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Pulmonary arterial hypertension associated with human immunodeficiency virus infection: study of 4 cases
Med Clin (Barc). 2016;146(8):350–353
www.elsevier.es/medicinaclinica
Clinical report
Pulmonary arterial hypertension associated with human immunodeficiency virus infection: study of 4 cases夽 ˜ d , Diana Valverde a,b,∗ Guillermo Pousada a,b , Adolfo Baloira c , Olalla Castro-Anón a
Departamento de Bioquímica, Genética e Inmunología, Facultad de Biología, Universidad de Vigo, Vigo, Spain Instituto de Investigación Biomédica de Ourense-Pontevedra-Vigo, Pontevedra, Spain Servicio de Neumología, Complexo Hospitalario Universitario de Pontevedra, Pontevedra, Spain d Servicio de Neumología y Unidad de Desórdenes del Sue˜ no, Hospital Universitario Lucus Augusti, Lugo, Spain b c
a r t i c l e
i n f o
Article history: Received 6 November 2015 Accepted 17 December 2015 Available online 24 June 2016 Keywords: Pulmonary arterial hypertension Human immunodeficiency virus Ambrisentan Mutational analysis
a b s t r a c t Background and objective: Pulmonary arterial hypertension (PAH) is a rare and progressive disease that can be inherited as autosomal dominant form. The BMPR2, ACVRL1 and ENG genes are main genes involved in the pathology. PAH associated to human immunodeficiency virus (HIV) is another rare disease with a low incidence, prevalence and survival. The main objective of this analysis was to study the clinical and molecular characteristics of PAH associated to HIV patients. Patients: We present 4 cases of HIV patients who developed PAH and have been treated with ambrisentan. Results: Pathogenic mutations have been identify in analyzed genes in 3 of the four analyzed patients. In addition, these patients present other changes classified as benign after a thorough in silico analysis. We identified some changes in genetic modifiers that predispose to these patients to more severe phenotype. Conclusions: The clinical analysis can help to define monitoring for these patients and the administration of appropriate treatment. These patients also have shown several pathogenic mutations. ˜ S.L.U. All rights reserved. © 2016 Elsevier Espana,
Hipertensión arterial pulmonar asociada a infección por el virus de la inmunodeficiencia humana: análisis de 4 casos r e s u m e n Palabras clave: Hipertensión arterial pulmonar Virus de la inmunodeficiencia humana Ambrisentan Análisis mutacional
Fundamentos y objetivos: La hipertensión arterial pulmonar (HAP) es una enfermedad rara y progresiva que se puede heredar de forma autosómica dominante. Los genes BMPR2, ACVRL1 y ENG son los principales relacionados con la enfermedad. La HAP asociada al virus de la inmunodeficiencia humana (VIH) es otra enfermedad rara con una incidencia, prevalencia y supervivencia muy bajas. El principal objetivo de este trabajo fue analizar las características clínicas y moleculares de pacientes con HAP asociada al VIH. Pacientes: Presentamos 4 casos de pacientes con VIH que han desarrollado HAP y han sido tratados con ambrisentan. Resultados: Se han identificado mutaciones patogénicas en los genes analizados en 3 de los 4 pacientes estudiados. Asimismo, estos pacientes presentan otros cambios clasificados como benignos tras un exhaustivo análisis in silico. Tras el análisis de los modificadores genéticos se han identificado cambios que predisponen a los pacientes a padecer un fenotipo más grave. Conclusiones: El análisis clínico nos ayudará a definir un seguimiento para estos pacientes y a la administración de un tratamiento adecuado. Asimismo, estos pacientes han mostrado un elevado número de mutaciones patogénicas. ˜ S.L.U. Todos los derechos reservados. © 2016 Elsevier Espana,
夽 Please cite this article as: Pousada G, Baloira A, Castro-Anón ˜ O, Valverde D. Hipertensión arterial pulmonar asociada a infección por el virus de la inmunodeficiencia humana: análisis de 4 casos. Med Clin (Barc). 2016;146:350–353. ∗ Corresponding author. E-mail address: [email protected] (D. Valverde). ˜ S.L.U. All rights reserved. 2387-0206/© 2016 Elsevier Espana,
