Williams Syndrome Predisposes to Vascular Stiffness Modified by Antihypertensive Use and Copy Number Changes in NCF1Novelty and Significance
Williams syndrome is caused by the deletion of 26 to 28 genes, including elastin, on human chromosome 7. Elastin insufficiency leads to the cardiovascular hallmarks of this condition, namely focal stenosis and hypertension. Extrapolation from the Eln+/− mouse suggests that affected people may also have stiff vasculature, a risk factor for stroke, myocardial infarction, and cardiac death. NCF1, one of the variably deleted Williams genes, is a component of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex and is involved in the generation of oxidative stress, making it an interesting candidate modifier for vascular stiffness. Using a case–control design, vascular stiffness was evaluated by pulse wave velocity in 77 Williams cases and matched controls. Cases had stiffer conducting vessels than controls (P<0.001), with increased stiffness observed in even the youngest children with Williams syndrome. Pulse wave velocity increased with age at comparable rates in cases and controls, and although the degree of vascular stiffness varied, it was seen in both hypertensive and normotensive Williams participants. Use of antihypertensive medication and extension of the Williams deletion to include NCF1 were associated with protection from vascular stiffness. These findings demonstrate that vascular stiffness is a primary vascular phenotype in Williams syndrome and that treatment with antihypertensives or agents inhibiting oxidative stress may be important in managing patients with this condition, potentially even those who are not overtly hypertensive.
Vascular stiffness is an independent risk factor for multiple negative cardiovascular outcomes in normally aging adults, including stroke, myocardial infarction, and sudden death.1 Whether vascular stiffness occurs in developmental cardiovascular disorders and portends similar adverse cardiovascular outcomes is not well documented. Previous investigations in mouse models have consistently linked haploinsufficiency for elastin, an extracellular matrix protein that provides recoil to elastic tissues, to increased vascular stiffness and hypertension.2 Comparable data on the effect of elastin deficiency on vascular stiffness in humans are lacking and, thus, warrant study in naturally occurring elastin deficiency disorders.
Loss of function defects in the human elastin gene (ELN) cause focal arterial stenosis, generalized vascular narrowing, and hypertension in a rare condition called nonsyndromic supravalvular aortic stenosis3–5 (SVAS; MIM No. 185500, prevalence 1:20 000). Deletion of an entire ELN allele as part of the contiguous gene deletion disorder, Williams syndrome (WS; MIM No. 194050, prevalence 1:8000–10 000), leads to the same vascular phenotype.6
To test the hypothesis that elastin insufficiency is associated with vascular stiffness in humans, we initiated the multi-institutional Williams Syndrome-Skin And Vessel Elasticity (WS-SAVE) study. Using applanation tonometry, we collected pulse wave velocity (PWV) measurements in the single largest WS cohort studied to date, consisting of 77 affected individuals aged 7 to 62 years. Vascular stiffness normally increases with age and with comorbid conditions such as hypertension and diabetes mellitus.7 Consequently, this robust sample size and broad age range allowed us to evaluate the presence of vascular stiffness in WS throughout the life span and also to identify covariates that modify arterial stiffness in this population. In particular, we investigate both pharmacological interventions and genetic alterations that influence arterial stiffness and identify treatments associated with lower PWV. Differences in the WS deletion affecting copy number (CN) for NCF1, a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunit, have been shown to affect hypertension risk in WS,8 but investigation of the gene’s effect on arterial stiffness has not been investigated. Because individuals with WS are at markedly increased risk of sudden death9,10 possibly due, in part, to increased vascular stiffness, the modifiers examined may be critical to improving their health and longevity. In addition, these findings could inform the management of other conditions that lead to pediatric vascular stiffness, such as diabetes mellitus and chronic renal failure.11,12
Subjects and Methods
Please see Methods in the online-only Data Supplement for additional details.
