Abstract
Rationale: The bone morphogenetic receptor type II gene is the major genetic determinant for the inherited form of pulmonary arterial hypertension. However, deleterious mutations of this gene are not observed in the majority of subjects who develop the condition spontaneously and familial disease displays age- and sex-dependent penetrance, indicating the requirement for additional environmental and/or genetic modifiers for disease development.
Methods: We investigated polymorphic variation of the serotonin transporter gene, a biological candidate for predisposition to this vascular disorder.
Results: No significant evidence of association between alleles of the serotonin transporter gene and pulmonary hypertension was detected, nor did we observe a relationship with age of onset in familial and idiopathic disease.
Conclusions: Variation of the serotonin transporter gene appears unlikely to confer significant susceptibility to pulmonary arterial hypertension. This study emphasizes the need for adequately powered cohorts for association analyses to identify not only genetic determinants of disease susceptibility but also inherited modifiers for disease development.
Pulmonary arterial hypertension (PAH) is a severe disorder characterized by a sustained elevation of pulmonary arterial resistance consequent on the vascular remodeling of small pulmonary arterioles. This process involves proliferation and hyperplasia of endothelial and smooth muscle cells with an increase in the extracellular matrix. The disease most frequently manifests in the fourth decade of life and in the absence of treatment leads to right heart failure (1, 2).
To aid clinical management, distinction has been made between familial (FPAH), idiopathic or sporadic disease (IPAH), and PAH associated with other diseases or exposure to exogenous agents, for example, appetite suppressants containing fenfluramine derivatives (APAH) (3). The genetic basis of familial PAH has been clarified. Heterozygous mutations of BMPR2 encoding a type II receptor of the transforming growth factor-β (TGF-β) cell-signaling superfamily may be detected in approximately 70% of analyzed kindreds (4–8). Importantly, BMPR2 mutations, either de novo or inherited, have also been shown to underlie a proportion of IPAH cases (8–11).
Although comprehensive epidemiologic studies are awaited, the penetrance of BMPR2 mutant alleles in families, although reduced, may be as high as 50% in some families (12). These findings imply the existence of additional genetic and/or environmental factors, necessary for disease pathogenesis (13). Reports linking the mitogen serotonin 5-hydroxytryptamine transporter (5-HTT) with PAH have generated attention. Internalization of indoleamine by 5-HTT mediates the stimulatory effects of serotonin (5-HT) on cell proliferation (14). Increased expression of the 5-HTT gene has been shown to drive growth responses of pulmonary artery smooth muscle cells derived from patients after 5-HT exposure, leading to speculation that the 5-HT pathway may act in antagonism to the predominantly growth-inhibitory effects of bone morphogenetic protein signaling (Figure 1). To examine the genetic basis of the differential growth effects of 5-HT on pulmonary artery smooth muscle cells, Eddahibi and coworkers investigated a functional 44-bp insertion/deletion polymorphism (5-HTTLPR) in the promoter of the gene (15). The insertion allele (I), corresponding to 16 units of a 20- to 23-bp tandem repeat (VNTR), increases transcript expression by as much as threefold over the 14-repeat unit deletion allele (D). The molecular basis of regulation of expression is as yet unclear (16). The 5-HTTLPR insertion allele was found to be significantly overrepresented (65%) among a cohort of 69 French subjects with IPAH by comparison with the frequency observed in control subjects (27%) (15). Taken together, these data have been interpreted to support a role for variation of 5-HTT conferring susceptibility for PAH.
Figure 1. Pulmonary artery smooth muscle cell shows opposing roles of the BMPR-II and serotonin (5-HT) pathways in the pulmonary vasculature. On binding extracellular ligand, the BMP receptor complex initiates signaling by phosphorylating a series of cytosolic mediators known as receptor (R-) Smads which, in association with Smad-4 and other DNA cofactors, regulate the transcription of genes that typically inhibit growth of pulmonary artery smooth muscle cells. By contrast, internalization of 5-HT by the transmembrane transporter 5-HTT leads to the downstream activation of mitogen-activated protein kinase (MAPK), in turn increasing levels of the GATA-4 transcription factor, which acts to stimulate the transcription of genes driving cell proliferation. 5-HTT = serotonin 5-hydroxytryptamine transporter; BMP = bone morphogenetic protein; ERK = extracellular signal–regulated kinase; P = phosphate; R-Smad 1/5/8 = receptor-regulated Smad 1/5/8 complex.
