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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. Disclosure
  8. REFERENCES

The endothelial protein C receptor (EPCR) has a critical role in the regulation of anticoagulant and anti-inflammatory functions of activated protein C (APC). Abnormalities in EPCR might be associated with an increased risk of thrombosis. In this respect, a 23 bp insertion in the exon 3 of the EPCR gene predicts a truncated protein which cannot bind APC. High levels of C-reactive protein (CRP), a strong predictor of cardiovascular events, are found both in the obese and in subjects with Prader-Willi syndrome (PWS). Several cardiovascular risk factors are already present in prepubertal PWS children, but it is uncertain which mechanism contributes to the increased risk of cardiovascular disease in PWS. We analyzed the distribution of 23 bp insertion in the EPCR gene in 81 overweight and obese PWS subjects, 52 adults and 29 children, and in 58 overweight and obese children and adolescents (controls). We found that 1/58 (1.7%) of the controls was heterozygous for the 23 bp insertion, while this mutation was never found in PWS subjects. Furthermore, we evaluated CRP levels, glucose, insulin, and lipid profile, and we found higher CRP values in PWS adults with respect to children with PWS and controls, and a better insulin sensitivity in all PWS subjects than in the controls. This study suggests that in PWS subjects there is no predisposition to develop thrombotic events in association with EPCR gene alteration and demonstrates substantial differences regarding metabolic and inflammatory profile between PWS and non-PWS obese children, with further impairment in adults with PWS.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. Disclosure
  8. REFERENCES

Activated protein C (APC) and its receptor, the endothelial protein C receptor (EPCR), represent the most important anticoagulant system in regulation of inflammation (1,2) and a major physiological control system of the vascular wall permeability during sepsis (3). The EPCR is a type 1 transmembrane glycoprotein highly expressed on the endothelium of large vessels, especially in arteries (4,5), which enhances the protein C activation by thrombin/thrombomodulin complex (4,6). Because of the important role of EPCR in the protein C anticoagulant pathway, it is possible to hypothesize that any functional mutation will lead to increased risk of thrombosis and hyperinflammatory response (7). It has been demonstrated that a 23 bp insertion in the exon 3 of the EPCR gene, which is located in 20q11.2 chromosome region, predicts a truncated protein that lacks part of the extracellular domain, and can neither bind APC nor be expressed on cell surface (8,9). This abnormality of the EPCR gene has been found in adult subjects with deep vein thrombosis (1.5%) and myocardial infarction (2%) (9), and in pediatric thrombotic cases (2.36%) (10). An homozygous 23 bp insertion of the EPCR gene may be associated with a tendency to sepsis and poor outcome in children(11). High levels of C-reactive protein (CRP), the main acute phase protein, have been demonstrated to be strong predictors of future cardiovascular events in subjects both with and without overt cardiovascular disease. Increased CRP serum levels have also been significantly correlated with weight excess, and overweight and obesity represent a strong risk factor for premature cardiovascular diseases as well as for recurrent venous thromboembolism (12,13,14). High levels of CRP have been also demonstrated in subjects with Prader-Willi syndrome (PWS) (15), a rare genetic obesity syndrome which has distinct physical, endocrine, and metabolic characteristics that are not common to severe obesity itself. Total fat mass is increased but unlike in obesity, visceral fat deposits are reduced (16) and insulin sensitivity is higher than in the obese (17). Regardless of their metabolic advantages, however, PWS patients are at risk of premature death (18,19). Complications conventionally related to obesity, including cardiovascular disease and respiratory insufficiency, are recognized as the main risk factors for sudden death during the life-span of patients with PWS (20,21). Nevertheless, it is not clear which components related to obesity may contribute to the increased risk of cardiovascular disease. In this context, evidence of microcirculatory dysfunctions as well as a trend toward increased mean intima—media thickness have been reported in young PWS adults (22). Moreover, several cardiovascular risk factors, such as abnormal levels of low-density lipoprotein cholesterol (LDL-C), apolipoprotein B, high-density lipoprotein cholesterol (HDL-C), triglycerides (TG), and lipoprotein (a), are already present in prepubertal children with PWS (23). All these findings are potentially associated with coronary artery disease and thus early sudden death. To date, however, only few cases of coronary artery and atherosclerotic heart diseases have been reported in PWS subjects (24,25,26), but this circumstance may be simply related to the early mortality of PWS, since ischemic heart disease is generally observed after the middle age. In addition, both superficial and deep venous thrombosis are frequently observed in PWS, particularly in patients who have stopped to walk for any reason (27).

