Genetics and Immunogenetic Aspects of Primary Pulmonary Hypertension

Genetics and Immunogenetic Aspects of Primary Pulmonary Hypertension* Robyn]. Barst, MD; and James E. Loyd, MD

Primary pulmonary hypertension (PPH), also referred to as unexplained or idiopathic pulmonary hypertension, is the clinical term used to describe a condition in patients for which we can find no underlying cause. Patients with PPH not uncommonly also have evidence of immune dysregulation: autoimmune disorders, drug therapy, or HIV infections. We will review these associations and possible relevant abnormalities in immune regulation with regard to how they may play a role in the pathogenesis of PPH. Autoantibody-HLA correlations have been observed in several subsets of PPH patients. In addition, a familial form of PPH has been described and characterized with linkage to chromosome 2q31-q32. The identification of a specific gene for PPH and the subsequent understanding of its effects will help us identify the basic cause of PPH. Furthering our understanding regarding the role(s) and significance of immunogenetic as well as genetic aspects of the pathogenesis and pathophysiology of PPH should also lead to improved therapeutic modalities for PPH. (CHEST 1998; 114:231S-236S)

IMMUNOGENETIC ASPECTS OF PRIMARY P ULMONARY HYPERTENSION

primary pulmonary hypertension (PPH) is a welldescribed clinical entity of unknown-etiology that is found in association with diseases and conditions that have immune dysregulation: autoimmune diseases, drug therapy, and HIV infection. The clinical association between autoimmunity and PPH, ie, the increased frequency of Raynaud's phenomenon and antinuclear antibodies, has prompted studies to determine whether patients with PPH can be divided into immunogenetic subsets. Pulmonary hypertension, often resembling PPH, can also be a clinical component of connective tissue diseases, including systemic lupus erythematosus, scle roderma, rheumatoid arthritis, dermatomyositis, and mixed connective tissue disease.l-5 Historically, the strongest immunogenetic associations have resulted from autoantibody correlations with human leukocyte antigen (HLA) class 2 alleles. 6 Levels of antinuclear antibodies are significantly increased in PPH,7 but only a few PPH patients have "marker" autoantibodies characteristic of a specific connective tissue disease. Patients with PPH have an increased frequency of the anti-KU *From the Division of Cardiology (Dr. Barst), Department of Pediatrics, Columbia University College of Physicians and Surgeons, New York; and the Division of Pulmonary and Critical Care Medicine (Dr. Loyd ), Vande rbilt University School of Medicine, Nashville, Tenn. Correspondence to: Robyn Barst, MD, Pediatric Pulmonary, Rm 102, Columbia-Presbyterian Med Ctr, 3959 Broadway, New York, NY 10032-1537

autoantibody,8 but this antibody is not specific for PPH. HLA class 2 alleles encoded within the major histocompatibility complex (MHC) appear to play important roles in autoimmune diseases.6 The associations of HLA with PPH and with connective tissue diseases associated with pulmonary hypertension have been varied and complex. Increased frequency of HLA-DR 52 has been found in patients with scleroderma and pulmonary hypertension9 and of HLA-DR3 and HLA-DR52 in children with PPH.l0 More recently, an increased frequency of HLA-DQ7 was reported in both children and adults with PPH .11 This association of PPH in children and adults with HLA-DQ7 is potentially an important finding, since this allele has been associated with the lupus anticoagulant in patients with systemic lupus erythematosus. Furthermore, HLA-DQ7 shares the same allele as the antiphospholipid antibodies (including the lupus anticoagulant). The possibility that autoimmune anticardiolipin antibodies may define a subset of patients with PPH requires examination in a larger cohort of patients. It is currently not known whether the autoimmune antiphospholipid antibodies are "marker" antibodies for thromboembolism and a hypercoagulable state, whether they have procoagulant activity by interfering with coagulation mechanisms, or whether they interact with relevant cell membranes, such as endothelial cells or platelets. Drawing the line between PPH and pulmonary hypertension secondary to connective tissues diseases is often problematic. PPH may be aforme fruste of an autoimmune disease. A PPH patient often fails to fulfill the established criteria for the CHEST I 114 I 3 I SEPTEMBER, 1998 SUPPLEMENT