Recruitment of cases and controls was performed as part of institutional review board–approved studies (WS cases through Washington University School of Medicine [WUSM] and the Massachusetts General Hospital, adult controls through WUSM, and pediatric controls through Semmelweis University). Each participant (adult controls) or their parent/caregiver (pediatric controls and all WS cases) consented to participation in the study. Procedures followed were in accordance with institutional guidelines. Historical, medical, and surgical data were obtained by the use of a questionnaire. Medical records, including echocardiogram, were reviewed to validate questionnaire data when possible. Participants with WS underwent cardiovascular physical examination, and WS phenotype was verified on physical examination by experienced clinical geneticists. Molecular confirmation of WS was achieved either by review of clinical testing results (fluorescent in situ hybridization or microarray) or by research quantification of ELN CN (Figure S1 in the online-only Data Supplement).
Pulse Wave Velocity
Height and weight were formally measured, and individuals were then placed in a supine position and allowed to rest quietly. Blood pressure was manually assessed in both arms. PWV was determined from contour analysis of arterial waveforms recorded by applanation tonometry using a highly reproducible technique.7,13,14
NCF1 Gene and Pseudogene CN Determination
The WS critical region on chromosome 7 is flanked by 3 regions of low CN repeats,8,15 each containing the NCF1 gene or 1 of its 2 NCF1 pseudogenes (NCF1B and NCF1C; Figure S2). The absence of 2 base pairs (ΔGT) at the beginning of exon 2 in the pseudogenes distinguishes them from the NCF1 gene.16 To calculate relative CN for the NCF1 gene to its pseudogenes, both the genes and pseudogenes were amplified together using polymerase chain reaction primers surrounding the ΔGT region. After polymerase chain reaction amplification, the product was gel purified and Sanger sequenced. At the ΔGT, the gene and pseudogene sequences diverge, and the relative peak heights of the next 27 bases are determined from the tracings as previously described.17 NCF1 gene and pseudogene CN were assigned using the ratio table in Table S1, and NCF1 gene number was used in subsequent statistical analysis.
PWV in WS Versus Matched Controls
Because PWV normally increases with age,7,14,18 our initial analysis evaluated the pediatric (n=36) and adult (n=41) cohorts separately. Cases and controls were matched, and PWV readings were compared using results from paired t tests. To determine whether PWV increases with age at the same rate in participants with WS versus controls, we also performed regression in the full-matched data set (n=77) with age as the covariate.
Covariate Analysis (PWV)
To identify additional features associated with higher PWV in the WS population, we performed regression on PWV data from participants with WS using diabetes mellitus, hypertension, antihypertensive medication, or NCF1 CN status as the covariate. For the NCF1 CN analysis, because of the observed protective effect of antihypertensives on PWV in WS (Figure 2B) and others,19 individuals known to use these medications were excluded from this analysis.
Covariate Analysis (Hypertension)
The Fisher exact test was used to compare the prevalence of hypertension in WS individuals with NCF1 gene CN of 1 versus ≥2. Hypertension was defined as any participant who had received a diagnosis of hypertension (treated or untreated). For each analysis, only participants with the necessary data components were analyzed, and n for each study is reported. All statistical analyses were performed using Prism statistical software.
A total of 103 individuals with WS (aged 7–62 years) were consented for the WS-SAVE study. Quality PWV measurements were obtained in 77 of the 103 (74.8%) participants with WS. Rates of vascular disease in those with successful versus unsuccessful PWV measurements were similar for most phenotypes assessed (see Table S2 for P values), with the unsuccessful cohort having a higher percentage of women, individuals with elevated body mass index (>35), and SVAS repair. No participants were fully excluded from the study, but individuals were excluded from portions of the analysis in which relevant data were not available. For example, history of hypertension was assessed in 101 of 103 participants. Similarly, DNA was of sufficient quality to calculate NCF1 CN in 101 of 103 individuals.
Adult controls (n=41; aged 21.5–62.4 years) were ambulatory subjects selected from participants of institutional review board–approved cardiovascular studies at WUSM. Subjects were excluded for any of the following: left ventricular systolic dysfunction (ejection fraction <55%), reported or suspected coronary artery disease, pulmonary hypertension, stage C or worse chronic kidney disease, reported infection by the HIV, and reported sickle cell disease. Adults were matched to participants with WS, in aggregate, for body mass index, hypertension diagnosis, use of antihypertensives, and diabetes mellitus, in addition to age and sex (paired t tests revealed nonsignificant results for these variables; Table S3).