[More] [Minimize]It has become widely accepted that assessment of association studies must be guarded because of the prevalence of false-positive results. Validation may come from replication studies that may confirm or reject association results (17). Here, we have examined the role of variation of 5-HTT in PAH predisposition by investigating (1) a cohort of subjects comprising 259 IPAH cases and (2) 133 subjects each with either a family history of PAH and/or identified germ line mutations. Furthermore, we added 136 subjects diagnosed with APAH, representing a total disease study population of 528 subjects.
Ethical permission for these studies was provided by the local ethics committee or institutional review board. All patients provided written informed consent. The diagnosis of pulmonary arterial hypertension was confirmed through clinical evaluation, chest radiography, electrocardiography, Doppler echocardiography, right heart catheterization, and/or histologic analysis of the pulmonary vasculature derived from postmortem material. The FPAH and IPAH mutation–positive cohort comprised 133 subjects, 82 of whom had a recorded family history of PAH whereas the remainder were diagnosed as idiopathic but had been shown to harbor a heterozygous germ line mutation in the BMPR2 gene. For the purposes of analysis patients were classified as idiopathic when disease was sporadic and deleterious mutation was not detected after comprehensive screening of all coding regions and exon–intron boundaries as previously described (6, 8). The APAH cohort (n = 136) was composed of subjects with connective tissue disease (n = 59), thromboembolic disease (n = 42), congenital heart disease (n = 15), portal hypertension (n = 11), and HIV infection (n = 9). Age-of-onset data were not available for the majority of this group. Measurements of mean pulmonary arterial pressure, cardiac output, and pulmonary vascular resistance were available for up to 86 FPAH/IPAH mutation–positive, 215 IPAH, and 76 APAH cases. In 15 FPAH cases, the presence of disease was confirmed by histologic analysis of postmortem material. Patients were ascertained by specialist centers for the management of PAH from Germany (n = 109), Poland (n = 16), the United States (n = 61), and the United Kingdom (n = 316). All patients and control subjects (n = 353) were of self-identified European ancestry.
Genotyping of 5-HTTLPR was performed as described (15). Briefly, the polymorphic site (deletion, 484 bp; insertion, 528 bp) was amplified under standard cycling conditions (annealing temperature, 61°C). A second VNTR in intron 2 of 5-HTT was amplified with the primer sequences 5′-GGTCAGTATCACAGGCTGCGAGTAG-3′ and 5′-TGTTCCTAGTCTTACGCCAGTGAAG-3′ under standard conditions (annealing temperature, 56°C). Amplification products were electrophoresed on 2.5% agarose (NuSieve; FMC Bioproducts, Rockland, ME). Only carriers of the common STin2.10 and STin2.12 alleles were included in subsequent data analysis. Of the two other identified alleles, STin2.7, was not observed in the study population whereas carriers of STin2.9 were seen infrequently (n = 7) (18). A comparison of genotype and allele frequencies between patients and control subjects was performed, using χ2 contingency tables. Pairwise comparisons of clinical data were performed for each of the three case groups, using the Mann-Whitney U test. Relative risk figures were determined with two-way contingency tables (http://members.aol.com/johnp71/ctab2×2.html). Power calculations were assessed at an α (significance) level of 0.05, using the Genetic Power Calculator (http://statgen.iop.kcl.ac.uk/gpc/qtlassoc.html), on the basis of relative risk for the 5-HTTPLR I/I genotype from data published previously (15). Linkage disequilibrium (D′) between the two polymorphisms from unphased genotype data was determined with Haploview 3.2 (19).
The hemodynamic features at the time of diagnosis were summarized for each PAH category (Table 1). The FPAH/IPAH mutation–positive case group differed significantly from the idiopathic (mutation negative) and APAH cohorts, likely reflecting more severe disease.
Mean and SD | |||||
|---|---|---|---|---|---|
| PAH Category | mPAP (mm Hg) | CO (L/min) | PVR (dynes · s · cm−5 · m2) | ||
| FPAH/IPAH mutation positive | 59.67 ± 13.2*† | 3.44 ± 1.14§‖ | 1,470 ± 540**†† | ||
| IPAH | 56.63 ± 15.0‡ | 3.94 ± 1.58¶ | 1,172 ± 597‡‡ | ||
| APAH | 50.76 ± 16.3 | 3.7 ± 1.22 | 1,260 ± 682 | ||
To assess the relationship of polymorphic variation across the 5-HTT gene, we used all available genotypes to measure the extent of linkage disequilibrium (LD) between 5-HTTPLR and the intron 2 variant in both case and control populations. Haplotypes (frequency) I/STin2.12 (29%), D/STin2.12 (35.5%), I/STin2.10 (26.4%), and D/STin2.10 (9.1%) yielded a D′ value of 0.42, with a score of 1 representing absolute linkage disequilibrium. D′ scores were similar for patient and control groups.