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diagnosis of a connective tissue disease, even when accompanied by an autoantibody or dysregulation of a complement component or immunoglobulin isotype. Clustering of autoimmune diseases or antibodies in certain kindreds is a common finding in autoimmunity. The observation that two families in whom a child was diagnosed as having PPH had one or more family members with a defined autoimmune disease supports the hypothesis that immune mediation may be involved in the pathogenesis of some patients with PPI-1.1 2 Most autoimmune diseases are associated with increased frequencies of certain HLA-DR, DQ, or DP (class 2) alleles. l-ILA genotyping, either by direct sequencing of the individual genes or the use of multiple sequence-specific oligonucleotide probes, has provided the technology that has aided discrete correlations of disease susceptibility with nucleic acid sequences. These associations are often increased by making autoantibody-l-ILA correlations. These molecules, present on antigen-presenting cells, are encoded within the MHC locus on chromosome 6. It is presently not known whether their role in antigen presentation and T -cell selection within the thymus is responsible for the l-ILA association. Increased susceptibility to autoimmune diseases could be result from genes closely linked to the l-ILA region. The l-ILA susceptibility genes are permissive for disease, subject to additional genetic and environmental modifying influences. Several recent studies have reported autoantibody-l-ILA correlations in subsets of PPH. AntiI-IMG (high-mobility group) antibodies have been reported in systemic lupus erythematosus, druginduced autoimmunity, scleroderma, and juvenile rheumatoid arthritis. 13 · 15 Antibodies to the nucleosome-associated protein HMG-14 and I-ll-histone and the immunogenetic correlation between antibodies to HMG-14117 and HLA-DQ6 have also been observed in a subset of adults with PPI-1. 16 The failure to find antibodies to any type of HM G protein in children with PPH may reflect the decreased frequency of autoantibodies in children compared with adults, or immaturity and/or anergy of the immune system in these children. Our clinical experience has been that children with PPH, although initially antinuclear antibody negative, develop antinuclear antibodies. This seroconversion to positive antinuclear antibodies is compatible with maturation of the immune system. In addition, the observation of a high incidence of maternal antinuclear antibodies implicates an environmental association, either before, after, or during pregnancy.l 2 The presence of antibodies to HM G-14117 proteins, which bind nucleosomal core histones of transcriptionally active chromatin, has suggested to several investigators that 2325

