A case–control study of candidate vasoactive mediator genes in primary Raynaud's phenomenon

A. E. Smyth, A. E. Hughes1, I. N. Bruce and A. L. Bell

Department of Rheumatology, Musgrave Park Hospital, Stockman's Lane, Belfast BT9 5LQ and
1 School of Clinical Medicine and Division of Molecular Medicine, The Queen's University of Belfast, Belfast, UK

Correspondence to: A. E. Smyth, Department of Rheumatology, Musgrave Park Hospital, Belfast BT9 5LQ, UK.


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives. To elucidate possible genetic factors involved in the pathogenesis of primary Raynaud's phenomenon (RP) and to determine the demographic features.

Methods. The allele frequencies of known polymorphisms in four vasoactive candidate genes, eNOS, BKRG, ET-1 and the ETA receptor genes, were compared in a phenotypically homogeneous group of patients with primary RP and a normal control population.

Results. In patients with primary RP, there was a higher reporting of both a family history of RP than in controls (45.3% vs 3.1%; P<0.0001) and a personal history of migraine (32.6% vs 7.2%; P<0.0001). No significant differences in allele frequencies of the candidate genes were found.

Conclusions. These findings support the concept that genetic susceptibility exists in primary RP. The high prevalence of migraine suggests that primary RP is part of a more widespread disorder of vascular tone. These findings do not suggest that common molecular variants of these candidate genes are involved in primary RP.

KEY WORDS: Primary Raynaud's, Candidate genes, Familial, Endothelin-1, Nitric oxide, Bradykinin


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Raynaud's phenomenon (RP) is characterized by episodic digital vasospasm provoked by cold and/or emotional stress. In the absence of an identifiable associated condition, such as scleroderma, it is known as primary RP. There is an increased prevalence of RP in family members of affected individuals in a range of 14–36% [14]. A familial predisposition in primary RP has been suggested by anecdotal accounts and epidemiological studies consistent with the concept of genetic susceptibility. Case-controlled studies using a candidate gene approach provide a means of elucidating the genetic component of a multifactorial disease. Likely candidate genes can be identified on the basis of available clinical and scientific evidence.

The underlying pathogenic process in RP remains largely unclear. The vascular endothelium plays a major role in the regulation of vascular tone and recent studies suggest endothelial dysfunction in RP [5]. Bedarida et al. [5] suggested that the impaired venodilatation to the endothelial-dependent vasoactive mediator bradykinin results from a diminution of nitric oxide release from the vascular endothelium. The endothelial-dependent vasoconstrictor endothelin-1 (ET-1), exerting its vasoconstrictor effect mainly via the ET-A receptor subtype, may also play a pathogenic role in primary RP [6].

Our aim was to study the clinical and demographic features of `familial' RP in a homogeneous Caucasoid population. We also determined whether previously described polymorphisms of potential candidate genes, i.e. endothelial nitric oxide synthase (eNOS) [7], ET-1, the ET-A receptor [8] and the bradykinin receptor genes [9], occurred with increased frequency in subjects with primary RP compared to unaffected controls.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patients and controls
Patients with suspected RP were recruited from hospital out-patients, general practice and self-referral following an advertising campaign. RP was diagnosed by a positive response to both a previously validated questionnaire inquiring about cold sensitivity and digital colour changes [10] and Maricq's colour charts [11]; these were administered as part of a full clinical assessment. This assessment followed a standard protocol which enquired about medical history, including the presence of a family history of RP, age of disease onset, seasonal variation in symptoms, smoking history, skin ulceration, current or previous therapeutic interventions and personal or family history of migraine headache. Migraine was diagnosed on the basis of two attacks of throbbing headache with at least two of the following: unilateral location, moderate or severe intensity, or aggravation by routine physical activity and with at least one of the following accompaniments: nausea, photophobia or vomiting. We also accepted the diagnosis of migraine if the patient had two or more attacks of throbbing headache preceded, or accompanied, by fully reversible neurological symptoms. Exclusion criteria were a co-s existent disease state, i.e. diabetes, hypertension, ischaemic heart disease, or the chronic use of any medication.

We classified patients as primary RP if they fulfilled LeRoy's proposed criteria for primary RP [12], i.e. (i) episodic digital colour changes evoked by cold and/or emotion; (ii) absence of digital scars or gangrene; (iii) a normal erythrocyte sedimentation rate (ESR; <20 mm/h, Westergren technique); (iv) negative antinuclear antibodies (ANA; titre<1:100 for all epitopes); (v) normal chest and hand radiographs; (vi) normal nailfold capillary morphology on microscopy. In addition, negative anticentromere, antitopoisomerase I and antibodies to other extractable nuclear antigens were confirmed in all subjects.

Control subjects were recruited from hospital employees. All controls were aged >25 yr to allow sufficient time for RP to become manifest. They were otherwise matched to patients for sex, smoking status, ethnicity and geographical location. Normal controls required a negative response to all three screening methods for RP (questionnaire, colour charts and clinical assessment); the same exclusions regarding any co-existent disease applied. Subjects all gave written informed consent and the study was approved by the local research ethics committee.