Association of the functional insertion/deletion polymorphism in the 5-HTT gene promoter was examined for each of the familial/idiopathic mutation–positive, idiopathic, and associated PAH cohorts (Table 2) together with the intron 2 VNTR in a subset of case and control subjects (Table 3). In the FPAH/IPAH mutation–positive cohort, no significant deviation from control 5-HTTPLR or intron 2 VNTR allele frequency (Af) and genotype frequency (Gf) was observed (5-HTTPLR Gf, p = 0.85; intron 2, p = 0.99). Relative risk (RR) for the I/I genotype was 1.060 (95% confidence interval [95% CI], 0.774–1.432). We found no evidence of association for either polymorphism in the IPAH panel (5-HTTPLR Gf, p = 0.43; intron 2, p = 0.99) and the 5-HTTPLR genotype I/I was not associated with change in risk for disease development (RR = 1.050; 95% CI, 0.859–1.271). Finally, analysis of the panel of APAH cases revealed no evidence for association at either 5-HTT locus (5-HTTPLR Gf, p = 0.89; intron 2, p = 0.98) and no evidence of increased risk susceptibility in carriers of the I/I genotype (RR = 1.027; 95% CI, 0.752–1.384). Although allele frequencies of 5-HTT polymorphisms are known to differ between subjects of white and Asian ancestry, minimal variation has been observed within white populations (20). Distributions of allele frequency in the control panel were similar to those observed for white subjects in previous reports (20) and all the populations under analysis conformed to Hardy-Weinberg equilibrium for each polymorphism.
5-HTT Promoter Genotype | |||||||||
|---|---|---|---|---|---|---|---|---|---|
| Number of Subjects (%) | Allele Frequency | ||||||||
| PAH Category | I/I | I/D | D/D | Relative Risk* (95% CI) | I | D | |||
| FPAH/IPAH mutation positive (n = 133) | 43 (32.3) | 60 (45.1) | 30 (22.6) | 0.980 (0.717–1.321) | 0.55 | 0.45 | |||
| IPAH (n = 259) | 84 (32.4) | 122 (47.1) | 53 (20.5) | 1.072 (0.898–1.267) | 0.56 | 0.44 | |||
| APAH (n = 136) | 43 (31.6) | 62 (45.6) | 31 (22.8) | 1.027 (0.752–1.384) | 0.54 | 0.46 | |||
| Control subjects (n = 353) | 108 (30.6) | 157 (44.4) | 88 (24.9) | NA | 0.53 | 0.47 | |||
5-HTT Intron 2 VNTR Genotypes | ||||||||
|---|---|---|---|---|---|---|---|---|
| Number of Subjects (%) | Allele Frequency | |||||||
| PAH Category | STin2.10/2.10 | STin2.10/2.12 | STin2.12/2.12 | STin2.10 | STin2.12 | |||
| FPAH/IPAH mutation positive (n = 45) | 6 (13.3) | 21 (46.7) | 18 (40.0) | 0.37 | 0.63 | |||
| IPAH (n = 80) | 13 (16.3) | 35 (43.8) | 32 (40.0) | 0.38 | 0.62 | |||
| APAH (n = 136) | 22 (16.2) | 58 (42.6) | 56 (41.1) | 0.38 | 0.62 | |||
| Control subjects (n = 150) | 25 (16.7) | 64 (42.7) | 60 (40.0) | 0.38 | 0.62 | |||
Patient ages at onset for the familial/IPAH mutation–positive and idiopathic cohorts were similar (p = 0.17), revealing a normal distribution with a mean in the fourth decade of life (FPAH/IPAH mutation positive: 32.5 yr; range, 0.4–70 yr; IPAH: 40.2 yr; range, 2–74 yr). To determine whether 5-HTTPLR genotypes may influence the age at which disease manifests, subjects from these two cohorts were partitioned according to genotype status, namely homozygote or heterozygote, and mean age at disease onset was calculated. No significant deviation was observed in the average onset age of patients in either the FPAH/IPAH mutation–positive cohort (I/I, 31.7 yr; I/D + D/D, 34.3 yr) or the IPAH cohort (I/I, 39.7 yr; I/D + D/D, 40.3 yr). To investigate a role of the 5-HTT genotype in the marked sex bias of the disease, both cohorts were also subdivided by sex and variation of 5-HTTPLR compared with control subjects (data not shown). In both patient groups, sex-specific 5-HTTPLR genotype distributions were concordant with control subjects.