the nucleosome or nucleosomal subunit, and not DNA or histone alone, is the immunogen in autoimmune disease. This suggestion, if substantiated, supports the hypothesis that a subset of patients with PPH have an autoimmune disease and an increased frequency of a particular l-ILA class 2 allele. Antibodies to mediators of endothelial dysfunction might also define immunogenetic responses. Endothelial markers of fibrinolysis, such as tissue plasminogen activator (tPA) and plasminogen activator inhibitor, have been reported to be increased in PPH patients,l 7 .18 and antibodies to fibrin-bound tPA have been found in patients with systemic lupus erythematosus 19 and scleroderma. 20 In scleroderma, the highest frequency of fibrin-bound tPA antibodies was found in the subgroup with CREST (calcinosis, Raynaud's disease, esophageal dysmotility, sclerodactyly, and telangiectasia) variant and pulmonary hypertension. 20 The possibility that antibodies to these or other markers of fibrinolysis might define a PPH subset prompted an immunogenetic study of the antifibrin-bound tPA response. We observed an increased frequency of autoantibodies to fibrinbound tPA in patients with PPH and a selected immunogenetic response to this protein marker of fibrinolysis. 21 In addition, there was a significant correlation between fibrin-bound tPA antibodies and HLA-DQ7 in these PPH patients. Whether these PPH patients with fibrin-bound tPA antibodies will develop systemic lupus erythematosus or scleroderma later in life remains unknown. The role of endothelial dysfunction in the pathogenesis of PPH remains unclear. Patients with PPH have increased endothelin, reduced excretion of a stable metabolite of protacyclin, decreased endothelial-derived nitric oxide synthetase, as well as increased levels of fibrinopeptide A and increased plasma levels of tPA.22 -24 The elevated levels of tPA normalize concomitant with the administration of prostacyclin. Survival has also increased with longterm anticoagulation, as well as long-term vasodilator therapy, particularly IV prostacyclin. Despite these findings, the precise pathophysiologic role of fibrinbound tP A antibodies in patients with PPH and in connective tissue diseases is not known. One would anticipate that antibodies against a protein marker of fibrinolysis may favor fibrin deposition in vivo. The potential pathogenetic role of autoantibodies to fibrin-bound tPA in plasminogen activation and fibrinolysis may also have therapeutic implications as we learn more about the various subsets of PPH patients. Clausell et al25 reported an increased production of fibrinonectin in donor coronary artery smooth muscle cells compared with those of the host, and the mechanism appeared to be associated with exPathogenesis of Primary Pulmonary Hypertension

pression of MHC on endothelial cells and induction of specific cytokines. Asimilar pathophysiologic condition may prevail in PPH if endothelial cells show similar patterns of activation. Inflammatory cells, however, have not been identified in the walls of arteries in PPH patients, but the process may be too advanced by the time of tissue diagnosis. However, inflammatory cells in pulmonary arteries have been described in various autoimmune disorders. Another potential subset of PPH is HIV-associated PPH. The pathogenesis of HIV-related PPH is unclear and does not appear to be due to endothelial infection with HIV-1. Investigating a possible link between HIV-PPH and autoimmunity, we typed HIV-PPH patients and observed an increased frequency of HLA-DR6. 26 This observation further supports the hypothesis that there are various subsets in PPH that have an autoimmune component to the pathogenesis of the disease. The HIV-1 envelope glycoprotein-120 stimulates monocytic endothelin-1 production, which is not detectable in monocytes from healthy control subjects. Monocytes are a target for the HIV virus and macrophage-monocytes produce multifunctional lymphokines. The possibility that monocytic or endothelial-derived endothelin plays a role in HIV-associated PPH needs to be addressed. Pulmonary hypertension has also been associated with a number of drugs and environmental agents. The role of a direct toxic agent vs an immunemediated event has not been defined in these associations (to our knowledge), but drugs and environmental agents can induce autoantibodies and autoimmune diseases. The increased incidence of PPH associated with appetite-suppressant drugs raises the question whether these patients also have an immunogenetic predisposition to develop pulmonary vascular disease. 27 We are currently investigating this question, looking for specific autoantibodies, as well as HLA class 2 associations as we have previously done with the various subgroups of PPH patients as discussed above.

FAMILIAL PRIMARY PULMONARY HYPERTENSION PPH in families was recognized in the early 1950s by Dresdale and colleagues, 28 •29 within a few years of their first clinical description of PPH. Since then, families with PPH have been reported from countries all around the world. The symptoms, clinical manifestations, and outcome appear to be identical regardless of whether PPH occurs sporadically or in families. It occurs in either gender at virtually any age, but it affects women nearly twice as commonly as men.