Laboratory methods
Genomic DNA was extracted from peripheral venous blood using a guanidine hydrochloride-based method and amplified by a separate polymerase chain reaction (PCR) for each candidate gene.

eNOS microsatellite repeat. PCR was performed using 50 µg of genomic DNA, 10 pmol of each primer, 3 µM MgCl2 , 0.l µM dNTPs, 2 µl of 10x buffer and 1 U of Taq polymerase in a final volume of 20 µl. One primer was 32P end labelled. The PCR conditions have been described elsewhere [7]. PCR products were analysed on a 6% denaturing polyacrylamide gel; allele sizes 134–190 bp, 21 alleles were identified ranging from 14 to 40 repeats.

ET-I and ETA receptor gene. The PCR for each gene was performed using 50 µg of genomic DNA, 20 pmol of each primer, 1.5 µM MgCl2 , 0.1 µM dNTPs, 2 µl of 10x buffer and 0.8 U of Taq polymerase in a final volume of 20 µl. ET-l samples were subjected to 4 min denaturation at 95°C; then to 35 cycles of 1 min at 95°C, l min at 65°C and 1 min at 72°C; and 10 min at 72°C. ET-A samples were subjected to 4 min denaturation at 95°C; then to 35 cycles of 1 min at 95°C, 1 min at 74°C and l min at 72°C; and 10 min at 72°C. A single-base insertion in exon 1 of the ET-1 gene is known to abolish a BsiYI restriction site; a silent polymorphism in codon 323 of the ET-A gene creates an AflII restriction site. Polymorphisms of ET-l and FT-A were identified on 2% agarose gels following restriction enzyme digestion of PCR products [8].

BKRG. PCR was performed using 50 µg of genomic DNA, 5 pmol of each primer, l.5 µM MgCl2 , 0.2 µM dNTPs, 2 µl of 10x buffer and 0.25 U of Taq polymerase in a final volume of 20 µl. Samples were subjected to 5 min denaturation at 95°C; then to 40 cycles of 1 min at 94°C, 45 s at 53°C and 30 s at 72°C. The T to C transition in the core promoter was detected by single-strand conformation polymorphism analysis and subsequent silver staining [9].

Statistical analysis
Statistical analysis was performed using the {chi}2 and unpaired Student's t-test. P significant was taken as 0.05.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
One hundred and twenty-two patients with a presumed diagnosis of primary RP were interviewed; 20 patients had RP, but had either a definite connective tissue disease or did not fulfil LeRoy's criteria, and a further seven did not have RP. Ninety-five patients with primary RP (17 males, 78 females) were therefore studied. Their characteristics are presented in Table 1Go. Fifty-six (58.9%) were recruited from general practice, 20 (21%) from hospital out-patients and 19 (20%) by self-referral following an advertising campaign. Patients and controls were matched for sex and smoking status. There was no difference in mean age, although there was variance as all controls were aged >25 yr. The mean recalled age at disease onset was 14 yr (interquartile range of 7–24.8 yr) and the mean disease duration was 20.9 yr. Forty-three of the 95 patients with RP (45.3%) had a first-degree relative with RP, compared to 3.1% of the control population (P<0.0001). Forty-three subjects had at least one first-degree relative with RP. Thirty-two reported RP in a parent, 19 in a sibling, 13 in their offspring. Data on the total number of relatives who were unaffected were not available and therefore it was not possible to estimate the sibling risk ratio. A history of migraine headache was obtained in 31 of the 95 patients (32.6%) and 7 (7.1%) controls (P<0.0001). When cases of primary RP were divided into those with a family history of RP (`familial') and those without (`sporadic' cases), 46.5% of `familial' RP had a personal history of migraine vs 21.2% in the `sporadic' group (P=0.016). A family history of migraine headache in a first-degree relative was present in 37 (38 9%) of RP patients; these data were not available for the control population. Nine (9.5%) patients with primary RP had had previous surgical sympathectomy and 35 (36%) reported current use of medical therapy for RP. Sixty-six (69.5%) were affected throughout the year and 29 (30.5%) were affected solely in winter months.


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TABLE 1.  Characteristics of primary RP compared to the control population
 
The frequency distributions of the ET-1, ET-A receptor and the BKRG genotypes are shown in Tables 2–4GoGoGo. The genotype frequencies did not show any departure from Hardy–Weinberg equilibrium. No difference was found between the patient and the control population. The intronic CA microsatellite polymorphism for the eNOS gene again did not give significant differences between the two groups in the 21 alleles identified in this cohort of patients and controls (Table 5Go).


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TABLE 2.  BKRG genotype frequency in primary RP compared to the control population
 

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TABLE 3.  ET-1 genotype frequency in primary RP compared to the control population
 

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TABLE 4.  ETA receptor genotype frequency in primary RP compared to the control population
 

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TABLE 5.  Endothelial NOS microsatellite allele frequency in primary RP and controls
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
This is the first study to analyse the contribution of candidate genes in primary RP. A high reported prevalence of a family history of RP was found in first-degree relatives of subjects with RP (45.3%), suggesting a significant underlying genetic basis. This population-based association study analysing the candidate genes, ET-1, the ETA receptor, eNOS and the bradykinin receptor genes, has demonstrated an absence of association with primary RP in a Northern Ireland population.