Pediatric controls (n=36; aged 7.6–21.2 years) were enrolled as part of an international study establishing reference values of PWV in children and adolescents.18 Children with known history of hypertension or diabetes mellitus were excluded. Children were matched by age (mean difference±SD [WS-C], 0.04±0.19 years), sex, and height (mean difference [WS-C] −6±7 cm) to WS youth.
Clinical Characteristics of the WS-SAVE Cohort
To determine whether the increased arterial stiffness noted in elastin-insufficient mice20–22 extrapolates to humans with elastin insufficiency, we attempted PWV in a cohort of 103 individuals with WS. Clinical features of the 77 subjects successfully completing the PWV portion of the study are included in Table S4. Sixty-nine percent of those with successful PWV measurements have a history of vascular stenosis (any location). Fifty-four percent had SVAS, but only 10% had SVAS requiring surgical intervention. Forty percent of the cohort reported a history of hypertension, but only 25% had hypertension that was treated with antihypertensive medication. Ten percent reported a diagnosis of diabetes mellitus. Stroke (any type) was reported in 6% (3/48) of all individuals asked about this phenotype; among the subset with a successful PWV measurement, 2 of 36 (6%) reported a history of stroke.
Children and Adults With WS Show Stiffer Vessels Than Matched Controls
PWVs from the 77 individuals with WS were compared with pediatric and adult controls. When the pediatric subset was compared in paired t tests, those with WS were found, on average, to have significantly higher PWV (mean±SD, 6.1±1.0 [WS] versus 5.1±0.8 m/s [control]; P<0.0001; Figure 1A). Increased PWV was obvious in even the youngest participants with WS (see Figure S3A for raw WS data plotted against healthy population control means).14,18 In adults, paired t tests again showed higher PWV in WS adults (7.5±1.8 m/s, WS) versus controls (6.9±1.1 m/s, controls; P=0.02; Figure 1B).
PWV normally increases with age.7,18 To evaluate for possible differential effects of aging on PWV in WS, regression was performed using the full WS data set and matched controls (n=77 each). This analysis showed higher PWVs in participants with WS across the whole age distribution (P<0.0001 for elevation), with no statistical difference in the rate of PWV increase with age compared with controls (P=NS for slope; Figure S3B).
PWV: WS Clinical Phenotypes Associated With Vascular Stiffness
We sought to determine whether hypertension and diabetes mellitus, previously linked to increased vascular stiffness in non-Williams cohorts,7,23,24 were associated with higher PWV in WS. Regression analyses showed no significant relationship between PWV and hypertension (treated and untreated; Figure 2A) or diabetes mellitus (Figure S4). We did, however, see relative protection from increased PWV in WS individuals taking antihypertensives (P=0.001; Figure 2B). Of note, 58% (21/36) of those with PWV >1 m/s higher than the population mean for age and sex14,18 had no history of hypertension, whereas 36% (4/11) of those with reported untreated hypertension did not show elevated PWV.
Decreased NCF1 Gene CN Protects From Vascular Stiffness in WS
We investigated whether NCF1 CN was also associated with changes in vascular stiffness, as predicted by quantitative trait analysis in the Eln+/− mouse.25 Regression analysis showed higher PWVs in participants with WS who had ≥2 copies of NCF1 rather than 1 (P=0.05 for elevation; Figure 3). There was no statistical difference in the rate of PWV increase with age (P=NS for slope) in individuals with CN=1 or 2 in this analysis. Interestingly, removal of the single participant with the highest PWV (denoted by #) increases the difference in PWV observed between CN=1 and CN=2 individuals (P=0.005 for elevation), with a trend toward better protection from increasing PWV with older age (P for slope improves from 0.6 to 0.1; Figure S5).