Incidence In the National Institutes of Health registry for PPH,3o a natural history study that enrolled 187 PPH patients from 32 US centers, 12 of the registry patients (6%) had a first-order relative with PPH. It seems likely that the proportion of PPH that is familial may be underestimated due to poor recognition of a familial basis. Because the number of affected individuals in many families may be relatively few (low penetrance) and the gene may be passed serially through unaffected individuals who never develop PPH (incomplete penetrance), the relationship of diseased members may be distant and difficult to recognize. A registry of PPH families at Vanderbilt University has records of 60 US families, in which 175 patients among 1,859 members have developed PPH. Associated Ahnomwlities A variety of interesting associated abnormalities have been reported in PPH families. Antinuclear antibodies occur commonly in PPH, and an increased risk of autoimmune diseases has been associated with genes closely linked to HLA. Morse et al,3 1 reported an HLA association of familial PPH in 15 members from four families with PPH. Eight patients were HLA DRw52, and seven were HLA DR3, DRw52, DQw2, suggesting that there is a susceptibility factor for PPH located within or near the MHC locus on chromosome 6p21.3. Platelet abnormalities could be pathogenetically related to pulmonary hypertension in many ways, including the release of substances that are vasoactive or that stimulate proliferation. Herve et aP2 described a 46-year-old man who developed pulmonary hypertension in the setting of an inherited bleeding disorder, platelet delta storage pool disease, associated with a high level of 5-hydroxytryptamine in plasma. Familial pulmonary hypertension associated with an abnormal hemoglobin (Warsaw), which has an abnormality of the !3-chain that is associated with lower oxygen affinity, was first reported by Rich and Hart,33 and later with another hemoglobin abnormality (Washtenaw) by Wille et al. 34 Defective fibrinolysis was reported in one family, 35 but subsequent studies in another family3 6 had normal results. It is suspected that there may be an important pathogenetic relevance of these interesting associations that has yet to be fully elucidated. Lung Vascular Pathology It is not known whether the different subsets of PPH described by pathologists actually represent CHEST /114 I 3/ SEPTEMBER, 1998 SUPPLEMENT

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different basic disease processes or are simply different manifestations of a single underlying mechanism . PPH in families is of special value for understanding pathogenetic mechanisms of disease because it is the only circumstance in which different individuals with PPH (within each family) can be reasonably presumed to have the same basic pathogenesis. The most common pathologic subsets of PPH are plexogenic PPH, defined by concentric intimal fibrosis and plexogenic lesions, and thromboembolic PPH, characterized by vessels with eccentric intimal fibrosis. Because there must be a single pathogenetic basis within each family, the variety of pathologic lesions in all patients in that family should reflect only that underlying mechanism. In one study, lung specimens from 23 affected members of 13 PPH families were recovered and analyzed. 37 This study categorized and analyzed 2,516 vessels, with a mean of 109 vessels per patient (range, 38 to 264). It found marked heterogeneity of the types of vascular lesions within and among families , including the frequent coexistence of thrombotic and plexiform lesions. It suggests that the most common pathologic subsets, plexogenic and thromboembolic PPH, are different pathologic manifestations of the same process, and not different disease processes. In one PPH family, the most common lesion type differed in each of three patients; pathologic subsets should not differ among members of the same family unless the subsets have the same basic pathogenesis. Other reports of the familial occurrence of uncommon and rare subsets of PPH, pulmonary vena-occlusive disease38·39 and pulmonmy capillary hemangiomatosis 40 have also been described.

finding that subsequent generations develop PPH at successively younger ages, a phenomenon known as genetic anticipation.42 These unusual features of familial transmission of PPH, including genetic anticipation, incomplete penetrance, and variable age of onset are all typical of a molecular mechanism of disease called trinucleotide repeat expansion. It was first reported in 1991 as the basis for the fragile X syndrome, and is now believed to be the basis of 10 different neurologic diseases. 43·44 To date and to our knowledge, trinucleotide repeat expansion is the only biological mechanism that has been reported to cause genetic anticipation. PPH Gene Linkage Searches with microsatellite markers spread at intervals across the human genome have been performed in PPH families by two different teams and have linked a PPH locus with definitive logarithm of odds (LOD) scores to chromosome 2q31. Six PPH families in one study with a LOD score45 >7 and two PPH families with LOD score46 >3 confirm with certainty that this region of chromosome 2 holds a gene(s) for PPH. PPH was subsequently diagnosed in a presumed-to-be-unaffected sister of a patient in one of these families with familial PPH after identifying that the disease-associated haplotype (cosegregating with the disease ) in the three affected family members was also present in the family member thought to be asymptomatic. 47 To date and to our knowledge, there has been no report of familial PPH linked to any other chromosomal location.