G. Pousada et al. / Med Clin (Barc). 2016;146(8):350–353
Introduction Pulmonary arterial hypertension (PAH) (PAH; OMIM # 178600, ORPHA 422) is a rare, progressive disease with reduced incidence and prevalence in the Spanish population, but with poor prognosis in terms of quality of life, morbidity and mortality.1 Its aetiology is diverse, producing a large variability, both clinically and genetically, and hindering the management of these patients, as well as a systematic diagnostic study.2,3 The first case of PAH associated with human immunodeficiency virus (HIV PAH), was described in 1987, with more cases being reported later. Today, HIV-PAH is considered a rare disease whose prevalence is estimated at up to 0.5%.4 These patients present with plexiform lesions similar to those of other patients with associated PAH.4–6 Diastolic dysfunction characteristic of HIV patients could contribute to increased intracardiac pressure, exacerbating the PAH phenotype.5 Viral antigens have been shown to stimulate abnormal pulmonary endothelial cell growth and proliferation, resulting in an increase in apoptotic markers and secretion of endothelin1, creating an imbalance between apoptosis and proliferation.5 Since the onset of the first symptoms until patients with HIV-PAH are diagnosed, an average of 6 months elapsed, while in patients with idiopathic PAH, the time involved is an average of 2½ years, approximately.6 The HIV-PAH patients have a 3-year survival of 84%, which is reduced to 28% in patients with severe PAH.6 59% of cases of HIV-PAH patients are using drugs intravenously.6,7 As for the genetic basis of PAH, the main gene involved is the Bone Morphogenetic Protein Receptor type II (BMPR2) located on chromosome 2q33.8 Since then, more genes have been described, such as Activin A Receptor Type II-Like 1 (ACVRL1) or Endoglin (ENG).8,9 Genetic modifiers that interact with the genes involved in the pathogenesis of PAH have also been associated, modifying the phenotype to produce vasoconstriction and proliferation (TRPC6, AGTR1, EDN1, . . .) or vasodilation (NOS2).10,11 In this paper we describe the clinical characteristics and genetic alterations of key genes and genetic modifiers of 4 patients with HIV-PAH. Clinical observation Three men and 1 woman with a mean age at diagnosis of 44 years (42–47) are included. Hepatitis C virus infection coexisted in 2 patients. The onset of symptoms of PAH occurred after an average of 6 years (3–15) following the first positive serology for HIV. In one case it was a chance discovery when performing an echocardiogram after suspected pulmonary embolism, as the patient did not report dyspnoea. In other cases, the average time elapsed since the patient experienced any symptoms until diagnosis of PAH was 1 month. Three of the patients had been addicted to intravenous drugs. All were under antiretroviral treatment. Pulmonary embolism
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was excluded in the 4 cases. Dyspnoea was the main symptom. Two patients were in functional class (FC) II and the other 2 in FC III. The echocardiogram showed severe dilation of right cavities with deviated septum to the left in all cases. The mean pulmonary arterial pressure measured by catheterization was 50 mmHg (35–57), cardiac index 2.43 l/m/m2 (1.71–3.2), portal blood pressure 11 mmHg (10–12) and pulmonary vascular resistance 780 ds/cm−5 /m2 (400–1120). The vasodilator test was negative in all patients. The average distance covered in the 6-min walk test was 350 m (225–556). In one patient, treatment was initiated with sildenafil. This had to be withdrawn due to interactions with antiretroviral drugs and was substituted with ambrisentan. Ambrisentan was chosen for the remaining