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Division of Endocrinology (J.L.M.O., C.M.-S., A.V.d.O.B., A.H.O.S., C.M.P., C.R.P.O., R.M.C.P., T.d.A.R.V, C.T.F., M.H.A-.O.), Federal University of Sergipe, Aracaju, 49060-100 Sergipe, Brazil; São Lucas Hospital (J.A.B.-F., R.X.F.), Aracaju, 49085-310 Sergipe, Brazil; and Division of Endocrinology (R.S.), Johns Hopkins University School of Medicine, Baltimore Maryland 21287
Address all correspondence and requests for reprints to: Roberto Salvatori, M.D., Division of Endocrinology, Johns Hopkins University, 1830 East Monument Street, #333, Baltimore, Maryland 21287. E-mail: salvator{at}jhmi.edu.
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Abstract |
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Objective: The aim of this study was to determine the consequences of lifetime isolated GHD (IGHD) on the metabolic and cardiovascular status of adult members of a large Brazilian cohort with severe IGHD due to a homozygous mutation in the GHRH receptor gene.
Design: Twenty-two GH naive adult dwarfs (10 men and 12 women; aged 44 ± 12 yr) were compared with 22 healthy volunteers (10 men and 12 women; aged 45 ± 12 yr) living in the same area.
Results: GHD subjects had increased abdominal obesity, higher total and low-density lipoprotein cholesterol, and higher C-reactive protein than controls. They did not have an increase in carotid wall thickness, and there was no evidence of premature atherosclerosis as evaluated by exercise echocardiography.
Conclusions: In this homogeneous cohort residing in a rural area of Brazil, lifetime, untreated severe IGHD is not associated with evidence of premature atherosclerosis despite unfavorable cardiovas- cular risk profile.
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Introduction |
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We have identified a large extended pedigree with approximately 100 individuals (over several generations) affected by familial IGHD, residing in Itabaianinha County, in the northeastern Brazilian state of Sergipe. They carry a homozygous null mutation in the GHRH receptor (GHRHR) gene. This is the largest cohort of patients with IGHD described to date. The adult patients have proportionate dwarfism (–4 to –8 SD score for height) and otherwise normal pituitary function. The vast majority of the adult patients have never received GH replacement therapy. Their serum IGF-I levels are lower than the ones reported to be associated with increased risk of cardiovascular disease (12, 13, 14, 15, 16, 17). Therefore, this is an ideal population to define the impact of severe, long-term IGHD on cardiovascular pathophysiology. We demonstrated previously that adult patients from this cohort present a cluster of atherosclerotic cardiovascular risk factors characterized by central obesity, increase in systolic blood pressure (SBP), and elevated levels of total and LDL-C (3). In this study, we test the hypothesis that the severe and untreated GHD status is associated with premature atherosclerosis (18).
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Subjects and Methods |
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Twenty-two GH naive adult patients with genotype-proven homozygous GHRHR mutation (10 men and 12 women; aged 44 ± 12 yr, range of 25.2–66.1 yr; 91% >30 yr old) (GHD group) and 22 normal, healthy, nonobese volunteers homozygous for the wild-type GHRHR allele (10 men and 12 women; aged 45 ± 12 yr, range of 21.3–74.8 yr; 95% >30 yr old) [control (CO) group], all living in Itabaianinha County, were studied. Normal GH secretory status of the CO group was confirmed by measurement of serum IGF-I level as reported previously (12). Patients were recruited through the local Dwarfs Association after a detailed explanation of the protocol. CO subjects were recruited by an advertisement placed in the local community. None of subjects had any evidence, based on medical history, of active cardiovascular or other systemic diseases. Eight of the GHD patients (36%) were receiving captopril therapy for hypertension and had been instructed previously to follow a reduced-salt diet by their treating physicians. None of the CO subjects had hypertension. Both the University of Sergipe and the Johns Hopkins University Institutional Review Boards approved these studies, and all subjects gave written informed consent.
Laboratory assessment
All subjects were admitted to the clinical research center at approximately 0800 h after an overnight fast. Blood samples to measure total cholesterol, LDL-C, high-density lipoprotein cholesterol (HDL-C), triglycerides, glucose, insulin, IGF-I, and CRP were collected. Total cholesterol level was determined by the CHOD-PAP method (Roche Diagnostics, Mannheim, Germany), and the LDL-C concentration was calculated indirectly (Friedwald formula). The HDL-C was separated using the phosphotungstic acid/magnesium chloride method. Glucose and triglycerides were measured by the enzymatic colorimetric test (GPO-PAP method; Roche Diagnostics). Insulin was measured by the RIA (Diagnostic Systems Laboratories, Webster, TX); IGF-I was measured by the immunoradiometric assay-5600, with double extraction and an assay sensitivity of 0.8 ng/ml (Diagnostic Systems Laboratories). Insulin resistance was estimated using the homeostasis model assessment of insulin resistance (HOMAIR), with the following formula: fasting serum insulin (µU/ml) x fasting plasma glucose (mmol/liter)/22.5 (19). A highly sensitive latex-based immunoassay was used to determine the level of CRP in blood. The cardiovascular risk was estimated by the Framingham score based on age, blood pressure, total cholesterol, HDL-C, diabetes, and smoking (20).
