Abstract
Aims:
Since desmin expression is diminished in vascular smooth muscle cells during reparative processes, we wanted to study whether a common intragenic single nucleotide polymorphism at nucleotide position 828 (rs1058261) of the DES gene associates with hypertension, cerebrovascular complications, and all cardiovascular events in the Tampere adult population cardiovascular risk (TAMRISK) study.
Materials and Methods:
A Finnish periodic health examination cohort of 336 subjects with diagnosed hypertension and 473 controls were analyzed. Samples were genotyped for polymorphism using TaqMan techniques. Prevalence of ischemic heart diseases, incidence of cerebrovascular diseases, and transient cerebral ischemic attacks (TIAs) were obtained by self-report and the National Hospital Discharge Registry (HILMO).
Results:
There was no association of any of the rs1058261 genotypes with hypertension at the age of 50. When the subjects were followed to the age of 60, after adjustment for gender and body mass index, subjects with the genotype CC had higher incidence of cerebrovascular events (cerebrovascular diseases and TIA) (4.1%) compared with the T allele (1.6%) (p = 0.046). In addition, those with CC genotype had a higher incidence of all combined cardiovascular events (12.8%) compared with subjects with the T allele (8.5%) (p = 0.028).
Conclusions:
Our findings suggest that variations in the DES gene may be involved in cardiovascular disease.
Introduction
D
Desmin is encoded by the DES gene located on chromosome 2q35. DES encompasses nine exons within an 8.4-kb region and codes for 476 amino acids (Li et al., 1989). Since recognition of the involvement of the DES gene in human disease in 1998 (Goldfarb et al., 1998), the number of publications on different DES mutations and associated phenotypes has increased steadily (van Spaendonck-Zwarts et al., 2011). We wanted to study whether a common intragenic single nucleotide polymorphism (SNP) at nucleotide position 828 (rs1058261) of the DES gene (Fichna et al., 2014) associates with hypertension, cerebrovascular complications, and all combined cardiovascular events in a Finnish population from the Tampere adult population cardiovascular risk (TAMRISK) study.
Materials and Methods
Subjects
TAMRISK study data were collected from periodic health examinations (PHEs) done for 50-year-old men and women living in Tampere, a city in southern Finland with 220,000 inhabitants (Maatta et al., 2015). At the PHEs in 2003 by a public health nurse, height (cm) and weight (kg) were recorded from which the body mass index (BMI) was calculated. Blood pressure measurement (mmHg) was done using a calibrated mercury sphygmomanometer. Serum glucose (mmoles/L), total cholesterol, HDL-cholesterol, and triglycerides (from which LDL-cholesterol was calculated by the Friedewald formula) were measured after an overnight fast by standard techniques. An interview was conducted using a structured questionnaire about health and health-related behavior. Buccal swabs for DNA extraction and a permission form to use PHE information and national registry data were collected by mail separately from the physical examination during years 2006-2010. Using the patient's national identity code, data on hospitalizations, including ICD-10 codes for discharge diagnoses, were obtained from the National Hospital Discharge Registry (HILMO) maintained by the National Institute of Health and Welfare. Prevalence of ischemic heart diseases (I20-I25), incidence of cerebrovascular diseases (I60-I69), and transient cerebral ischemic attacks (TIAs) (G45) were followed up from 2005 to 2014 until the subjects were on average 60 years old. In the follow-up of genotyped subjects, there were 67 with ischemic heart disease, 15 who had a diagnosis of cerebrovascular disease, and 8 with TIAs. The subjects with cerebrovascular disease and TIAs were combined for the group with cerebrovascular events. All participants gave informed consent, and the Ethics Committees of the Tampere University Hospital and the City of Tampere approved the study.
Cases (n = 336) were subjects who had hypertension at the age of 50 years (as diagnosed by a physician), and for each case, at least one normotensive control subject (n = 473) with the same sex, and similar smoking habits, was chosen in order of admission from the PHE cohort (n = 6000). The present study population at the age of 50 years thus included 809 subjects.
Genotyping
DNA was extracted from buccal swabs using a commercial kit (Qiagen, Inc., Valencia, CA). An intragenic SNP at nucleotide position 828 (rs1058261) of the desmin gene was chosen (Fichna et al., 2014). For DNA genotyping, PCR was performed in a final volume of 5 μL containing 10 ng of sample DNA, 0.05 μL of custom SNP-specific assay mix, and 2.18 μL of TaqMan Universal PCR Master Mix. The assay mix used was C__11735969_20. Amplification proceeded for 40 cycles of 15 s at 95°C and 60 s at 60°C. Genotyping followed the Applied Biosystems (Pleasanton, CA) protocol. Automatic genotype call was performed after PCR by scanning plates on the 7900 HT Fast Real-Time PCR, which provides the SDS2.3 software (Applied Biosystems).
