Genetic and Clinical Characteristics of 103 Patients With Primary Pulmonary Hypertension
by Eric W. Glissmeyer, Greg T. Havlena, B.S., Jon Schmidt, John Carlquist, PhD., Bonnie Baxter, PhD.,and C. Greg Elliott, MD.
Background Mutations in the gene that codes for bone morphogenetic protein receptor two (BMPR2) are present in many patients with primary pulmonary hypertension (PPH). The purpose of this study is to identify novel mutations in BMPR2 and report clinical characteristics of PPH patients heterozygous for mutations in BMPR2.
Methods High-resolution melting curve analysis and subsequent gene sequencing were used to scan the BMPR2 gene of 103 patients affected by either sporadic or familial PPH. Clinical data from study patients were obtained from 26 treatment centers and used to confirm PPH.
Results Twenty-three of 103 (22%) patients exhibited mutations in BMPR2. Seven novel mutations were identified, four of which cause a single amino acid alteration in the gene product and two cause truncation. One causes no change in the gene product and is likely a polymorphism. Hemodynamic data showed PPH patients without mutations in BMPR2 to be more likely to respond to vasodilator challenge than PPH patients with mutations in BMPR2 (P= 0.011). PPH patients with mutations in BMPR2 appear to have similar severity of disease as those PPH patients negative for mutations in BMPR2.
Conclusions Seven novel BMPR2 mutations exist in this cohort of 112 PPH patients. The severity of disease observed in patients with PPH does not appear to be affected by the presence of mutations in BMPR2; however, such mutations do appear to affect the likelihood of vasoreactivity in the PPH patient.
Genetic and Clinical Characteristics of 103 Patients With Primary Pulmonary Hypertension
Primary pulmonary hypertension (PPH) is a rare disorder with an estimated incidence of 1 to 2 cases per million per year.1 It is characterized pathologically by plexiform lesions of proliferating endothelial cells in pulmonary arteries, which lead to elevated pulmonary artery pressures followed by right heart failure and death.2,3 The disease can occur throughout life but has a mean age at onset of 36 years, and affects twice as many females as males. Without treatment, the median survival is less than 3 years after diagnosis.4 Recently, however, survival has improved significantly with lung transplantation and the advent of long-term prostacyclin therapy.5,6 PPH cases are classified as either sporadic or familial. Sporadic cases are those for whom no other member of the immediate family has been affected. Familial cases are heritable, autosomal dominant disorders that show incomplete penetrance. Mutations in the gene encoding bone morphogenetic protein receptor two (BMPR2) have been identified in patients with familial PPH and sporadic PPH, but are only rarely observed in patients with other forms of pulmonary hypertension.7 The clinical phenotype of sporadic and familial PPH patients has been described.3 However, that of PPH patients with mutations in BMPR2 is not well defined. We hypothesized that this cohort of 103 PPH patients harbors novel mutations in BMPR2 that can be identified by high-resolution melting curve analysis. In addition to addressing this hypothesis, this study seeks to answer the following questions: 1) Do all familial PPH patients exhibit mutations in BMRP2? 2) Is there a difference in vasoreactivity between PPH patients without mutations in BMPR2 and those with mutations in BMPR2? 3) What differences between the previous two groups are observed with respect to age of onset of disease and hemodynamic data at diagnosis?
Patient selection: Patients were diagnosed at one of the following 26 medical centers in the United States, Canada and Australia: LDS Hospital SLC, UT (33), Rush (15), Mayo (11), University of Alabama at Birmingham (4), UCLA (4), University of Maryland (4), Vanderbilt (4), University of Colorado (3), Duke (3), Baylor (2), UCSD (2), Cleveland Clinic (2), Columbia Presbyterian (2), Stanford University (2), UCSF (1), Mercy Medical Center, Buffalo NY (1), Methodist Medical Center, Peoria IL (1), University of Michigan (1), Monash Medical Center, Victoria Australia (1), O'Connor Hospital, San Jose CA (1), University of Pennsylvania (1), Saddleback Memorial Hospital, Laguna Hills CA (1), Tampa General Hospital (1), UTSA (1), University of Washington (1), Women's College Hospital, Ontario Canada (1). All patients provided written informed consent for participation according to a protocol that was approved by the institutional review board of the LDS Hospital. Medical records were obtained and reviewed by Dr. Elliott to confirm the diagnosis of sporadic or familial PPH. Clinical data of those patients with sporadic PPH demonstrated the following:
- Mean pulmonary artery pressure > 25mmHg at rest.
- Pulmonary artery wedge pressure < 15 mmHg.
- Echocardiogram demonstrating neither primary cardiac disease nor congenital heart disease other than patent foramen ovale.
- Pulmonary embolism ruled out by lung perfusion (VQ) scan or pulmonary angiogram when VQ scan was indeterminate.
- Chest disease ruled out by clinical evaluation, chest radiograph, and pulmonary function testing.
- Central hypoventilation ruled out by a normal arterial PCO2.
Clinical data of those patients with familial PPH demonstrated the above findings as well as pedigree evidence of inherited disease.
