts, which permitted distinction only between valves having two as opposed to three aortic sinuses. From the sixth to the 11th generations, we added dissection and histological methods to the use of casts, using these two techniques exclusively subsequent to the 12th generation. The techniques used for the numbers of animals examined are shown in Table 1. We assessed the condition of the aortic valves in the outbred Syrian hamsters using stereomicroscopy.

Table 1.

Number of specimens and techniques

Inbred generation Corrosion-cast technique Dissection Histology
1–5 106 0 0
6–11 356 159 53
12–39 0 1099 76
Total 462 1258 129
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Nomenclature

When describing our anatomic findings, we have followed the nomenclature suggested by Angelini et al. (1989), and used subsequently by McKay et al. (1992), Sans-Coma et al. (1993b) and Sutton et al. (1995). So as to prevent any confusion, we provide the following observations. It is now well recognised that the atrioventricular valves are best analysed in terms of a valvar complex made up of the annulus, the leaflets, the tendinous cords and the supporting papillary muscles (Perloff & Roberts, 1972). It is equally advantageous to analyse the arterial valves in terms of a comparable morphological complex. With regard to the arterial valves, or arterial roots (Anderson, 2000), the components are the leaflets, the valvar sinuses, the subvalvar fibrous interleaflet triangles and the supporting ventricular structures, with the distal extent of the valvar complex marked by the circular sinutubular junction (Sutton et al. 1995). Several of these descriptive terms remain potentially contentious. In our opinion, it is preferable to describe the working units of the arterial valvar complex as the leaflets, rather than the ‘cusps’. Such usage is supported by the vernacular meaning of ‘cusp’ as a point or elevation. If used in this fashion, then each of the leaflets has two such cusps, this in itself making the word itself unsuitable for describing the entirety of the individual semilunar leaflets (Frater & Anderson, 2010). In this respect, it is equally clear that the leaflets themselves are semilunar, with the overall valves guarding the ventriculo-arterial junctions best described as being arterial. Following this reasoning, we use the terms trifoliate and bifoliate, rather than tricuspid and bicuspid, to describe the overall arrangement of the aortic valves, although the latter terms are still in common use.

The term ‘genetically alike’ is also in need of clarification. Animal strains that have been inbred for many generations have almost identical genomes. In a full-sib mating system, total isogeny is theoretically reached at the 150th inbred generation (Falconer, 1996; Benavides & Guénet, 2003). It is generally accepted, however, that from the 20th generation onwards, the probability of homozygosity is so high that individuals can be regarded as genetically alike, or virtually isogenic (Benavides & Guénet, 2003). In this study, we have used the term ‘genetically alike’ to designate the inbred hamsters belonging to generations 20–39. Our main purpose in doing so was to avoid potential confusion derived from the use of the term isogenic, which, in denoting a much stricter genetic condition, is better applied to describe the status of monozygotic twins than siblings produced from different zygotes.

Coefficient of inbreeding

The coefficient of inbreeding (F) of the Syrian hamsters, defined as the probability of two alleles having identical descendents, was calculated according to Falconer (1996) for a full-sib mating system.

Statistical analysis

We used the χ2 test for determining independence of data cross-tabulated in two-way contingency tables. A probability of 0.05 or less was regarded as evidence of a significant difference with regard to the null hypothesis.

Results

As in man (Sutton et al. 1995), the normal aortic valve of the Syrian hamster has three leaflets, each leaflet guarding one of three aortic sinuses, best described as being right, left and non-coronary, with three fibrous subaortic interleaflet triangles interposed between the adjacent sinuses. We found no differences related to gender with regard to the occurrence of VABs, so we pooled the data for males and females.

Variation in phenotype

Overall, we studied 1387 aortic valves from hamsters obtained by inbreeding in generations 6–39 by dissection or histological techniques. In 80 of them, there was a normal trifoliate design (Fig. 1A). An additional 588 valves were also trifoliate, but the right and left coronary aortic leaflets were fused in more-or-less cephalo-caudal direction. In 325 of these valves, the fusion involved less than half of the distance between the most cephalic and most caudal margins of the valve (Fig. 1B). In the other 263 instances, the fusion affected more than half of this distance (Fig. 1C). In these aortic valves with fused right and left coronary aortic leaflets, the interleaflet triangle between the right and left sinuses was decreased in size concomitant with the degree of the fusion. When the leaflets were totally fused, the interleaflet triangle was lacking (Fig. 1D). The remaining 719 hamsters displayed BAVs with leaflets made up, on the one hand, of the fused right and left coronary aortic leaflets and, on the other hand, the non-coronary leaflet. In 409 of these valves, a raphe was seen in the aortic sinus supporting the fused coronary aortic leaflets (Fig. 1E). The raphe varied in size, ranging from one that encroached toward the fused leaflets to one confined to the aortic wall. No raphe was observed in the remaining 310 bifoliate valves (Fig. 1F). As far as could be ascertained using our described techniques, there was no evidence pointing to the possibility that the fusion of the leaflets was of acquired origin.

Fig. 1.

Fig. 1

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Scanning electron micrographs of aortic valves from Syrian hamsters. The specimens were opened through the non-coronary sinus to expose the anterior aspect of the valve. (A–D) The variability of valves with three leaflets and sinuses; (E and F) BAVs. (A) A TAV with no fusion of the leaflets, and a well-developed interleaflet triangle (asterisk) between the right and left aortic sinuses. (B) A trifoliate valve having fusion of the right and left coronary aortic leaflets, but with the zone of fusion extending less than half of the distance between the most cephalic and most caudal margins of the valve; (C) a valve with fusion affecting more than half of this distance. (D) The arrangement with complete fusion of the right and left coronary aortic leaflets. Note that, in (B and C), the interleaflet triangle between the right and left aortic sinuses is decreased in size, according to the degree of the leaflet fusion. The triangle is lacking in the variant shown in (D). (E) A BAV with a raphe (arrow) located in the aortic sinus supporting the fused right and left aortic coronary leaflets; (F) a BAV devoid of any raphe. L, left coronary leaflet; R, right coronary leaflet; RL, conjoined right–left leaflet. Scale bar: 200 μm.