The aim of this study was to analyze the 23 bp insertion in the exon 3 of the EPCR gene in a cohort of PWS adults and children with PWS, as well as in a control group of obese and overweight children, to evaluate the frequency of this alteration in these two types of obesity. In addition, we investigated the predisposition to develop thrombotic events in association with biochemical and inflammatory profile of PWS children in comparison to those obtained in the control group. Finally, measurement of the metabolic and inflammatory markers was performed in the group of PWS adults, to evaluate the development of these risk factors in different ages.

Methods and Procedures

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. Disclosure
  8. REFERENCES

Subjects

Eighty-one subjects with PWS, 52 adults (34 females, mean age 28.9 ± 0.8 years) and 29 children with PWS (16 females, mean age 10.4 ± 0.7 years) were included in this study (Table 1). All patients showed the typical PWS clinical phenotype (28).

Table 1.  Clinical and biochemical features of the PWS and control subjects
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Concerning genetic abnormalities distribution, cytogenetic analysis was performed in all subjects and 54 of them had interstitial deletion of the proximal long arm of chromosome 15 (del15q11-q13). Twenty-five subjects presented uniparental maternal disomy for chromosome 15 (UPD15), one patient had an imprinting defect, while a positive methylation test (meth+) was demonstrated in the remaining individual but the underlying genetic defect was not identified. Forty PWS subjects (17 adults) were treated with growth hormone from at least 6 months at the moment of the study.

Physical examination included determination of height and weight in fasting conditions and after voiding. Standing height was determined by a wall-mounted Harpenden Stadiometer (Zamboni, Bologna, Italy). Weight measure was performed with subjects in minimal clothes, with a scale accurate to the nearest 0.1 kg. BMI was defined as weight in kilograms divided by the square of height in meters. The published international standards for sex-and age-specific BMI centiles were used for subjects aged 2–18 years (29). We defined as overweight and obese those patients with a BMI more than the centile curves, that at age 18 passed through the cut-off point of 25 and 30 for adult overweight and obesity, respectively. In addition, BMI standard deviation score (SDS) of subjects younger than 18 years was derived from the published Center for Disease Control and Prevention (CDC) standards (30). Finally, the BMI cut-off points of 25–30 to define overweight and >30 to define obesity were used for individuals >18 years of age. According to the BMI cut-off points, 18 PWS subjects were overweight (6 adults) and 63 PWS patients were obese (46 adults).

As controls, we evaluated a group of 58 children and adolescents, matched to children with PWS for gender, age, and BMI SDS (Table 1), with secondary causes of obesity excluded.

Mean BMI was 44.1 ± 1.7 kg/m2 (range 25.3–76.1) in PWS adults while BMI SDS ranged from 0.82 to 4.92 in children with PWS (mean ± s.e.: 2.36 ± 0.19) and from 1.60 to 5.25 in control subjects (mean ± s.e.: 2.60 ± 0.07).

The entire study protocol was approved by the ad hoc ethical committee of the Istituto Auxologico Italiano Foundation. Written informed consent was obtained from patients or from their parents, when necessary.