Molecular methods: Blood samples of consenting patients were drawn and DNA extracted from lymphocytes. Primers were designed to amplify exons 1-12 of the BMPR2 gene via PCR. Exons larger than 200 base pairs were amplified in multiple segments (i.e. exon 8-1 and 8-2). Comprehensive mutation scanning was done using high-resolution melting curve analysis, a technique developed by Idaho Technology, Inc. It employs an instrument called the HR-1TM, which employs a newly developed non-specific fluorescent dye called LCGreenTM that binds duplex DNA during PCR. The HR-1TM measures changes in the fluorescent signal from the amplicon as it is thermally denatured. The transition from duplex to single stranded DNA is accompanied by a rapid decrease in fluorescence at the specific melting temperature of the amplicon, creating a characteristic melting profile (Fig. 1). Comparison of melting profiles from multiple samples differentiates those harboring a mutation from samples of consensus wild type sequence. Samples in which a mutation was identified by melting curve analysis were sequenced by standard direct DNA sequencing.
Figure 1. HR-1TM Melt Curve Example.
Melting curves from a single exon are displayed. The more pronounced curve is the result of stacked identical melting curves from wild type patients. The single deviant curve illustrates a more rapid decrease in fluorescence, indicating a mutation causing an interruption in the nucleotide base pairing of DNA.
We identified 23 of 103 PPH patients with mutations in BMPR2 (22%). Seven novel mutations were identified, four of which cause a single amino acid alteration in the gene product (missense mutations) and two cause truncation (nonsense mutations). One novel mutation found causes no change in the gene product and is likely a polymorphism (Fig. 2). This cohort of 103 PPH patients contained 17 individuals characterized as having familial PPH, of whom 10 showed mutations in BMPR2 by melting curve analysis. Seven individuals with a family history of PPH do not appear to have mutations in BMPR2.
Figure 2. Novel mutations found in the BMPR2 gene of patients with PPH.
The cohort of 103 PPH patients contained 23 patients with germline mutations in BMPR2. Patients 1, 2, 9, and 11 exhibit missense mutations that cause a single amino acid change in the primary protein structure. Patient 8 exhibits an insertion at cDNA position 1113 that causes a stop codon in exon 9. The mutation observed in patient 10 causes the formation of an immediate stop codon. Seven of the 23 patients showed mutations previously identified. Four other patients showed mutations in exons 2 (two patients), 3, and 9 that have yet to be sequenced. Melting curve analysis correctly identified one patient that has a previously identified mutation in exon 3, but sequencing failed to confirm the mutation.
The results of acute vasodilator testing were obtained on those from the cohort who had such testing performed. Vasodilator testing is usually done at the time of diagnostic right heart catheterization when the patient is challenged with one of a number of vasodilator medications. The results of this testing aid in the prediction of patient response to various therapies used in the treatment of PPH. As used in this study, "vasoreactivity" or a positive response to vasodilator challenge is defined as a reduction in mean pulmonary artery pressure (MPAP) greater than 20% from baseline measurements, accompanied by a drop in pulmonary vascular resistance (PVR). Hemodynamic data showed 0 of 12 PPH patients with mutations in BMPR2 to be vasoreactive (Fig. 3). However, 40%, or 17 of 42 PPH patients without mutations in BMPR2 were vasoreactive, suggesting that it is likely that PPH patients at large will respond differently to acute vasodilator challenge based upon whether or not they harbor mutations in BMPR2 (P= 0.011). In other clinical data, however, significant differences are not observed between PPH patients with mutations in BMPR2 and those negative for mutations in the gene (Fig. 4). Age at onset of disease is determined by the patient's age at time of diagnostic right heart catheterization. These two groups do not differ significantly with respect to their age at onset of disease (P= 0.60) and baseline MPAP at time of diagnosis (P= 0.42). Mutation positive PPH patients did not include the patients with the silent mutation (i.e. polymorphism) in exon 5.
Figure 3. Vasoreactivity differences between PPH patients with and without mutations in BMPR2.
No PPH patients with mutations in BMPR2 had a >20% reduction in MPAP in response to acute vasodilator challenge. One PPH mutation + patient, however, had a 19.6 % reduction in MPAP. Two PPH mutation- patients considered non-vasoreactive also showed near vasoreactivity (19.3% and 18.7% reductions in MPAP). These two groups of PPH patients are significantly different (P= 0.011) with respect to their response to acute vasodilator challenge. Acute vasodilator challenge was performed at 19 different centers using the following therapies: prostacyclin (23 cases), nifedipine (15), inhaled nitric oxide (6), adenosine (6), aceto-nitrile (1), nitroprusside (1), phentolamine (1), prostaglandin E2 (1).
Figure 4. Similarities seen in age at onset of disease and MPAP for PPH patients with and without mutations in BMPR2.
No statistically significant differences are observed between the two groups with respect to age at onset of disease (P= 0.60) and MPAP at time of diagnostic right heart catheterization (P= 0.42).