Phenotypic variation related to degree of inbreeding

Prior to detailing our findings in the successive generations of our inbred Syrian hamster family, we report briefly our observations on the condition of the aortic valve in the 92 hamsters from the closed outbred colony, in this way providing the basis for comparison with the results of our subsequent inbreeding. We have considered five groups of valves, namely TAVs with no fusion of the coronary leaflets, TAVs with less than half of leaflet fusion, TAVs with more than half fusion, BAVs with a raphe, and BAVs lacking a raphe. Of our outbred hamsters, 29 (32%) had a TAV with no fusion of the leaflets, 52 (56%) a TAV with a slight fusion, seven (8%) a TAV with an extensive fusion, three (3%) a BAV with a raphe, and the remaining hamster (1%) had a BAV devoid of any raphe.

The variation in the incidence of these different morphologies in the successive generations of our inbred family is shown diagrammatically in Fig. 2a. We have excluded the data relating to inbred generations 1–5, as they were obtained from internal casts. As can be inferred from the diagram, the incidence of each morphological group has varied markedly across generations. Overall, however, an increase in the incidence of BAVs, to the detriment of TAVs with no or slight fusion of the leaflets, occurred as homozygosity increased. This was particularly obvious from the 30th generation onwards. To make this fact more evident, we pooled the values of the TAVs with no or slight fusion into a single group, and those of the two subsets of BAVs into another group, maintaining the TAVs with extensive fusion of the leaflets as a third, separate group. Our reasons for proceeding in this fashion reflect the fact that the incidence of TAVs with no or slight fusion of the leaflets has decreased across the inbred generations. In addition, according to the findings in outbred hamsters, both these patterns can be regarded as variants of the normal valvar phenotype in the Syrian hamster (see also Fernández et al. 2000). TAVs with significant fusion of the coronary aortic leaflets show the smallest variation across generations, and are representative of an intermediate valvar phenotype, which cannot be included within the notion of valvar normality in the hamster (see the values obtained from outbred hamsters and also Fernández et al. 2000). The incidence of the two subsets of BAVs, both being genuine representatives of the bifoliate condition, increased appreciably from generations 6 to 39. The diagram resulting from this simplification is shown in Fig. 2b. Of note is that all hamsters belonging to the 14th generation possessed a BAV (Fig. 2). The offspring resulting from their matings, however, possessed both TAVs and BAVs.

Fig. 2.

Fig. 2

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Incidence, in percentages, of the different valvar morphologies in the inbred generations 6–39. (a) The variation in the incidence of the five groups of valves defined in the text, namely: trifoliate aortic valves with no leaflet fusion (TAV); trifoliate aortic valves with slight fusion (< 50%) of the coronary leaflets (TAVf); trifoliate aortic valves with extensive (> 50%) fusion of the coronary leaflets (TAVF); bifoliate aortic valves with raphe (BAVR); and bifoliate aortic valves with no raphe (BAV). (b) The variation after pooling the values of the trifoliate valves with no (TAVs) or slight (TAVfs) fusion of the coronary leaflets into a single group (T), the values of the bifoliate valves with (BVRs) or without raphe (BAVs) into another group (B), maintaining the trifoliate valves with extensive fusion of the leaflets (TAVFs) as a third, single group (TF). See text for further explanation.

To seek for any relationship between the incidence of the different morphological variants and the degree of inbreeding, we used the five groups of valves as defined to produce the diagram in Fig. 2a. In addition, we pooled the data of the different generations into four groups, specifically generations 6–10, 11–20, 21–30 and 31–39 (Table 2), thus facilitating their subsequent statistical treatment. The groups were established according to the probability of homozygosity deduced from the respective ranges of the coefficient of inbreeding. So, the probability of homozygosity for a given locus, expressed as a percentage, was < 90% in generations 6–10, between 90 and 99% in generations 11–20, between 99 and 99.8% in generations 21–30, and between 99.9 and 99.98% in generations 31–39.

Table 2.

Aortic valve condition according to the inbreeding coefficient (F) of the hamsters

TAV
 TAVf
 TAVF
 BAVR
 BAV
Inbred generations F n (%) n (%) n (%) n (%) n (%) tn
6–10 0.734–0.886 15 (10.4) 57 (39.9) 16 (11.2) 35 (24.5) 20 (14.0) 143
11–20 0.908–0.986 24 (5.3) 123 (27.0) 79 (17.4) 128 (28.1) 101 (22.2) 455
21–30 0.989–0.998 39 (7.7) 105 (20.9) 109 (21.7) 148 (29.4) 102 (20.3) 503
31–39 0.999–0.9998 2 (0.7) 40 (14.0) 59 (20.6) 98 (34.3) 87 (30.4) 286
tn 80 325 263 409 310 1387
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TAV, trifoliate aortic valve with no leaflet fusion; TAVf, trifoliate aortic valve with slight (< 50%) fusion of the coronary leaflets; TAVF, trifoliate aortic valve with extensive (> 50%) fusion of the coronary leaflets; BAVR, bifoliate aortic valve with raphe; BAV, bifoliate aortic valve with no raphe; n, number of specimens; tn, total number of specimens.