Methods

After an overnight fast, blood samples were taken in PWS and obese subjects to obtain serum and plasma. Blood glucose, total cholesterol, HDL-C, LDL-C, and TG concentrations were measured using an automated analyzer (Roche Diagnostics, Mannheim, Germany). Serum insulin was measured by a chemiluminescent assay (DPC, Los Angeles, CA). Insulin resistance was measured by homeostasis model assessment (HOMA-index), calculated as insulin (µU/ml) × blood glucose (mmol/l)/ 22.5. CRP levels were measured by an ultrasensitive immunoturbidimetric assay (CRP Latex HS; Roche Diagnostics).

DNA of all subjects was extracted from whole blood using QIAamp DNA Blood Midi Kit (Qiagen, Hilden, Germany). Exon 3 and intron/exon boundaries were amplified using a forward primer (5′-ctctctgcacagtcccctga-3′) and a reverse primer (5′-aattcccgcagttcataccg-3′) designed to amplify a region of 260 bp. Amplification was performed for 35 cycles with an annealing temperature of 54°C. The amplification product was evaluated by running on 3% agarose gel. A wild-type control was included in each run (Figure 1a).

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Figure 1. Insertion of 23 bp in the EPCR gene. (a) PCR amplification of the EPCR gene exon 3 in a wild-type subject (lane 1) and in the obese subject showing the 23 bp insertion (lane 2). (b) Electropherogram of the EPCR gene exon 3 of the obese with a 23 bp insertion.

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To verify the presence of insertion, the two DNA bands visible on Tris/Borate/EDTA (TBE) agarose gels were excised and purified with Quantum Prep Freeze 'N Squeeze DNA Gel Extraction Spin Columns (Bio-Rad, Hercules, CA). The recovered DNA was used for PCR and direct sequencing (Figure 1b).

Direct sequencing was performed after purifying PCR products with QIAquick PCR purification kit (QIAGEN-D40724; Qiagen). Both strands were sequenced using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA) and analyzed on an ABI PRISM 310 automated sequencer (Applied Biosystems).

Statistical analysis

Data are expressed as mean ± s.e., Statistical analysis was performed by t-test for unpaired data, and using analysis of variance for parametric and nonparametric data (Mann—Whitney test), where appropriate. Significance was set at P < 0.05.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. Disclosure
  8. REFERENCES

The analysis of the EPCR gene showed that 1/58 (1.7%) of obese subjects was heterozygous for the 23 bp insertion (Figure 1b). This mutation was never found in the group of PWS subjects.

The main clinical and biochemical features of the patients are summarized in Table 1. Children with PWS were compared with controls. BMI SDS was similar between children and adolescents with PWS and control subjects. Pediatric PWS subjects showed lower glucose, insulin, HOMA-index, and CRP levels and higher HDL-C values with respect to controls (P < 0.0001). No differences were present between children with PWS and controls for total cholesterol, LDL-C, and TGs.

As expected, PWS adults showed higher glucose, triglycerides, LDL-C, and CRP levels as well as lower HDL-C values than children with PWS (P < 0.005; P < 0.0001, P < 0.02, P < 0.0001, and P < 0.02, respectively). On the contrary, insulin levels and HOMA-index were similar in the two PWS groups.

Concerning metabolic parameters, no significant difference was detected between PWS patients with and without growth hormone therapy, both in children and in adults (data not shown). Similarly, CRP levels were similar in treated or untreated children (0.39 ± 0.16 mg/l vs. 0.81 ± 0.36 mg/l, respectively; P = 0.2) and adults (1.00 ± 0.24 mg/l vs. 1.69 ± 0.24 mg/l, respectively; P = 0.08) with PWS. No gender difference in the metabolic profile and CRP levels was detected in PWS subjects, with the exception of higher total cholesterol values in females than in males in the adult cohort (180.5 ± 5.9 mg/dl vs. 156.2 ± 7.2 mg/dl, respectively; P < 0.02).