NCF1 Gene CN and Hypertension in WS
To determine whether the deletion of NCF1 affected hypertension in our cohort, we compared the frequency of hypertension between WS individuals with 1 versus ≥2 NCF1 genes. In our total cohort of 99 individuals with WS in whom both NCF1 CN and hypertension status were known, a single copy of NCF1 was associated with protection from hypertension (P=0.03; Figure S6). This association persisted even when restricting the analysis to those aged <18 years (P=0.04; data not shown) in which the WS-related component of hypertension may be more dominant. These results confirm the previously reported protective association of reduced NCF1 CN and risk of hypertension in WS.8
In a developing blood vessel, collagen is responsible for tensile strength, whereas elastin provides recoil capability. When the elastin gene is mutated or deleted, the resulting elastin insufficiency leads to multiple cardiovascular abnormalities. Most consistently described in these populations are focal arterial stenoses and hypertension.5,6,26,27 However, the severity of the vascular features in WS is variable. The frequency of WS-associated vascular symptoms in WS-SAVE is similar to previous reports,6,28 with history of SVAS in 55% and hypertension in 40%. Importantly, only 25% of the cohort reported using antihypertensive medication for blood pressure control. Decision to treat was made by each individual’s primary medical team and may reflect overall population undertreatment or possible hesitancy to treat hypertension in syndromic individuals in whom high blood pressure measurements are often felt to represent anxiety in the medical setting. Interestingly, studies on this question have shown a paucity of true white coat hypertension in this population.28
Previous investigations of vascular stiffness in WS yielded contradictory results, ranging from increased arterial stiffness29,30 to normal or even paradoxically reduced values31,32 in small studies consisting of 3 to 29 participants. Consistent with these reports and with other cardiovascular features in WS, our larger analysis found considerable interpersonal PWV variability. However, even with this variability, individuals with WS had significantly stiffer vessels than matched controls. As in the Eln+/− mouse, this stiffness is apparent from the earliest ages studied20,33 and progresses with increasing age at the same rate as controls, suggesting that elastin insufficiency causes early-onset and possibly congenital arterial stiffness. Eln+/− mice generally become hypertensive in the neonatal period, a process suggested to be a physiological response to the altered vascular mechanics brought on by elastin insufficiency.21 In many of our cases, vascular stiffness was present without hypertension, and conversely, in some cases, hypertension was present without stiffness, suggesting that the 2 features may be related but independent effects of elastin insufficiency. For some of the studied individuals, increased PWV is the only known cardiovascular manifestation of the disorder. The development of hypertension in WS is likely multifactorial with influences due to vascular stiffness, complex effects of genetic background, and other features of WS such as renal artery stenosis and small-for-gestational-age birth.28
Our data show that vessels of pediatric participants with WS are more uniformly stiff than matched controls. Greater variability is seen in older participants with WS, with evidence of protection in those on antihypertensive medications. The mean PWV observed in pediatric participants with WS is 6.1 m/s, approaching that seen in predialysis youth with end-stage renal disease (6.6 m/s)11 and youth with type 2 diabetes mellitus (6.4 m/s12, both with similar age demographics). Interestingly, in the end stage renal disease study, no improvement resulted from hemodialysis, suggesting a final common pathway to these conditions ending in destruction of elastic matrices and lasting alteration of the biomechanical properties of the vessel wall.
The fact that we did not see a primary hypertension-by-PWV effect in regression analysis may result from an admixture of untreated hypertensive individuals (who generally have higher PWVs) with treated hypertensive WS participants (in whom our data show lower PWVs). This mixing may ultimately act to normalize the PWV for the hypertensive group, causing it to appear similar to the nonhypertensive subset. Further subgroup analysis, however, was underpowered. The use of antihypertensive medication showed greater protection in older individuals with WS, possibly suggesting cumulative effects of longer treatment duration; however, longitudinal evaluation is needed to confirm this.