From the diagrams in Fig. 2, and the values shown in Table 2, we inferred that the incidence of both TAVs with no fusion of the leaflets and TAVs with partial fusion decreased as the coefficient of inbreeding, and hence the degree of homozygosity, increased, while the incidence of the remaining phenotypic variants progressively increased. To determine whether the differences between the values in Table 2 reached statistical significance, we carried out a chi-square contingency test, using as the null hypothesis the notion that variations in the incidence of different aortic valvar morphologies are independent from the degree of inbreeding of the hamsters. In other words, we started from the assumption that differences in the incidence of the five valve types in the four groups of inbred generations were random. We show the results of the test in Table 3. The computed value of the chi-square statistic is 89.442, with 12 degrees of freedom. The null hypothesis, therefore, is rejected, with a degree of significance for P of < 0.001. As is also shown in Table 3, the major departure from homogeneity is due, first, to the relatively high incidence of TAVS with no or slight fusion of the leaflets in generations 6–10 when compared with their relatively low incidence in generations 31–39; second, to the relatively low incidence of TAVs with extensive fusion of the leaflets in generations 6–10; third, to the high incidence of BAVs with raphe in generations 21–30; and, fourth, to the high incidence of BAVs devoid of any raphe in generations 31–39 when compared with their relatively low incidence in generations 6–10.

Table 3.

Contingency table of aortic valve condition according to the inbreeding coefficient (F), and results of the χ2 contingency test

Inbred generations F TAV TAVf TAVF BAVR BAV tn Σχ2
6–10 0.734–0.886 15 (8.3) 57 (33.5) 16 (27.1) 35 (42.2) 20 (32.0) 143
χ2 5.408* 16.485*** 4.546* 1.228 4.500* 32.167***
11–20 0.908–0.986 24 (26.2) 123 (106.6) 79 (86.3) 128 (134.2) 101 (101.7) 455
χ2 0.185 2.523 0.617 0.286 0.005 3.616
21–30 0.989–0.998 39 (29.0) 105 (117.9) 109 (95.4) 148 (112.4) 102 (112.4) 503
χ2 3.448 1.411 1.939 11.275*** 0.962 19.035***
31–39 0.999–0.9998 2 (16.5) 40 (67.0) 59 (54.2) 98 (84.3) 87 (63.9) 286
χ2 12.742*** 10.880*** 0.425 2.226 8.351** 34.624***
tn 80 325 263 409 310 1,387
Σχ2 21.783*** 31.299*** 7.527* 15.015** 13.818** 89.442***
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TAV, trifoliate aortic valve with no leaflet fusion; TAVf, trifoliate aortic valve with slight (< 50%) fusion of the coronary leaflets; TAVF, trifoliate aortic valve with extensive (> 50%) fusion of the coronary leaflets; BAVR, bifoliate aortic valve with raphe; BAV, bifoliate aortic valve with no raphe; n, number of specimens; tn, total number of specimens. The expected values are given in parentheses.

*

P < 0.05

**

P < 0.01

***

P < 0.001.

Results of crosses

Based on the preceding statistical results, we analysed further the results of the crosses between siblings belonging to generations 21–39, that is between very closely related individuals, each with a high probability (99–99.98%) of homozygosity. In order to simplify the analysis, we pooled the valves into the three categories used to prepare the diagram shown in Fig. 2b, namely, TAVs with no or slight fusion of the right and left coronary leaflets, TAVs with significant fusion of leaflets, and BAVs with or without raphe. This simplification was particularly necessary for TAVs with no or slight fusion of the leaflets, as none of the siblings of generations 21–30 used for crossing had an aortic valve with a purely trifoliate design.

The results of crosses are given in Table 4. To test whether differences were statistically significant, we performed a chi-square contingency test using the null hypothesis that the valvar morphology of the parents and the incidence of the different anatomic variants in the offspring were independent events. We show the results of the test in Table 5. The computed value of the chi-square statistic is 10.571, with 10 degrees of freedom. The null hypothesis, therefore, is accepted with a value for P of > 0.30.

Table 4.

Results of crosses between hamsters belonging to the inbred generations 21–39 (probability of homozygosity 0.989 or higher)

T
 TF
 B
Cross Crosses n Offspring n Embryos/litter n (%) n (%) n (%)
T × T 9 55 6.1 17 (30.9) 6 (10.9) 32 (58.2)
T × TF 15 85 5.7 25 (29.4) 18 (21.2) 42 (49.4)
T × B 39 198 5.1 44 (22.2) 48 (24.2) 106 (53.6)
TF × TF 4 21 5.2 6 (28.6) 5 (23.8) 10 (47.6)
TF × B 25 125 5.0 22 (17.6) 28 (22.4) 75 (60.0)
B × B 45 226 5.0 50 (22.1) 45 (19.9) 131 (58.0)
tn 147 710 164 150 396
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T, trifoliate aortic valve with no leaflet fusion or with slight (< 50%) fusion of the coronary leaflets; TF, trifoliate aortic valve with extensive (> 50%) fusion of the coronary leaflets; BAV, bifoliate aortic valve; n, number of specimens; tn, total number of specimens.

Table 5.

Contingency table of aortic valve morphology in offspring vs. types of crosses between hamsters belonging to generations 21–39 (probability of homozygosity 0.989 or higher), and results of the χ2 contingency test

Cross T χ2 TF χ2 B χ2 tn Σχ2
T × T 17 (12.7) 1.456 6 (11.6) 2.703 32 (30.7) 0.055 55 4.214
T × TF 25 (19.6) 1.488 18 (18.0) 0.000 42 (47.4) 0.615 85 2.103
T × B 44 (45.7) 0.063 48 (41.8) 0.920 106 (110.5) 0.183 198 1.166
TF × TF 6 (4.9) 0.247 5 (4.4) 0.008 10 (11.7) 0.247 21 0.502
TF × B 22 (28.9) 1.647 28 (26.4) 0.100 75 (69.7) 0.403 125 2.150
B × B 50 (52.2) 0.093 45 (47.7) 0.153 131 (126.1) 1.190 226 0.436
tn 164 150 396
Σχ2 4.994 3.884 1.693 10.571
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T, trifoliate aortic valve with no leaflet fusion or with slight (< 50%) fusion of the coronary leaflets; TF, trifoliate aortic valve with extensive (> 50%) fusion of the coronary leaflets; BAV, bifoliate aortic valve; tn, total number of specimens.