The identification of seven novel BMPR2 mutations confirms our hypothesis that this cohort of 103 PPH patients harbors novel mutations in BMPR2 that can be identified by high-resolution melting curve analysis. All novel BMPR2 mutations identified in this cohort of 103 PPH patients are located within kinase domains of the protein, with exception of the silent polymorphism located within the transmembrane region (exon 5). The majority of mutations identified to date in the BMPR2 gene of PPH patients are located in the ligand-binding extracellular domain and the intracellular kinase domain of the BMPR2 protein.8 It is thought that mutations in other domains, specifically the cytoplasmic tail portion, may not be as likely to cause disruption of the TGF-b signaling pathway, for which BMPR2 is a cellular membrane receptor.
Scanning for mutations in BMPR2 using the HR-1TM proved to be a cost-effective molecular biology technique. The reagent and material cost of scanning the BMPR2 gene of one subject was roughly $23, whereas direct sequencing of the BMPR2 gene of one subject would have cost over $300. The designs of this study did not allow for the evaluation of sensitivity and specificity of the HR-1TM; however, these have been previously determined by Idaho Technology, Inc and are 100% for DNA fragments up to 300 base pairs.9 All BMPR2 gene fragments scanned in this study using the HR-1TM were less than 300 base pairs in length.
Seven individuals in our study cohort with documented familial PPH do not show mutations in BMPR2. A research group led by Matthias Rindermann reported a similar finding in 2003.10 They identified three families with no BMPR2 mutation that showed evidence for linkage to a more proximal location on chromosome 2q31 (odds ratio for linkage 1.1x106:1) called PPH1. The seven familial PPH individuals without mutations in BMPR2 may harbor mutations in PPH1, in genes that code for other proteins that are involved in the TGF-b signaling pathway, or in other genes that may be associated with the development of pulmonary hypertension.
Response to acute vasodilator challenge is the only clinical finding that displayed statistically significant difference between the phenotype of PPH patients with mutations in BMPR2 and the phenotype of PPH patients without such mutations. This finding may be explained by different causes of pulmonary vascular resistance in these two groups. The pulmonary vascular endothelium is thought to proliferate and narrow the lumens of pulmonary arteries of patients with mutations in genes that code for proteins involved in the TGF-b signaling pathway. It is unlikely that vasodilator therapy will benefit the PPH patient whose pulmonary arteries are not constricted, but occluded by marked endothelial cell proliferation. The increased vasoreactivity observed in PPH patients without BMPR2 mutations may in part be due to the nature of vasoconstriction that can be relieved by vasodilator therapy.
The vasoreactivity frequency observed in PPH patients without mutations in BMPR2 is 40%, and significantly greater than that found in PPH patients with mutations in BMPR2 (P= 0.011). When only patients with sporadic PPH are considered, 45% of sporadic PPH patients without BMPR2 mutations are vasoreactive. However, the frequency of vasoreactivity in such sporadic PPH mutation negative patients is not significantly greater than that observed in sporadic PPH mutation positive patients (17 of 55 mutation negative and 0 of 6 mutation positive patients were vasoreactive P= 0.17). Nevertheless, the 45% vasoreactivity of sporadic PPH patients observed in this cohort is greater than that reported by others. Sitbon and colleagues reported 13.3% vasoreactivity in 430 sporadic PPH patients at the world symposium on pulmonary hypertension in June 2003.11 Their cohort of 430 sporadic PPH patients may have included patients with mutations in BMPR2. If all sporadic patients with acute vasodilator testing in our study are combined, their frequency of vasoreactivity is 39%, which is still greater than that reported by Sitbon, et al. The higher frequency of vasoreactivity reported in this study may be explained by the unique patient makeup of this cohort of 103 PPH patients. Seventy of the 103 subjects in this cohort were enrolled at Pulmonary Hypertension Association (PHA) meetings in Stone Mountain Georgia (1994 & 1996), Chicago (2000) and Irvine California (2002). Patients well enough to attend these conferences may be more likely to be vasoreactive, since vasoreactivity correlates with increased survival in the PPH patient.12 We attempted to test this hypothesis by examining the vasoreactivity data of patients treated at only one pulmonary hypertension center. The PH clinic at LDS Hospital in SLC, UT was the one center with the greatest number (33) of patients included in this cohort of 103. The vasoreactivity data from sporadic PPH patients treated at LDS Hospital reveal vasoreactivity in 2 of 13 PPH patients or 15.4%. Therefore, the sporadic PPH patients included in this study that were treated at LDS Hospital were not significantly more likely to be vasoreactive than European sporadic PPH patients reported by Sitbon, et al (P= 0.69).
Research performed at LDS Hospital, Salt Lake City Utah and in cooperation with Westminster College, Salt Lake City Utah. Funded by a grant from the Deseret Foundation (447).
This research represents the collective effort of many families affected by pulmonary hypertension and their courage to face this devastating disorder. Their willing participation in research allows us to work towards a better understanding of the mechanisms of PPH and eventually find a cure.
From LDS Hospital and Westminster College, Salt Lake City Utah. Address correspondence to firstname.lastname@example.org.
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