Anthropometric measurements
After blood drawing, the subjects had a light breakfast without coffee, and anthropometric measurement was performed. SD score height for age (SDS h/a) was calculated by using the National Center for Health Statistics (http://www.cdc.gov/nchs) data. Body mass index (BMI) was calculated by dividing body weight (in kilograms) by the square of the height (square meters), and the percentage of fat mass was measured using the near infrared interactance method (21).
Assessment of carotid intima-media thickness
Longitudinal ultrasonographic scans of the carotids artery were all performed by the same trained observer (R.X.F.). An ATL Ultramark (Advanced Technology Laboratories, Bothell, WA) equipped with a 10.0 MHz linear-array transducer was used. Subjects were examined in the supine position and the neck in slight hyperextension. The far wall of the distal 1.0 cm of both common carotid arteries immediately proximal to the origin of the bifurcation was studied (22). In each examination, the sonographer used different scanning angles (anterior and posterior) to identify the greatest intima-media thickness (IMT), defined as the distance between the junction of the lumen and intima and that of the media and adventitia. Three measurements of IMT were obtained on the right and left carotid arteries and averaged. In addition, both the right and left common, internal, and external carotid arteries were examined in multiple projections to identify the presence of atherosclerotic plaques, defined as an isolated focal thickening of the intima-media layer with a thickness of 1.3 mm. The statistical power of detecting IMT changes of similar degree to the ones observed by other authors in adult GHD subjects (23) that is provided by a sample size of 22 subjects per group is more than 0.999.
Resting echocardiography
Echocardiographic studies were performed with a commercial machine (HP-Sonos 5500; Hewlett Packard, Palo Alto, CA) according to standard procedures. M-mode echocardiography of the LV was performed according to the American Society of Echocardiography recommendations. Only frames with optimal visualization of interfaces and simultaneously visible septum, LV internal diameters, and posterior wall were used for calculations. LV mass was calculated according to Devereux et al. (49) and normalized according to body surface area and height. Relative wall thickness was calculated as [2 x posterior wall thickness]/LV internal radius.
Exercise echocardiography
The pre-exercise standing SPB, diastolic blood pressure (DBP), and heart rate (HR) were measured just before the exercise test. Patients underwent symptom-limited treadmill exercise testing according to the standard Bruce protocol. Two-dimensional echocardiographic images were obtained from the parasternal and apical windows before and immediately after exercise. Both digitized and VHS recorded images were used for interpretation of the studies. Regional wall motion was assessed semiquantitatively by the same experienced echocardiographer (J.L.M.O.). Wall motion at rest and with exercise was scored on a five-point scale: 1, normal; 2, mild hypokinesis (decrease of movement and systolic thickening); 3, severe hypokinesis; 4, akinesis (absence of movement and systolic thinning); and 5, dyskinesis (paradoxical outward movement and possible systolic thinning) according to a 16- segment model. Wall motion score index was determined at rest and peak exercise as sum of the segmental scores divided by the number of visualized segments (24). The development of new or worsening wall motion was considered indicative of myocardial ischemia. A wall motion abnormality present at rest and unchanged with exercise was classified as "fixed." Exercise echocardiography results were defined as abnormal if there was ischemia or fixed wall motion abnormalities. The exercise electrocardiogram (ECG) was considered positive for ischemia if there was horizontal or down-sloping ST segment depression of 1 mm at 80 msec after the J point, nondiagnostic if the baseline ST segment was abnormal, and negative for ischemia in the absence of these criteria (25).
Age distribution of GHD individuals
We previously identified 71 living GHD subjects spanning several generations of an extended pedigree of 1570 individuals. We were able to determine the exact ages of 1205 normal-stature people and 63 GHD individuals from this extended pedigree who currently reside in Itabaianinha County, and we stratified them according to age. In addition, we obtained oral information about the cause of death of 28 dwarfs from their family members.
Statistical analysis
Statistical analysis was performed using the statistical software SPSS/PC 11.5. Values are expressed in mean ± SD, and the Mann-Whitney U test was used because of the skewness of IGF-I and height distributions in the GHD group. Probability values less than or equal to 0.05 were considered statistically significant.
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Results |
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As shown in Table 1
, age, sex, and BMI did not differ between groups. As expected, height, weight, SDS h/a, and serum IGF-I levels were significantly lower in the GHD group (Fig. 1).