The expected values are given in parentheses.

Discussion

It is now over 40 years since Waller et al. (1973) described a variety of morphologies in human aortic valves with two and three leaflets, outlining thereby the notion that BAVs should be regarded as an expression of a phenotypic continuum. At that time, however, interest was more focused on solving the ancient controversy concerning the congenital as opposed to the acquired origin of such valves (Osler, 1886; Lewis & Grant, 1923; Koletsky, 1941), rather than in evaluating the significance of the observations of Waller et al. (1973) from the aetiological stance (see Roberts, 1970). Knowledge about congenital anomalies of the aortic valve has increased significantly in recent years, and currently the bifoliate condition of the human aortic valve tends to be regarded in the context of a phenotypic continuum that ranges from valves with a single leaflet to valves with four leaflets (Collins et al. 2008; Mangini et al. 2010). Within this continuum, BAVs themselves show a wide morphological variation, making it necessary, though not without difficulties, to achieve a classification of the distinct variants that may be satisfactory from the clinical, surgical and aetiological views. In recent times, however, there is a marked tendency to accept that there are three main types of BAVs, namely those with fused right and left coronary aortic leaflets, those with right and non-coronary fusion, and those with left and non-coronary fusion (Russo et al. 2008; Schaefer et al. 2008).

The valves having two leaflets displayed both by the Syrian hamsters of our inbred family and our outbred hamsters correspond to the type characterized by the fusion of the right and left coronary aortic leaflets. The Syrian hamster, therefore, cannot be regarded as a true animal model for the overall spectrum of humans with BAVs. It will serve only to provide insights into different aspects of patients with fusion of the right and left coronary aortic leaflets, which interestingly are the most frequent in man (Russo et al. 2008; Schaefer et al. 2008; Calloway et al. 2011; Laforest et al. 2011). Likewise, mice with knock-out of the gene for endothelium nitric oxide synthase should be used only to gain information about BAVs with fusion of the right and non-coronary aortic leaflets (Fernández et al. 2009). Indeed, it was a comparative study between these two animal models that led to the conclusion that these two most frequent types of aortic valves having two leaflets are distinct morphogenetic entities (Fernández et al. 2009).

A high proportion (56%) of the outbred Syrian hamsters had a TAV with a slight fusion of the right and left coronary leaflets, so that this arrangement can be considered within normality in this rodent species. TAVs with an extensive fusion of the coronary leaflets also occurred in the outbred hamsters, but their frequency was far less high (8%). Minimal fusions in one of the three commissures seem to be relatively common in human TAVs, although in smaller proportions than in the Syrian hamster. Roberts & Ko (2005) reported a frequency of such fusions in 18% of 417 TAVs from adults undergoing isolated valvar replacement for aortic stenosis, with or without associated aortic regurgitation. TAVs with an extensive leaflet fusion have been reported in man (Roberts, 1970; Waller et al. 1973), but, to our knowledge, their incidence remains to be estimated.

Another aspect worthy of comment before discussing our specific current findings concerns the difference between humans and Syrian hamsters having two aortic valvar leaflets regarding the gender ratio. It is well known that, in man, there is a marked male gender predilection for BAV (reviewed in Collins et al. 2008), whereas in the Syrian hamster, the gender ratio does not significantly differ from equity. The cause of the departure from the ratio of equity in man remains unknown. Our findings throw no light on this question. They do, nonetheless, highlight an important difference between man and Syrian hamster, a difference that cannot be ignored when seeking comparisons between the aetiological factors implicated in the formation of aortic valves with two leaflets.

Almost 20 years ago, we reported the variation in the incidence of BAVs from the first to the ninth generation of our inbred Syrian hamster family (Sans-Coma et al. 1993b). In addition, we showed the results of crosses between siblings belonging to that family, and pedigrees resulting from the combined crosses between these hamsters and hamsters belonging to another inbred family with a low incidence of BAVs. Although the morphology of numerous valves could not then be assessed in detail, because they had been examined using a corrosion-cast technique, our findings already indicated that the BAVs of our hamsters should be regarded as expressions of a trait, the variation of which takes the form of a continuous phenotypic range, thus behaving as a quantitative trait.

As shown by both the values given in Table 1 and the diagrams in Fig. 2, in our large cohort of Syrian hamsters, the incidence of the different morphological variants varied markedly in the inbred generations 6–39. Yet, the aortic valves with a purely trifoliate design, and those with relatively slight fusion of the coronary aortic leaflets, significantly decreased as the inbreeding coefficient increased. Concomitantly, the incidence of TAVs with extensive fusion of the coronary leaflets moderately increased, while that of the two bifoliate patterns increased significantly (Table 3). Moreover, the results of crosses between siblings of the generations 21–39 (Table 5) matched with those obtained in our previous work (Sans-Coma et al. 1993b) by crossing siblings with a lower degree of inbreeding. In both instances, the results showed that there was no statistically significant association between the phenotypes of the valves in the parents and those of their offspring.