patients, at a dose of 5 mg/d. A favourable response was observed in 3 patients, being necessary to add nebulized iloprost in one of them. After 3, 4 and 6 years of follow up, they continue alive in FC II. The other case responded well initially, staying in FC II for a year but showing progressive worsening of the underlying disease and died from sepsis within 20 months of PAH diagnosis. These results are shown in Table 1. Genetic study The study was approved by the Galician Regional Research Ethics Committee, in harmony with the ethical and clinical practices of the Government of Spain and the Declaration of Helsinki. All patients signed an informed consent for genetic testing. After the genetic analysis of these 4 patients, mutations were detected in genes BMPR2, ACVRL1 and ENG. Two of the patients have a pathogenic mutation in the BMPR2 gene, 2 patients have mutations in the ENG gene and only one patient is carrying a pathogenic mutation in the ACVRL1 gene. Case 1 presented a mutation in BMPR2 (p.H688Q) and ENG (p.R554C) genes, both classified as pathogenic after the in silico analysis of the changes. Case 2 showed two pathogenic mutations, one in the ACVRL1 (P.S232T) gene and another in the ENG (P.F474Y) gene. Case 3 presented a pathogenic mutation in the BMPR2 (P.V341M) gene. Finally, case 4 revealed a mutation in the ENG (P.G191N) gene, however, we cannot say whether this mutation is pathogenic, since although the in silico analysis classified it as pathogenic other studies classified it as polymorphism (Table 2). Except the p.R554C of the ENG gene, all other detected mutations are preserved throughout evolution, as these are not altered after having compared the wild-type sequence of the altered point with 10 different species, showing a high homology with other species. Therefore, the changes observed are rare variants. On the contrary, the point where the p.R554C mutation is located presents no homologies between different species (Fig. 1). Regarding the genetic modifiers, 3 of the 4 cases studied show the c.1-1853 1897del44 polymorphism in the SLC6A4 gene and the
Table 1 Clinical and haemodynamic characteristics in patients with HIV-PAH included in this analysis. Clinical and haemodynamic characteristics
Sex Age at diagnosis (years) MPAP (mmHg) SPAP (mmHg) PVR (mmHg l−1 m−1 ) CI (lm−1 m−2 ) 6MWT (m) Exitus Treatment
Patients with PAH associated with HIV Patient 1
Patient 2
Patient 3
Patient 4
Female 44 56 88 10 1.82 260 No Ambrisentan
Male 45 57 90 14 1.71 270 Yes Ambrisentan
Male 47 52 35 9 3.21 235 No Ambrisentan
Male 42 75 50 8 1.93 556 No Ambrisentan
HIV-PAH, pulmonary arterial hypertension associated with HIV; CI, cardiac index; MPAP, mean pulmonary artery pressure; SPAP, systolic pulmonary arterial pressure; PVR, pulmonary vascular resistance; 6MWT, 6-min walk test.
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G. Pousada et al. / Med Clin (Barc). 2016;146(8):350–353
Table 2 Molecular characterization of key genes and genetic modifiers of patients with HIV-PAH included in this analysis. Genes and genetic modifiers
BMPR2 ACVRL1 ENG c.1-1853 1897del44-SLC6A4 c.5665G>T-EDN1 c.1-361A>T-TRPC6a c.1-254C>G-TRPC6a c.1-218C>T-TRPC6a c.1166A>C-AGTR1 CCTTT-NOS2b
Patients with PAH associated with HIV Patient 1
Patient 2
Patient 3
Patient 4
(p.H688Q) – (p.R554C) S/L G/T A/T C/G C/T A/C 9/16
– (p.S232T) (p.F474Y) S/S G/T A/A C/C C/C A/C 12/10
(p.V341M) – – L/L G/T A/T C/G C/T A/C 9/11
– – (p.G191N) L/L G/T A/A C/G C/C A/A 8/12
HIV-PAH, pulmonary arterial hypertension associated with human immunodeficiency virus. a The 3 polymorphisms of the TRPC6 gene are associated with a worse prognosis, being the carriers of the 3 polymorphisms more susceptible to severe disease. b It has been shown that patients with a number of CCTTT repeats in the NOS2 gene ≤12 have their vasodilator capacity reduced compared to patients who have at least one allele with more than 12 repetitions.