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. Disclosure
  8. REFERENCES

To our knowledge, this is the first study to examine the 23 bp insertion in exon 3 of the EPCR gene in PWS subjects and in obese and overweight children and adolescents. We have found that this mutation is absent in a large number of PWS, including adults and children with PWS. On the other hand, our results demonstrate that in childhood obesity the 23 bp EPCR gene alteration has a frequency similar to other diseases (9,10,11). Altogether, our data suggest that no predisposition to develop thrombotic events in association with EPCR gene alteration is observed in PWS subjects. Complications associated with obesity are recognized as the main risk factors for death during the life-span of patients with PWS, particularly respiratory insufficiency and cardiovascular disease (21,31). However, PWS is associated with a healthier metabolic profile, probably correlated to the lower amount of visceral fat and the higher insulin sensitivity than in patients with simple obesity (14,15). Consequently, other mechanisms may be involved in the cardiovascular complications of these patients, including inflammation. In this context, it has been recently demonstrated that PWS is associated with increased activation of the innate immune system compared with simple obesity, which may be either a specific genetic feature of PWS or linked to the commonly associated obstructive sleep apnea syndrome (32). By contrast, high serum levels of CRP have been found in both PWS and obese adult subjects suggesting that inflammation might result in a similar increased risk of cardiovascular morbidity and mortality in both diseases (22,33). In addition, a recent study has shown that a low-grade systemic inflammation is more evident in obese adults with PWS than in obese adults without PWS having a similar level of insulin resistance and insulin-like growth factor-1 (IGF-1) plasma concentration (33). However, inflammation seems to be less related to obesity in PWS as demonstrated by normal levels of tumor necrosis factor-α (TNF-α), a cytokine secreted by visceral adipose tissue, found in PWS subjects (34).

In this study, we have confirmed that children and adolescents with PWS have a better metabolic profile than that one observed in control subjects. However, our results partly vary from what recently reported in the literature, derived from different groups of subjects, showing that young obese PWS presented a frequency of metabolic syndrome and its components very close to those of obese controls (35). The cause of this discrepancy may be related to the different degree of obesity of our PWS and control subjects, given that the current study included both overweight and obese subjects.

Differently from what observed in adult patients (22,33), we have found that children with PWS had lower CRP levels than control group. The finding of low CRP levels in our cohort of PWS subjects is not in accordance with previous data which demonstrated the presence of similar level of CRP in PWS children and obese controls (15). The reason for these discordant results could be related to the small number of subjects taken into consideration by these authors (nine PWS and three obese). Moreover, we found that adult PWS had high levels of CRP in respect to PWS children and adolescents. Thus, it is conceivable that the increased risk of cardiovascular disease due to the inflammatory state linked to obesity becomes more important with the aging PWS population. Although there is no close relationship between the EPCR gene abnormalities and CRP, it has been demonstrated that high levels of this protein can exert a downregulation of the EPCR receptor in a time and concentration-dependent manner, contributing to the prothrombotic effect (36,37).

As expected, children with PWS had a healthier metabolic profile in respect to adult patients with PWS, with the exception of similar insulin levels and HOMA-index.

These findings confirm that PWS subjects are more insulin sensitive than subjects affected by nongenetic obesity (20), supporting the hypothesis that a lower risk for insulin resistance than would be expected based on the degree of overweight could be a characteristic of the whole PWS population.

In conclusion, this study did not reveal any alteration in the exon 3 of the EPCR gene in a large group of PWS, suggesting a lack of relationship between cardiovascular disease and protein C activation capacity. We have demonstrated, however, that there are considerable differences regarding the metabolic profile and CRP levels between overweight and obese PWS children and those with nongenetic weight excess. Furthermore, we have found that the inflammatory state in children with PWS and PWS adults is significantly different and becomes more evident with age. Altogether, these findings seem to indicate that chronic inflammation might be a contributing factor to the raised morbidity and mortality of cardiovascular origin in adult patients with PWS.

REFERENCES

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. Disclosure
  8. REFERENCES