Taken together, therefore, our previous (Sans-Coma et al. 1993b) and the present findings are consistent with the notion that the morphological variants of the phenotypic continuum of aortic valves in our inbred Syrian hamsters are subject to a polygenic mode of inheritance, with reduced penetrance (65% in generations 31–39) and variable expressivity (BAVs with no raphe or with a more or less developed raphe) of the bifoliate phenotype. This denotes complex inheritance, as is the case of human BAVs (Emanuel et al. 1978; Huntington et al. 1997; Cripe et al. 2004; Ellison et al. 2007; Loscalzo et al. 2007; McBride & Garg, 2011).

The hamsters belonging to the inbred generation 31–39 could be considered almost isogenic. Their coefficient of inbreeding was 0.999 or higher. The probability for isoallelic locuses, potentially implicated in the formation of aortic valves with fused coronary leaflets, therefore, was also very high. On this basis, we expected that these hamsters would display similar aortic valvar morphology. In contrast, we observed all the identified morphological variants among them (Table 2), though five-sixths had defective valves, either trifoliate ones with extensive fusion of the coronary aortic leaflets, found in one-fifth, or BAVs with a conjoined coronary aortic leaflet accounting for two-thirds. The remaining valves showed a tricuspid design, but in most cases the coronary leaflets were slightly fused.

The high proportion of genetically alike hamsters having aortic valves that diverge strikingly from the trifoliate prototype denotes that the genotype of these animals greatly predisposes to the development of anomalous aortic valve. There is embryological evidence that BAVs with fused coronary leaflets are the outcome of an anomalous septation of the proximal portion of the embryonic outflow tract, which is likely due to a distorted behaviour of neural crest cells (Fernández et al. 2009). As a result of the anomalous septation, the right and left valvar primordiums become more or less extensively fused, a fact that has been adduced as a key factor for the fusion of the coronary aortic leaflets (Sans-Coma et al. 1996). We now hypothesise that the genotype shared by our hamsters affects normal behaviour of the neural crest cells.

Morphological variation in genetically alike animals, as we have detected in the aortic valve of our hamsters, is not a novelty. There is enough evidence that isogenic populations can show a considerable phenotypic variation between individuals for a given trait (reviewed in Veitia, 2005; Wong et al. 2005; Vogt et al. 2008; Seewald et al. 2010). The phenotype results from the interaction between genotype and environmental factors, along with an additional third component (Gärtner, 1990) of stochastic nature that causes random variation. The end result is the so-called intangible variation, or developmental noise (reviewed in Peaston & Whitelaw, 2006; Vogt et al. 2008). Recent work in this field has implicated epigenetic mechanisms as a main cause of paradoxical findings in inbred animals when phenotypic differences occur in the absence of recognisable environmental differences (Wong et al. 2005; Peaston & Whitelaw, 2006; Vogt et al. 2008; Delcuve et al. 2009). In this setting, however, a limitation of our current study is the lack of any molecular evidence to sustain the conjectures that have been inferred from the results of inbreeding. We recognise that further studies are needed to fill this gap. In this context, a factor that should not be overlooked in future research on the aetiology of BAVs is the possible involvement of genetic modifiers, which can affect both penetrance and expressivity of a given trait (Nadeau, 2001). Recent work has shown the impact of genetic modifiers in cardiac development (Winston et al. 2010) and the progression of disease in murine models of cardiomyopathy (Wheeler et al. 2009).

Despite the preceding methodological constraints, our findings indicate that a single genotype may account for the whole range of valvar variants. This, together with current knowledge above on phenotypic variation, raises the possibility that homozygous individuals with genetic determinants presumably responsible for the formation of aortic valves with two leaflets can also produce a valve having a trifoliate design. This allows understanding of the apparently contradictory findings concerning the structure of aortic valves in monozygotic twins, namely the presence of a valve with two leaflets in each twin (Brown et al. 2003), as opposed to the occurrence of a BAV in one twin but a TAV in the other (Lewis & Henderson, 1990). In addition, it might explain, first, the relatively uncommon occurrence of BAV in more than one family member, despite the estimated frequency of 1–2% for the defect in the general human population (Glick & Roberts, 1994); and second, its low recurrence in first-degree relatives (Calloway et al. 2011).

In man, aortic valvar dysfunction, such as aortic stenosis (Chan et al. 2001; Freeman & Otto, 2005) and ascending aortic dilation (Ward, 2000; Fedak, 2008; Nistri et al. 2008; Tadros et al. 2009) are significantly associated with the bifoliate condition of the aortic valve. Our findings in Syrian hamsters throw no new light on the pathogenesis of such clinical complications, as none of the specimens we examined showed signs of calcification or aortic enlargement. In supporting the notion that BAVs and TAVs with fusion of coronary leaflets relay on the same genotype, however, the information gained from our hamsters might be useful for outlining accurate strategies to prevent future pathologies. Indeed, our findings should encourage clinicians to pay particular attention to TAVs with fused coronary leaflets as such congenitally defective valves, especially those with an extensive fusion of coronary leaflets, may harbour an inherent risk of calcification and stenosis and a predisposition to aortic dilation similar to that already recognised for BAVs with fused coronary leaflets.

In conclusion, our findings indicate that the genotype of the present genetically alike or virtually isogenic Syrian hamsters produced by systematic inbreeding greatly predisposes to the development of anomalous aortic valves, specifically bifoliate valves with fused right and left coronary aortic leaflets or trifoliate valves with extensive fusion of the coronary leaflets. They also denote, however, that the same underlying genotype may account for the whole range of valvar morphological variants, suggesting that factors other than the genetic determinants of the aortic valvar condition, operating during embryonic life and creating the so-called intangible variation or developmental noise, play an important role in the definitive anatomic configuration of the valve.