c.1021G > A (p.V341M) – BMPR2
c.572G > A (p.G191D) ENG
c.2064C > G (p.H688Q) – BMPR2
c.694T > A (p.S232T) – ACVRL1
c.1402G > C (p.F474Y) ENG
c.1660C > A (p.R554C) ENG
Fig. 1. Electropherograms of different mutations identified in patients with HIV-PAH and their orthologues. (1) Homo sapiens (sp. | P17813 # 1); (2) Homo sapiens mutated (sp. | P17813 # 1); (3) Mus musculus (sp. | Q63961 # 1); (4) Rattus norvegicus (sp. | Q6Q3E8 # 1); (5) Macaca mulatta (sp. | F7BB68 # 1); (6) Sus scrofa (sp. | P37176 # 1); (7) Oryctolagus cuniculus (sp. | G1SSF2 # 1); (8) Canis familiaris (sp. | F1P847 # 1); (9) Bos taurus (sp. | Q1RMV1 # 1); (10) Equus (sp. | W046 F6 # 1); (11) Loxodonta africana (sp. | G3SR82 # 1); (12): Ailuropoda melanoleuca (sp. | G1 M9D6 # 1).
c.5665G>T polymorphism of the EDN1 gene appears in all cases. On the contrary, as can be seen in Table 2, polymorphisms associated with the TRPC6 gene show variability among patients. The c.1166A>C polymorphism of the AGTR1 gene appears in 3 of the 4 patients included. Finally, in all cases, the CCTTT repeat polymorphism of the NOS2 gene shows at least one allele with less than 12 repetitions.
Discussion We present 4 patients with HIV-PAH, describing their clinical features, therapeutic response and genetic study of the major genes associated with PAH and genetic modifiers. Since the first case of HIV-PAH was described, there has been speculation about its prevalence, which is variable in the different populations analyzed.4,12
G. Pousada et al. / Med Clin (Barc). 2016;146(8):350–353
To date very few series of HIV-PAH cases have been published because of its rarity.13 Today PAH is considered as one of the most serious complications HIV patients can have. This involvement occurs in all stages of the disease and does not appear to be related to the degree of immune deficiency.14 However, HIV-PAH presents clinical features, pathological manifestations and imaging that is very similar to that of patients with idiopathic PAH, making their differentiation difficult when based on its clinical signs and symptoms.13 There is a female predominance in patients with PAH, however, there are some differences between the various types of PAH. In idiopathic PAH, the ratio of women affected with respect to men is 1.7:1 in the USA register and 1.6:1 in the National Register of France.7,15 In the case of patients with HIV-PAH, 55% are male,7 with a ratio of men to women of 3:1. This may be due to the prevalence of drug addiction in men over women. Three of our patients showed a satisfactory progression during follow-up. By contrast, the fourth patient died after a progressive worsening. All patients were treated with ambrisentan. Data on specific treatment efficacy for PAH associated with HIV are limited. To date there have been no reported studies aimed at these patients.13 In addition to the rarity of the disease, another reason for this may be that patients with HIV-PAH are excluded from clinical trials because of the possibility that therapies for PAH interfere with antiretroviral drugs and treatment compliance is not optimal. Meaningful conclusions about treatment efficacy cannot be drawn from our small series. The published mean survival after 3 years of follow up is around 21%.5,16 However, in our patients it reached 75%. One of the most relevant data of this study is the presence of several pathogenic mutations in 2 patients analyzed and a pathogenic mutation in another patient, identified in the BMPR2, ACVRL1 and ENG genes. These amino acid change mutations alter the protein sequence and may produce a protein misfolding, altering its subcellular localization, or cause alterations in mRNA processing.8,17–19 Another patient presented a p.G191N change, but we cannot confirm its involvement in the development of PAH as it has been described by some authors as polymorphism, although in silico analyses classify it as pathogenetic.19,20 All patients had a change in genetic modifiers with potential vasoconstrictor effect in the pulmonary arteries, possibly favouring a more severe PAH phenotype.10,11 In conclusion, we show a number of HIV-PAH cases treated with ambrisentan, the majority of which showed an improvement. To date this is the first Spanish study where the mutational frequency of patients with HIV-PAH was analyzed, confirming the importance of such studies. Our results highlight the importance of genetics in HIV-PAH onset, together with the HIV effects themselves. Funding This study has been funded with the projects IN-202-05 of SOGAPAR, CO-0118-2012 of Actelion Pharmaceuticals and INBIOMED 2009-063 of Xunta de Galicia.