Statistical analyses
Logistic regression, one-way ANOVA, or T-test for continuous variables and chi-square test or Fisher's exact test for categorical variables were applied for comparison of cases, controls, and genotype groups. Analyses were carried out using SPSS 23.0 for Windows (SPSS, Inc., Chicago, IL).
Results
Clinical characteristics of the case group of 336 hypertensive subjects and 473 controls at the age of 50 years have been previously described (Kunnas et al., 2012). Samples were available and genotyping for rs1058261 was successful for 809 subjects: 336 cases and 473 controls (351 women and 458 men). The genotype frequencies were CC: 421 (52.0%), CT 329 (40.7%), and TT 59 (7.3%). The genotypes were in Hardy-Weinberg equilibrium (chi-square = 0.23, p > 0.05).
There was no association of rs1058261 genotypes with hypertension when all genotypes were compared with each other (p = 0.235); when the CC genotype was compared with combined CT+TT genotypes (p = 0.987); or when the TT genotype was compared with combined CT+CC genotypes (p = 0.102).
When the subjects were followed up to the age of 60, subjects with genotype CC had higher incidence of cerebrovascular events (4.0%) compared with the T allele (1.5%) (p = 0.031) (Table 1). When incidence of cerebrovascular events was adjusted by BMI and gender, the OR for CC genotype was 2.63 (p = 0.046, CI: 1.018-6.78) compared with T allele carriers.
Chi-square test, Fisher's exact test, one-way ANOVA, or T-test.
Logistic regression adjusted by BMI and gender.
p values <0.05 are in bold.
BMI, body mass index; SD, standard deviation.
At the age of 60, those with genotype CC also had higher incidence of all combined cardiovascular events (12.6%) compared with subjects with the T allele (8.5%) (p = 0.052) (Table 1). When adjusted by BMI and gender, the OR for CC genotype was 1.72 (p = 0.028, CI: 1.06-2.79) compared with T allele carriers.
DES rs1058261 did not associate with other background characteristics of the study population (Table 1).
Discussion
The DES intragenic SNP (C>T) at nucleotide position 828 (rs1058261) is a synonymous codon that does not lead to amino acid change. However, it may be possible that rs1058261 is in linkage disequilibrium with a functional polymorphism that could lead to pathological changes at the protein level since we report that this variation is associated with cerebrovascular complications and all combined cardiovascular events. At the age of 60, those with genotype CC had higher incidence of these events compared with subjects with the T allele. However, there was no association of this SNP with hypertension. Previously, the C allele of the benign DES rs1058261 was associated with myofibrillar myopathies in two Polish families having other pathological mutations in the DES gene (Fichna et al., 2014). It is thought that intimal SMCs in native atherosclerotic plaque are derived mainly from the medial arterial layer. Smooth muscle involvement is not reported as the main manifestation in DES-related myopathy, but some manifestations that indicate smooth muscle involvement have been reported, such as swallowing difficulties (Goldfarb et al., 1998).
Numerous research groups have used a variety of in vivo and in vitro model systems and clinical studies to demonstrate the conversion of normally contractile vascular SMCs during phenotypic modulation to a less differentiated state capable of proliferation, migration, and extracellular matrix secretion. These changes in the vascular SMC phenotype are thought to underlie many vascular occlusive diseases (Nguyen et al., 2013). Phenotypic switching of the SMC plays a major role in a number of major diseases in humans, including atherosclerosis and hypertension (Owens et al., 2004). Phenotype of the arterial SMC in various stages of modulation or repair during fetal development, aging, and atherosclerosis is characterized by synthetic SMCs that are rich in synthetic organelles and are desmin negative (Nikkari, 1991). Thus, variation in the expression of desmin may be considered important in the repair and function of the arterial wall. This may be influenced by the SNP DES rs1058261 since it was associated with cardiovascular events.
A weakness of the present study is the relatively small population with few cases of cerebrovascular complications. Nevertheless, these cases were manifested at a relatively young age with possible genetic predisposition. In conclusion, we report for the first time that the DES gene may be involved in cardiovascular disease as well, in addition to muscular disease (Goldfarb et al., 1998).
Footnotes
Acknowledgments
The expert technical assistance by Mirka Pietiläinen and Nina Peltonen is gratefully acknowledged. This study was supported by grants from competitive research funding of the Pirkanmaa Hospital District.
Author Disclosure Statement
No competing financial interests exist.