Because vascular stiffness is strongly associated with negative cardiovascular consequences, such as myocardial infarction and stroke,1 further investigation into the predictive power of PWV for clinical outcome in individuals with WS is warranted. In our study, 48 participants were surveyed about their stroke history, and 3 of 48 (6%), aged 22, 32, and 32 years, reported a history of stroke. The prevalence of age-adjusted stroke for control individuals aged 18 to 45 years is 0.6% to 0.7%34 compared with 12% (3/25) when we consider only the subset of participants with WS in this demographics. In 2 of 3 WS participants with stroke, we obtained successful PWV measurements, and both were elevated (+0.6 and +1.4 m/s relative to age- or sex-matched controls), although both participants were receiving antihypertensive medications at the time of PWV measurement. Currently, the best estimate of risk of sudden death in WS is 25- to 100-fold increased relative to the general population.10 It is likely that this risk does not apply equally to all individuals with WS, and the use of PWV may enable individualized risk assessment. Full details about the nature of strokes in these participants are not available, and further investigation into this area is warranted.
Because of its status as a variably deleted gene in WS, NCF1 CN was investigated and found to be a significant modifier of arterial stiffness in WS. Data from the current cohort also confirm the reduced risk of hypertension associated with NCF1 CN=1 originally described by Del Campo et al.8 Although our study found generally lower PWVs in WS participants with only 1 copy of NCF1 relative to those with ≥2, initial analysis revealed no difference in the rate of age-associated increase in PWV between the 2 groups. However, when a single outlier is removed, a trend toward progressive protection with advancing age is identified in those with CN=1. Because NCF1 is a member of the NADPH oxidase family and is involved in the generation of reactive oxygen species in tissues,35 its association with severity of vascular stiffness suggests a role for chronic oxidative stress in the pathology of vascular stiffness in WS. Additional studies with focused recruitment of older individuals with WS, although difficult, may help clarify this intriguing finding.
Limitations of our study include the possibility that because of the topic of the study, parents of individuals with significant vascular disease were more interested in participating. However, our study includes many subjects with no previously known vascular features of WS (who, in fact, have high PWV). Second, challenges inherent to WS limited the number of successful PWV studies we were able to obtain. Individuals with WS have an average intelligence quotient of 55 and are often anxious, making it difficult to obtain resting measurements in younger children with WS. High body mass index, in combination with these features, additionally complicated PWV acquisition in some adults. Reassuringly, phenotypes of participants on whom we failed to obtain PWV trended toward more severe vascular disease with higher rates of hypertension and stenosis seen in this group. These findings suggest that it is unlikely that the data loss excluded a milder cohort that might skew the PWV results. Finally, our studies are merely a single snapshot in time. To investigate the interaction between vascular stiffness and hypertension in elastin insufficiency, additional larger prospective studies are needed to document the natural evolution of these phenotypes.
This study successfully evaluated vascular stiffness in the largest cohort of individuals with WS reported to date and demonstrates that individuals with WS, regardless of their blood pressure, are at risk for increased aortic stiffness, a well-established surrogate for adverse cardiovascular outcomes in many disease processes. This increase in vascular stiffness is caused by elastin insufficiency, as evidenced by Eln+/−mouse studies20–22 and, importantly, is modified by antihypertensive use and NCF1 CN. PWV differences are detectable at the earliest ages studied and continue into adulthood, making WS and elastin insufficiency useful models in which to study the long-term effects of chronic and early-onset vascular stiffness. Overall, this study suggests that (1) monitoring of vascular stiffness is warranted in WS; (2) antihypertensive treatment might protect against vascular stiffness and consequent adverse cardiovascular events in individuals with WS, even without overt hypertension. Consequently, at a minimum, antihypertensives should be considered in all WS individuals with hypertension; and finally, (3) novel medications, aimed at the NADPH oxidase pathway, should be investigated as potential modulators of vascular disease severity in this condition with use potentially determined by NCF1 CN. To generate formal treatment guidelines, larger prospective studies are needed to quantify the effects of antihypertensive drug choice and duration in treating arterial stiffness in WS. In addition, more work is needed to better understand the prognostic relevance of early-onset vascular stiffness in those with WS, with special attention given to organ systems known to be adversely affected in aging adults with vascular stiffness, such as the kidneys, heart, and brain. Finally, medication by NCF1 CN studies are needed to determine which patients may most benefit from pharmacological intervention.