Acknowledgments

This study was supported by grants SAF2006-01548 (Ministerio de Educación y Ciencia, Spain), PI-0689/2010 (Consejería de Salud, Junta de Andalucía, Spain), P10-CTS-06068 (Consejería de Innovación, Ciencia y Empresa, Junta de Andalucía, Spain) and FEDER funds. We thank L. Vida and J. A. Zamora, Málaga, for their technical assistance, and G. Martín, Málaga, for his assistance in operating the scanning electron microscope.

References

  1. Anderson RH. Clinical anatomy of the aortic root. Heart. 2000;84:670–673. doi: 10.1136/heart.84.6.670. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Angelini A, Ho SY, Anderson RH, et al. The morphology of the normal aortic valve as compared with the aortic valve having two leaflets. J Thorac Cardiovasc Surg. 1989;98:262–367. [PubMed] [Google Scholar]
  3. Benavides FJ, Guénet JL. Manual de Genética de Roedores de Laboratorio. Principios Básicos y Aplicaciones. España: Universidad de Alcalá y SECAL; 2003. [Google Scholar]
  4. Braverman AC, Güven H, Beardslee MA, et al. The bicuspid aortic valve. Curr Probl Cardiol. 2005;30:470–522. doi: 10.1016/j.cpcardiol.2005.06.002. [DOI] [PubMed] [Google Scholar]
  5. Brown C, Sane DC, Kitzman DW. Bicuspid aortic valves in monozygotic twins. Echocardiography. 2003;20:183–184. doi: 10.1046/j.1540-8175.2003.03012.x. [DOI] [PubMed] [Google Scholar]
  6. Calloway TJ, Martin LJ, Zhang X, et al. Risk factors for aortic valve disease in bicuspid aortic valve: a family-based study. Am J Med Genet A. 2011;155:1015–1020. doi: 10.1002/ajmg.a.33974. [DOI] [PubMed] [Google Scholar]
  7. Chan KL, Ghani M, Woodend K, et al. Case-controlled study to assess risk factors for aortic stenosis in congenitally bicuspid aortic valve. Am J Cardiol. 2001;88:690–693. doi: 10.1016/s0002-9149(01)01820-3. [DOI] [PubMed] [Google Scholar]
  8. Clementi M, Notari L, Borghi A, et al. Familial congenital bicuspid aortic valve: a disorder of uncertain inheritance. Am J Med Genet. 1996;62:336–338. doi: 10.1002/(SICI)1096-8628(19960424)62:4<336::AID-AJMG2>3.0.CO;2-P. [DOI] [PubMed] [Google Scholar]
  9. Collins MJ, Butany J, Borger MA, et al. Implications of a congenitally abnormal valve: a study of 1025 consecutive excised aortic valves. J Clin Pathol. 2008;61:530–536. doi: 10.1136/jcp.2007.051904. [DOI] [PubMed] [Google Scholar]
  10. Cripe L, Andelfinger G, Martin LJ, et al. Bicuspid aortic valve is heritable. J Am Coll Cardiol. 2004;44:138–143. doi: 10.1016/j.jacc.2004.03.050. [DOI] [PubMed] [Google Scholar]
  11. De Mozzi P, Longo UG, Galanti G, et al. Bicuspid aortic valve: a literature review and its impact on sport activity. Br Med Bull. 2008;85:63–85. doi: 10.1093/bmb/ldn002. [DOI] [PubMed] [Google Scholar]
  12. Delcuve GP, Rastegar M, Davie JR. Epigenetic control. J Cell Physiol. 2009;219:243–250. doi: 10.1002/jcp.21678. [DOI] [PubMed] [Google Scholar]
  13. Durán AC, Sans-Coma V, Arqué JM, et al. Blood supply to the interventricular septum in rodents with intramyocardial coronary arteries. Acta Zool (Stockholm) 1992;73:223–229. [Google Scholar]
  14. Ellison JW, Yagubyan M, Majudmar R, et al. Evidence of genetic locus heterogeneity for familial bicuspid aortic valve. J Surg Res. 2007;142:28–31. doi: 10.1016/j.jss.2006.04.040. [DOI] [PubMed] [Google Scholar]
  15. Emanuel R, Whiters R, O'Brien K, et al. Congenitally bicuspid aortic valves. Clinicogenetic study of 41 families. Br Heart J. 1978;40:1402–1407. doi: 10.1136/hrt.40.12.1402. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Falconer DS. Introduction to Quantitative Genetics. San Francisco (USA): Benjamin-Cummings; 1996. [Google Scholar]
  17. Fedak PW. Bicuspid aortic valve syndrome: heterogeneous but predictable? Eur Heart J. 2008;29:432–433. doi: 10.1093/eurheartj/ehm609. [DOI] [PubMed] [Google Scholar]
  18. Fedak PW, Verma S, David TE, et al. Clinical and pathophysiological implications of aortic valve. Circulation. 2002;106:900–904. doi: 10.1161/01.cir.0000027905.26586.e8. [DOI] [PubMed] [Google Scholar]
  19. Fernández MC, Durán AC, Real R, et al. Coronary artery anomalies and aortic valve morphology in Syrian hamsters. Lab Anim. 2000;34:145–154. doi: 10.1258/002367700780457545. [DOI] [PubMed] [Google Scholar]
  20. Fernández B, Durán AC, Fernández-Gallego T, et al. Bicuspid aortic valves with different spatial orientation of the leaflets are distinct etiological entities. J Am Coll Cardiol. 2009;54:2312–2318. doi: 10.1016/j.jacc.2009.07.044. [DOI] [PubMed] [Google Scholar]
  21. Frater RWM, Anderson RH. How can we logically describe the components of the arterial valves? J Heart Valve Dis. 2010;19:438–440. [PubMed] [Google Scholar]