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The percentage of fat mass was higher in the GHD group (Table 1
). Total and LDL-C were also higher in the GHD group compared with the CO group, but HDL-C and triglycerides did not differ when comparing the groups. Fasting glucose, insulin, HOMAIR, and Framingham risk score were similar in the two groups (Table 1). CRP levels were significantly higher in the GHD group than in the CO group (Fig. 1).
Carotid IMT
Carotid IMT was similar in the GHD and CO groups (Fig. 1). A single plaque in the carotid artery was present in one GHD woman, aged 47 yr.
Resting echocardiography
Septal wall thickness, posterior wall thickness, LV mass index, and end-diastolic diameter were all significantly lower in the GHD group than in the CO group; relative wall thickness did not differ between groups (Fig. 1 and Table 2). To evaluate systolic function, we measured ejection fraction and fractional shortening. To assess diastolic function, peak velocity in early diastole to late diastolic flow velocity and deceleration time were measured. All of these values were similar between groups, indicative of normal resting systolic and diastolic function.
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During the Bruce protocol, no symptoms or ECG changes suggestive of myocardial ischemia were observed. The pre-exercise standing SBP, DBP, and HR were similar in the two groups. The increase in SBP was greater in the CO group than in the GHD group (P = 0.03). The magnitude of DBP and HR responses to treadmill exercise did not differ between the groups.
The exercise echocardiogram was normal in both groups. Rest and/or exercise wall motion score index did not detect any abnormality (Table 3).
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Data on age distribution are shown in Table 4. There is no evidence that the GHD subjects have reduced life expectancy compared with normal-stature members of the same pedigree. Reported death causes in GHD individuals were "natural death" (16 individuals, aged 72–80 yr), dehydration due to diarrheal diseases (six individuals, all aged <4 yr), accident (two individuals, 26 and 34 yr old), murder (two individuals, 28 and 32 yr old), sudden death (two individuals, 27 and 75 yr old), and cancer (one individual, 57 yr old). Official death certificates were not available.
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Discussion |
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Previously, we demonstrated that children, adolescents, and adult GHD individuals of the Itabaianinha kindred present the typical cluster of metabolic alterations, such as increase in fat mass, waist-to-hip ratio, total cholesterol, and LDL-C (1, 2, 3). We also reported that adult patients present an increase in SBP (3). For all of these reasons, it would be plausible to expect this population to be prone to premature atherosclerosis. Despite the increase in blood pressure and obesity, GHD subjects do not have cardiac hypertrophy. We speculate that the very low levels of IGF-I may counterbalance the effects of hypertension and obesity in the development of LV hypertrophy. Indeed, the GH/IGF-I axis contributes to the variability of LV mass associated with hypertension (37), and acromegaly causes LV hypertrophy (38).
In the present study, we found that, despite the dysmetabolic and proinflammatory profile (high LDL-C and total cholesterol and high CRP), the higher prevalence of treated hypertension (8 of 22 GHD vs. 0 of 22 in CO) and the increase in fat mass, GHD subjects have no evidence of premature atherosclerosis, as evaluated by carotid sonography and resting and exercise echocardiography. IMT was no different between GHD and CO subjects. None of the echocardiography parameters were different between GHD and CO subjects, with the exception of reduced increase in SBP in GHD subjects, likely due to the antihypertensive therapy that eight of them were receiving. None of the GHD or CO subjects had evidence of exercise-induced ischemia. Although we used an indirect method to access myocardial ischemia, the sensitivity of stress echocardiography for coronary artery disease is quite high (88%) (39). The lack of intima-media thickening is surprising, because several studies have shown an increase in IMT in non-GH-treated adult patients with acquired hypopituitarism and reported its normalization with GH therapy (23, 35). Another interesting difference between our subjects and subjects with adult-onset GHD is that the Itabaianinha dwarfs have normal HDL-C and no evidence of insulin resistance, whereas many (but not all) studies have shown that adults with acquired GHD have low HDL and decreased insulin sensitivity (40). The reasons for such discrepancies are not clear. Several differences exist between our GHD subjects and hypopituitaric GHD patients studied by other investigators. In addition to having an otherwise normal pituitary function, our subjects have serum IGF-I levels that are much lower than what is observed in patients with acquired hypopituitarism. In addition, our GHD individuals were never exposed to GH and, therefore, differ from patients with acquired GHD, who have normal GH secretion for a large part of their lives.