This study sought to examine the role of variation within the gene encoding the serotonin transporter in predisposition to and modification of the development of PAH. Pulmonary vascular lesions in PAH display markedly elevated levels of 5-HTT whereas explant-derived pulmonary vascular smooth muscle cells exhibit increased 5-HT uptake compared with non-PAH, implicating the serotonin transporter as a contributor to vascular remodeling (15). We sought to investigate the association of genetic variation of the 5-HTT gene with familial, idiopathic, and associated forms of the disorder by genotyping two common variable repeat sequences. The first is located within the promoter, 5-HTTLPR, and the other in intron 2 of the gene. In addition to the correlation of the 5-HTTLPR I/I genotype with IPAH, both polymorphisms have been previously shown to be associated with a number of behavioral disorders, including depression and schizophrenia susceptibility (15, 21, 22). An epidemiologic study testing the onset of depression in response to stress found that diagnosable depression and suicidality were more common amongst individuals heterozygous or homozygous for the 5-HTTLPR deletion allele (21). By contrast, a metaanalysis based on all published association studies between schizophrenia and variation in 5-HTT showed highly significant evidence for association with the STin2.12 allele of the intron 2 VNTR but none with 5-HTTPLR (22).
PAH is a rare disorder that shows monogenic inheritance in at least 6% of cases (1). Hence, in seeking to identify disease susceptibility alleles and, in particular, to achieve replication of previous evidence for allelic association, extensive collaboration is necessary to attain sufficient subjects with PAH. In this article, we have investigated cohorts with sufficient power (> 95% at a significance level of 0.05) to detect association for any allele conferring a relative risk comparable to that previously reported for 5-HTT in IPAH (15). We have now shown that allele and genotype frequencies do not differ between PAH cohorts and control subjects. These findings indicate that neither the promoter VNTR polymorphism known to underlie variability in serotonin transporter expression (16) nor a tandem repeat within intron 2 of the gene are likely to contribute to disease development and risk to subjects with the BMPR2 mutation or to independently confer susceptibility to idiopathic or associated forms of disease.
We also questioned whether inherited variation at the 5-HTTPLR locus might in part explain the variable age of onset, a characteristic of PAH within and between kindreds. We observed no significant difference between the average age of onset in FPAH/IPAH mutation–positive and IPAH subjects when differentiated by 5-HTTPLR genotype. Nevertheless, it remains possible that 5-HTT variants may alter this clinical parameter in other subsets of PAH. To date, the most discriminant risk factor for disease development known is sex, with females favored at a ratio of 2:1. We found a similar distribution of 5-HTTPLR alleles between males and females with PAH, in keeping with Hardy-Weinberg estimates. As linkage disequilibrium between 5-HTTLPR and the intron 2 VNTR is low (D′ = 0.42), these analyses likely represent a survey of relatively independent regions of 5-HTT and, together, suggest that it is unlikely to play a significant role in disease onset.
The mechanisms explaining the complex features of disease manifestation for inherited forms of PAH remain unclear. Although both environmental and genetic features are likely to contribute, the proportion of variance explained by either factor is unknown. As a consequence, careful attention to methodologic detail is required as more studies are proposed and undertaken to examine the role of genetic variation in uncommon but complex disorders such as PAH. Specifically, the potential for substantial bias in these studies due to hidden population stratification must be recognized together with assessment of population cohorts of sufficient size to provide more robust reliability in the evaluation of allelic frequency differences. Finally, initial reports of disease association will most satisfactorily be interpreted when accompanied by independent cohort replication.
In summary, reduced penetrance of BMPR2 disease-causing alleles together with variability in the age of onset suggest a role for genetic risk factors in the pathogenesis of PAH. Although a plausible candidate for susceptibility, the gene encoding the serotonin transporter appears unlikely to confer significant risk to disease development or modify age of onset.
The authors thank the many clinicians who have contributed to this work through the ongoing management of patients with PAH.
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