  22. Freeman RV, Otto CM. Spectrum of calcific aortic valve disease; pathogenesis, disease progression, and treatment strategies. Ciculation. 2005;111:3316–3326. doi: 10.1161/CIRCULATIONAHA.104.486738. [DOI] [PubMed] [Google Scholar]
  23. Garg V. Molecular genetics of aortic valve disease. Curr Opin Cardiol. 2006;21:180–184. doi: 10.1097/01.hco.0000221578.18254.70. [DOI] [PubMed] [Google Scholar]
  24. Garg V, Muth AN, Ransom JF, et al. Mutations in NOTCH1 cause aortic valve disease. Nature. 2005;437:270–274. doi: 10.1038/nature03940. [DOI] [PubMed] [Google Scholar]
  25. Gärtner K. A third component causing random variability beside environment and genotype. A reason for the limited success of a 30 year long effort to standardize laboratory animals? Lab Anim. 1990;24:71–77. doi: 10.1258/002367790780890347. [DOI] [PubMed] [Google Scholar]
  26. Glick BN, Roberts WC. Congenitally bicuspid aortic valve in multiple family members. Am J Cardiol. 1994;73:400–404. doi: 10.1016/0002-9149(94)90018-3. [DOI] [PubMed] [Google Scholar]
  27. Hinton RB, Martin LJ, Rame-Gowda MS, et al. Hypoplastic left heart syndrome links to chromosome 10q and 6q and is genetically related to bicuspid aortic valve. J Am Coll Cardiol. 2009;53:1065–1071. doi: 10.1016/j.jacc.2008.12.023. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Huntington K, Hunter AG, Chan KL. A prospective study to assess the frequency of familial clustering of bicuspid aortic valve. J Am Coll Cardiol. 1997;30:1809–1812. doi: 10.1016/s0735-1097(97)00372-0. [DOI] [PubMed] [Google Scholar]
  29. Koletsky SD. Congenital bicuspid pulmonary valves. Arch Pathol Lab Med. 1941;31:338–353. [Google Scholar]
  30. Laforest B, Andelfinger G, Nemer M. Loss of Gata5 in mice leads to bicuspid aortic valve. J Clin Invest. 2011;121:2876–2887. doi: 10.1172/JCI44555. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Lewis T, Grant RT. Observations relating to sub-acute infective endocarditis. Heart. 1923;10:21–99. [Google Scholar]
  32. Lewis NP, Henderson AH. Calcific aortic stenosis in twins; a clue to its pathogenesis? Eur Heart J. 1990;11:90–91. doi: 10.1093/oxfordjournals.eurheartj.a059598. [DOI] [PubMed] [Google Scholar]
  33. Loscalzo ML, Goh DLM, Loeys B, et al. Familial thoracic aortic dilation and bicommissural aortic valve: a prospective analysis of natural history and inheritance. Am J Med Genet. 2007;143A:1960–1967. doi: 10.1002/ajmg.a.31872. [DOI] [PubMed] [Google Scholar]
  34. Mangini A, Lemma M, Contino M, et al. Bicuspid aortic valve: differences in the phenotypic continuum affect the repair techniques. Eur J Cardiothorac Surg. 2010;37:1015–1020. doi: 10.1016/j.ejcts.2009.11.048. [DOI] [PubMed] [Google Scholar]
  35. Martin LJ, Ramachandran V, Cripe LH, et al. Evidence in favour of linkage to human chromosomal regions 18q, 5q and 13q for bicuspid aortic valve and associated cardiovascular malformations. Hum Genet. 2007;12:275–284. doi: 10.1007/s00439-006-0316-9. [DOI] [PubMed] [Google Scholar]
  36. McBride KL, Garg V. Heredity of bicuspid aortic valve: is familial screening indicated? Heart. 2011;97:1193–1195. doi: 10.1136/hrt.2011.222489. [DOI] [PubMed] [Google Scholar]
  37. McDonald K, Maurer BJ. Familial aortic valve disease: evidence for a genetic influence? Eur Heart J. 1989;10:676–677. doi: 10.1093/oxfordjournals.eurheartj.a059546. [DOI] [PubMed] [Google Scholar]
  38. McKay R, Smith A, Leung MP, et al. Morphology of the ventriculoaortic junction in critical aortic stenosis. Implications for hemodynamic function and clinical management. J Thorac Cardiovasc Surg. 1992;104:434–442. [PubMed] [Google Scholar]
  39. Mohamed SA, Hanke T, Schlueter C, et al. Ubiquitin fusion degradation 1-like gene dysregulation in bicuspid aortic valve. J Thorac Cardiovasc Surg. 2005;130:1531–1536. doi: 10.1016/j.jtcvs.2005.08.017. [DOI] [PubMed] [Google Scholar]
  40. Mohamed SA, Aherrahrou Z, Liptau H, et al. Novel missense mutations (p.T596M, and p.1797H) in NOTCH1 in patients with bicuspid aortic valve. Biochem Biophys Res Commun. 2006;345:1460–1465. doi: 10.1016/j.bbrc.2006.05.046. [DOI] [PubMed] [Google Scholar]
  41. Nadeau JH. Modifier genes in mice and humans. Nat Rev. 2001;2:165–174. doi: 10.1038/35056009. [DOI] [PubMed] [Google Scholar]
  42. Nistri S, Grande-Allen J, Noale M, et al. Aortic elasticity and size in bicuspid aortic valve syndrome. Eur Heart J. 2008;29:472–479. doi: 10.1093/eurheartj/ehm528. [DOI] [PubMed] [Google Scholar]
  43. Osler W. The bicuspid condition of the aortic valve. Trans. Assoc. Am. Physicians. 1886;2:185–192. [Google Scholar]