Epidemiological studies have suggested that reduced levels of IGF-I are associated with increased risk of cardiovascular mortality and ischemic heart disease in the general population (15, 16). Conversely, a recent study performed in 330 Japanese men suggested that increased levels of IGF-I or IGF binding protein-3 may accelerate atherosclerosis (41). IGF-I is a key peptide in regulating vascular physiology. Through both endocrine and autocrine/paracrine mechanisms, IGF-I modulates multiple physiological functions of the vasculature, including proliferation and migration of vascular smooth muscle cells (VSMCs), increase of cell survival after an ischemic insult, and inhibition of monocyte and VSMC apoptosis (42, 43). It has therefore been suggested that IGF-I may have a dual role in atherosclerosis pathophysiology: it may promote atherogenesis by increasing VSMC proliferation and migration or protect against it by increasing nitric oxide production, vascular compliance, and insulin sensitivity. The final effect of IGF-I on the vascular wall would be the net effect of the multiple atherogenic and antiatherogenic functions of this peptide. It is possible that different degrees of IGF-I deficiency may result in different end effects on the vascular wall. It is possible that the very severe IGF-I reduction may be protective against atherosclerosis, although a less dramatic reduction may be harmful.
In agreement with the lack of evidence of premature atherosclerosis, our age distribution data and data on reported causes of death, albeit limited, do not suggest reduced life expectancy (or peculiar pattern of mortality) in the GHD population when compared with normal-stature members of the same extended kindred. Our data are in accordance with a study of 1014 subjects with hypopituitarism, in which the excess respiratory and cardiovascular mortality did not seem to be associated with GHD. In this study, the only mortality-associated endocrine-axis deficiency was the untreated gonadotropin deficiency (44). The gonadal axis is normal in our GHD subjects.
One important limitation of our study is the relatively young age of our study subjects, who may have not yet manifest the consequences of risk factors. Nevertheless, 91% of our GHD group is older than 30 yr. They are exposed to cardiovascular risk at least since 5 yr of age (2). We do not know whether subjects from the same area with similar risk factors but no GHD have premature atherosclerosis. However, a recent study from the Bogalusa cohort has shown an increase in IMT in young patients (mean age of 32 yr) with metabolic syndrome (45). In addition, another study from the same cohort has shown that, in adults aged 25–37 yr residing in a semirural community, LDL-C level and BMI predicted IMT, demonstrating that premature atherosclerosis can be detected at a young age (46). We would therefore have expected to find increased IMT in subjects exposed throughout the life to a cluster of cardiovascular risk factors.
Another limitation is the specific geographic area and genetic homogeneity of this population. All of the subjects studied here come from Itabaianinha County in the northeast of Brazil, and one may argue that such population might have a lower prevalence of cardiovascular disease than more developed populations. To obtain jobs, most of the study subjects moved from the countryside to the municipal seat. They are in the process of transitioning to a more sedentary lifestyle. Moreover, the concept of atherosclerosis as a disease of developed countries is no longer accepted. The global burden of cardiovascular disease is increasing more rapidly in developing countries than in Western countries (47). In Brazil, based on the World Health Organization report, cardiovascular mortality in the general population in the year 2000 was higher than in the United States (48). Actualized data from DATASUS, the Brazilian health information system, has shown that, despite the wide differences in income and living conditions, in the entire country the leading cause of morbidity and mortality is cardiovascular disease.
Finally, the uniqueness of our model needs to be kept in mind in interpreting our results, because it may limit the applicability of our results to the general population. Our subjects belong to a highly inbred kindred (
25% of unions are consanguineous) (12). It is conceivable that they may share other genetic traits that may reduce their atherosclerosis risk. Future studies aimed at determining the prevalence of atherosclerosis in non-GHD members of the Itabaianinha kindred will be needed to clarify this point.
In conclusion, our data show that, in a homogeneous cohort of patients with a homozygous GHRHR mutation, severe, lifetime, untreated GHD is associated with a cluster of cardiovascular risk factors that could potentially promote premature atherosclerosis. However, we did not find evidence of increase in carotid IMT or premature myocardial ischemia in adult GH naive GHD subjects. Extremely low and lifetime (vs. moderately decreased and acquired later in life) serum IGF-I differentiate our patients from adults with acquired GHD. It is possible that acquired GHD may have a more dramatic impact on atherosclerosis than congenital GHD. Our data do not support the previous hypothesis that GHD causes premature atherosclerosis.
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Acknowledgments |
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Footnotes |
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R.S. consults for Teva Inc. and Pfizer Inc. and received lecture fees from Genentech. All other authors have nothing to declare.
First Published Online March 7, 2006
Abbreviations: BMI, Body mass index; CO, control; CRP, C-reactive protein; DBP, diastolic blood pressure; ECG, electrocardiogram; GHD, GH deficiency, GH deficient; GHRHR, GHRH receptor; HDL-C, high-density lipoprotein cholesterol; HOMAIR, homeostasis model assessment of insulin resistance; HR, heart rate; IGHD, isolated GHD; IMT, intima-media thickness; LDL-C, low-density lipoprotein cholesterol; LV, left ventricle; SBP, systolic blood pressure; SDS h/a, SD score height for age; VSMC, vascular smooth muscle cell.
Received November 28, 2005.
Accepted February 28, 2006.
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