The background for considering RP as a genetic disease comes from anecdotal and a limited number of epidemiological studies which report a family history of between 14 and 35% [14]. Of the four studies that have addressed the issue of `familial' RP, some have been limited in that patients were not classified as primary or secondary RP, or they did not compare patients with an unaffected population. Strengths of this study include the clear definition of RP subcategories, interview of the proband by a clinician and in addition the proband had witnessed a typical Raynaud's episode in their affected relative. The difference in family history between the groups is large and most of this difference is likely to be due to a familial predisposition to the disease. A diagnostic awareness bias in patients compared to controls is impossible to eliminate without direct assessment of relatives, which was not a feature of this study. The prevalence of a family history in the control population, while significantly less than in the patient group, was lower than many reported in population prevalence studies [13].

The prevalence of migraine headache was significantly different between the RP and control groups (33% and 6.9%, respectively; P<0.0001). As with RP, the pathogenesis of migraine is uncertain; these prevalence data would suggest that migraine might represent a different manifestation of a generalized disorder of vascular tone. Our data support those of Shaun et al. [14]; however, they found a much higher reported prevalence of migraine in their primary RP cohort (61%). We, however, confirmed the diagnosis as part of a clinical interview rather than relying on questionnaire responses alone, our estimated prevalence of migraine in the control group being similar to that reported in population surveys [15]. Interestingly, in our cohort, patients with `familial' RP had a significantly higher prevalence of migraine than those with `sporadic' RP (P=0.016), again suggesting that this generalized disorder of vascular tone is likely to have a genetic basis.

While evidence suggests a strong genetic component in the pathogenesis of primary RP, clearly the pattern of inheritance seen does not suggest the action of a single gene. Furthermore, it is clear that environmental influences such as smoking and cold exposure worsen the condition. It is, therefore, a complex disorder that is likely to be caused by the interaction of environmental factors with several different genes at different loci, each perhaps with an additive effect.

As primary RP has not been linked to a known subchromosomal region, the investigative strategy employed to identify allelic association was a position-independent candidate gene approach. The four candidate genes studied were identified based on the current concepts of disease aetiopathogenesis. ET-1 exerts its vasoconstrictor effects through distinct endothelial receptors, including the ET-A receptor, which has a high affinity for ET-1. Several studies have found ET-1 to be elevated in primary RP and some have found a further increase in response to cold challenge [6]. The elevated circulating ET-1 levels may reflect even higher local concentration of ET-1 and/or overexpression of receptor on microvessels which may contribute to an imbalance in the control of vascular tone in primary RP. Additionally, impaired release of nitric oxide from the endothelium has been suggested as a possible pathogenic mechanism in primary RP. Bedarida et al. [5] demonstrated impaired venodilatation to bradykinin, an endothelium-dependent vasodilator, in patients with primary RP. Therefore, the constitutive eNOS and the bradykinin receptor genes were also studied.

The polymorphisms analysed in the four candidate genes had previously been described. While the polymorphisms examined have not clearly been found to be functional, the markers employed were within introns and hence tightly linked. The allele frequencies were not significantly different between subjects with primary RP and their matched RP-negative controls. This suggests that genetic polymorphisms in these genes do not contribute to the aetiopathogenesis of primary RP. As with any genetic study, these findings may only apply to the population studied, and it is possible that variants of these genes may be involved in different populations or under different environmental conditions.

An allele can be said to be associated with the disease if it occurs at a significantly higher frequency in the affected group or, alternatively, an allele at a higher frequency in a normal population may have a protective effect. Several considerations must be taken into account, as a positive association does not always mean that variation in the gene has contributed to expression of the disease. An alternative explanation is that the marker polymorphism lies close to or is in linkage disequilibrium with the putative functional gene variant [16]. Even if a marker is very close to a pathogenic locus, there will not necessarily be an association as this may depend on the evolutionary history of the marker and the disease polymorphism. Additionally, `spurious' associations can occur due to population stratification, in that the marker alleles and the disease can have different frequencies in different subpopulations. Our study populations had clearly defined phenotypes, and both disease and control groups were of Northern Ireland parentage so that any observed difference would not have merely been due to ethnic variation.

In summary, we have found a high reporting of a family history of RP in patients with primary RP. There is also a higher prevalence of migraine among patients with primary RP, this being especially marked among those with a family history of RP. Overall, these results suggest that primary RP is part of a more widespread disorder of vascular tone with a significant genetic component to its pathogenesis. The specific genes involved remain to be elucidated; those tested in this study are unlikely to have a major involvement.


    Acknowledgments
 
Supported by a Royal Victoria Hospital clinical research grant and a project grant from the Raynaud's and Scleroderma Group, UK.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Submitted 19 August 1998; revised version accepted 14 May 1999.



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