Genetic Transmission Vertical transmission of PPH has been demonstrated in several PPH families , and has caused documented disease in as many as five successive generations in one Arizona family. Such vertical transmission is highly indicative of a single dominant gene. Father to son transmission of disease was demonstrated in seven cases, which excludes X chromosome linkage. The transmission patterns of familial PPH are compatible with autosomal dominant, but the analysis is complicated by incomplete penetrance, increased prevalence in women, and variable age of onset. 41 Cumulative mortality curves of deaths from PPH in men vs women are identical; they show that 20% of the mortality occurs below age 20 years, and 20% occurs above age 50 years. Another observation, that fewer male subjects than normal are born to parents who have the PPH gene, suggests the possibility that the gene could have a developmental influence. Of special interest is the 2345

Future Possibilities-More Specific Understanding of PPH Pathogenesis It is currently not known how many different diseases cause clinical PPH. Although valuable scientific information about biochemical and cellular mechanisms has been garnered in the last several years, the central basic cause remains unknown. The identification of a specific gene for PPH and the subsequent understanding of its effects appear to have the greatest likelihood of identification of the basic cause of PPH. The clinical diagnosis of PPH is made by excluding other causes of pulmonary hypertension; there is not even one pathognomonic finding. The identification of a specific gene or cause of PPH might provide a benchmark or gold standard to categorize this disease(s). Specific understanding about one actual cause of PPH should help determine how many diseases can cause PPH, as well as bringing focus to new treatments. Pathogenesis of Primary Pulmonary Hypertension

Future Possibilities-Genetic Testing The testing of individual members of PPH families to quantifY risk assessment for the later development of PPH may be of substantial value. However, the social and psychological impact of genetic testing can be immense, regardless of whether the results are positive or negative. The linkage of markers on chromosome 2q31 to the PPH gene could already be used to provide refined estimates of risk of disease for individuals in selected PPH families. However, because penetrance is so variable, possessing the marker for the gene does not necessarily mean that an individual will develop the disease. The incomplete penetrance discussed above, where an individual has progeny with PPH but does not have the disease, is a similar circumstance in which that individual is known to have the gene but does not have disease, and may never develop it. There is no consensus about a recommendation for such unaffected individuals known to have the PPH gene. Attempts to improve risk stratification for individual family members might best be postponed at present until the specific gene defect and its consequences are defined with sufficient certainty to provide substantial insight, rather than the imperfect knowledge provided at present. In most of the diseases caused by trinucleotide repeat expansion, the size of the expansion correlates with penetrance of the disease and with the age of onset. So if this molecular mechanism were found to be the cause of PPH, it should be possible to identify that the individual has the abnormal gene, and even to predict whether disease would be expected and at what age it might develop.

Future Possibilities-Basic Pathogenesis of Related Causes of Pulmonary Hypertension Many other causes of pulmonary hypertension are similar to PPH, both clinically and pathologically. Especially those disorders that cause plexogenic pulmonary hypertension, such as congenital heart disease or portal hypertension, seem likely to be based on similar pathogenetic mechanisms that remain to be fully defined. A firm understanding of the pathogenesis of PPH seems likely to have relevance in virtually every secondary cause of pulmonary hypertension.

Future Possibilities-More Specific Therapy/Gene Therapy Several new therapeutic agents have been developed that have relevance to many of the recently proved abnormalities of biochemical and cellular mediators in pulmonary hypertension. The identifi-

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Pathogenesis of Primary Pulmonary Hypertension