We thank the Williams Syndrome Association and the families who participated in the WS-SAVE study. We gratefully acknowledge the assistance of Dr Chengsheng Zhang in clarifying the copy number status of genes within the Williams region for select patients, Dr Mark Johnson for review of Williams echocardiograms, and Dr Richard Feinn for recommendations on statistical tests. We thank Dr Carmel McEniery for sharing updated normative data from the Anglo-Cardiff Collaborative Trial14 and Dr Mark Levin for his helpful review of the article.
Sources of Funding
Funding was provided to Dr Kozel by the Children’s Discovery Institute of Washington University and St. Louis Children’s Hospital. In addition, Dr Kozel received funding for the study through her appointment as a scholar of the Child Health Research Center in Developmental Biology (National Institutes of Health [NIH] K12-HD01487) and the Genetic Basis of Inflammatory Airway Disease (NIH K12-HL089968). Original pediatric control studies were supported by the Hungarian National Research Fund, OTKA 100909.
The online-only Data Supplement is available with this article at http://hyper.ahajournals.org/lookup/suppl/doi:10.1161/HYPERTENSIONAHA.113.02087/-/DC1.
- Received July 22, 2013.
- Revision received August 11, 2013.
- Accepted September 24, 2013.
- © 2013 American Heart Association, Inc.
- Mecham RP
- Kozel BA,
- Mecham RP,
- Rosenbloom J
- Boutouyrie P,
- Vermeersch SJ
- Wessel A,
- Gravenhorst V,
- Buchhorn R,
- Gosch A,
- Partsch CJ,
- Pankau R
- Covic A,
- Mardare N,
- Gusbeth-Tatomir P,
- Brumaru O,
- Gavrilovici C,
- Munteanu M,
- Prisada O,
- Goldsmith DJ
- Wadwa RP,
- Urbina EM,
- Anderson AM,
- Hamman RF,
- Dolan LM,
- Rodriguez BL,
- Daniels SR,
- Dabelea D
- Roesler J,
- Curnutte JT,
- Rae J,
- Barrett D,
- Patino P,
- Chanock SJ,
- Goerlach A
- Heyworth PG,
- Noack D,
- Cross AR
- Reusz GS,
- Cseprekal O,
- Temmar M,
- Kis E,
- Cherif AB,
- Thaleb A,
- Fekete A,
- Szabó AJ,
- Benetos A,
- Salvi P
- Wagenseil JE,
- Ciliberto CH,
- Knutsen RH,
- Levy MA,
- Kovacs A,
- Mecham RP
- Kozel BA,
- Knutsen RH,
- Ye L,
- Ciliberto CH,
- Broekelmann TJ,
- Mecham RP
- Eronen M,
- Peippo M,
- Hiippala A,
- Raatikka M,
- Arvio M,
- Johansson R,
- Kähkönen M
- Aggoun Y,
- Sidi D,
- Levy BI,
- Lyonnet S,
- Kachaner J,
- Bonnet D
- Le VP,
- Knutsen RH,
- Mecham RP,
- Wagenseil JE
- Lassègue B,
- Griendling KK
Novelty and Significance
What Is New?
The Williams Syndrome (WS)-Skin And Vessel Elasticity study is the largest multi-institutional study of vascular stiffness in Williams syndrome to date, revealing increased pulse wave velocity in individuals with this rare condition.
What Is Relevant?
Increased pulse wave velocity was identified in even the youngest Williams participants, suggesting that vascular stiffness is of early, if not congenital, onset. Stiffness seems to be independent of hypertension in WS, making vascular stiffness a new primary WS phenotype. Protection against increasing stiffness is afforded by antihypertensive medication and also by deletion of NCF1, an NADPH oxidase component and gene variably deleted in WS.
Elastin insufficiency causes increased arterial stiffness, a phenotype associated with multiple negative cardiovascular outcomes complicating WS. Elevated pulse wave velocity is seen less often in those with WS deletions also removing the NCF1 gene and in those on antihypertensive therapy. Our findings suggest the need for enhanced evaluation and anticipatory treatment of vascular disease in Williams syndrome.