  44. Peaston AE, Whitelaw E. Epigenetics and phenotypic variation in mammals. Mamm Genome. 2006;17:365–374. doi: 10.1007/s00335-005-0180-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Perloff JK, Roberts WC. The mitral apparatus. Functional anatomy of mitral regurgitation. Circulation. 1972;46:227–239. doi: 10.1161/01.cir.46.2.227. [DOI] [PubMed] [Google Scholar]
  46. Roberts WC. The congenitally bicuspid aortic valve. A study of 85 autopsy cases. Am J Cardiol. 1970;26:72–83. doi: 10.1016/0002-9149(70)90761-7. [DOI] [PubMed] [Google Scholar]
  47. Roberts WC, Ko JM. Frequency by decades of unicuspid, bicuspid, and tricuspid aortic valves in adults having isolated aortic valve replacement for aortic stenosis, with or without associated aortic regurgitation. Circulation. 2005;111:920–925. doi: 10.1161/01.CIR.0000155623.48408.C5. [DOI] [PubMed] [Google Scholar]
  48. Russo CF, Cannata A, Lanfranconi M, et al. Is aortic wall degeneration related to bicuspid aortic valve anatomy with valvular disease? J Thorac Cardiovasc Surg. 2008;136:937–942. doi: 10.1016/j.jtcvs.2007.11.072. [DOI] [PubMed] [Google Scholar]
  49. Sabet HY, Edwards WD, Tazelaar HD, et al. Congenitally bicuspid aortic valves: a surgical pathology study of 542 cases (1991 through 1996) and a literature review of 2,715 cases. Mayo Clin Proc. 1999;74:14–26. doi: 10.4065/74.1.14. [DOI] [PubMed] [Google Scholar]
  50. Sans-Coma V, Arqué JM, Durán AC, et al. The coronary arteries of the Syrian hamster, Mesocricetus auratus (Waterhouse, 1839) Ann Anat. 1993a;175:53–57. doi: 10.1016/s0940-9602(11)80239-6. [DOI] [PubMed] [Google Scholar]
  51. Sans-Coma V, Cardo M, Durán AC, et al. Evidence for a quantitative genetic influence on the formation of aortic valves with two leaflets in the Syrian hamster. Cardiol Young. 1993b;3:132–140. [Google Scholar]
  52. Sans-Coma V, Fernández B, Durán AC, et al. Fusion of valve cushions as a key factor in the formation of congenital bicuspid aortic valves in Syrian hamsters. Anat Rec. 1996;244:490–498. doi: 10.1002/(SICI)1097-0185(199604)244:4<490::AID-AR7>3.0.CO;2-Z. [DOI] [PubMed] [Google Scholar]
  53. Schaefer BM, Lewin MB, Stout KK, et al. The bicuspid aortic valve: an integrated phenotype classification of leaflet morphology and aortic root shape. Heart. 2008;94:1634–1638. doi: 10.1136/hrt.2007.132092. [DOI] [PubMed] [Google Scholar]
  54. Seewald AK, Cypser J, Mendenhall A, et al. Quantifying phenotypic variation in isogenic Caenorhabditis elegans expressing Phsp-16.2:gfp by clustering 2D expression patterns. PLoS ONE. 2010;5:e11 426. doi: 10.1371/journal.pone.0011426. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Siu SC, Silversides CK. Bicuspid aortic valve disease. J Am Coll Cardiol. 2010;55:2789–2800. doi: 10.1016/j.jacc.2009.12.068. [DOI] [PubMed] [Google Scholar]
  56. Sutton JP, III, Ho SY, Anderson RH. The forgotten interleaflet triangles: a review of the surgical anatomy of the aortic valve. Ann Thorac Surg. 1995;59:419–427. doi: 10.1016/0003-4975(94)00893-c. [DOI] [PubMed] [Google Scholar]
  57. Tadros TM, Klein D, Shapira OM. Ascending aortic dilatation associated with bicuspid aortic valve: pathophysiology, molecular biology and clinical implications. Circulation. 2009;119:880–890. doi: 10.1161/CIRCULATIONAHA.108.795401. [DOI] [PubMed] [Google Scholar]
  58. Veitia RA. Stochasticity or the fatal “imperfection” of cloning. J Biosci. 2005;30:21–30. doi: 10.1007/BF02705147. [DOI] [PubMed] [Google Scholar]
  59. Vogt G, Huber M, Thiemann M, et al. Production of different phenotypes from the same genotype in the same environment by developmental variation. J Exp Biol. 2008;211:510–523. doi: 10.1242/jeb.008755. [DOI] [PubMed] [Google Scholar]
  60. Waller BF, Carter JB, Williams HJ, et al. Bicuspid aortic valve. Comparison of congenital and acquired types. Circulation. 1973;48:1140–1150. doi: 10.1161/01.cir.48.5.1140. [DOI] [PubMed] [Google Scholar]
  61. Ward C. Clinical significance of the bicuspid aortic valve. Heart. 2000;83:81–85. doi: 10.1136/heart.83.1.81. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Wheeler FC, Tang H, Marks OA. Tnni3k modifies disease progression in murine models of cardiomyopathy. PLOS Genet. 2009;5(9):e1000647. doi: 10.1371/journal.pgen.1000647. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Winston JB, Erlich JM, Green CA. Heterogeneity of genetic modifiers ensures normal cardiac development. Circulation. 2010;121:1313–1321. doi: 10.1161/CIRCULATIONAHA.109.887687. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Wong AHC, Gottesman, Petronis A. Phenotypic differences in genetically identical organisms: the epigenetic perspective. Hum Mol Genet. 2005;14:R11–R18. doi: 10.1093/hmg/ddi116. Spec No 1. [DOI] [PubMed] [Google Scholar]

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