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Conflict of interest The authors declare that they have no conflicts of interest. Acknowledgements We would like to thank the patients who have given their consent to participate in this study. References 1. Hoeper MM, Bogaard HJ, Condliffe R, Frantz R, Khanna D, Kurzyna M, et al. Definitions and diagnosis of pulmonary hypertension. J Am Coll Cardiol. 2013;62:25S. 2. Peacock AJ, Murphy NF, McMurray JJV, Caballero L, Stewart S. An epidemiological study of pulmonary arterial hypertension. Eur Respir J. 2007;30:104–9. 3. Simonneau G, Gatzoulis MA, Adatia I, Celermajer D, Denton C, Ghofrani A, et al. Updated clinical classification of pulmonary hypertension. J Am Coll Cardiol. 2013;62:25s. 4. Araújo I, Enjuanes-Grau C, Lopez-Guarch CJ, Narankiewicz D, Ruiz-Cano MJ, Velazquez-Martin T, et al. Pulmonary arterial hypertension related to human immunodeficiency virus infection: a case series. World J Cardiol. 2014;6:495–501. 5. Barnett CF, Hsue PY. Human immunodeficiency virus-associated pulmonary arterial hypertension. Clin Chest Med. 2013;34:283–92. 6. Correale M, Palmiotti GA, lo Storto MM, Montrone D, Foschino Barbaro MP, di Biase M, et al. HIV-associated pulmonary arterial hypertension: from bedside to the future. Eur J Clin Investig. 2015;45:515–28. 7. Sanchez O, Marié E, Lerolle U, Wermert D, Israël-Biel D, Meyer G. Pulmonary arterial hypertension in women. Rev Mal Respir. 2010;27:e79–87. ˜ C, Cifrián JM, Valverde D. Novel mutations in 8. Pousada G, Baloira A, Vilarino BMPR2, ACVRL1 and KCNA5 genes and hemodynamic parameters in patients with pulmonary arterial hypertension. PLOS ONE. 2014;9:e100261. 9. Pffar N, Szamalek-Hoegel J, Fischer C, Hinderhofer K, Nagel C, Eklken N, et al. Hemodynamic and clinical onset in patients with hereditary pulmonary arterial hypertension and BMPR2 mutations. Respir Res. 2011;12:99–109. 10. Pousada G, Baloira A, Valverde D. Molecular and clinical analysis of TRPC6 and AGTR1 genes in patients with pulmonary arterial hypertension. Orphanet J Rare Dis. 2015;10:1. ˜ ˜ 11. Baloira-Villar A, Pousada-Fernández G, Vilarino-Pombo C, Núnez-Fernández M, Cifrián-Martínez J, Valverde-Pérez D. CCTTT pentanucleotide repeats in inducible nitric oxide synthase gene expression in patients with pulmonary arterial hypertension. Arch Bronconeumol. 2014;50:141–5. 12. Tcherakian C, Rivaud E, Zucman D, Metivier AC, Couderc LJ. Curing HIVassociated pulmonary arterial hypertension. Eur Respir J. 2012;39:1045–6. 13. Janda S, Quon BS, Swiston J. HIV and pulmonary arterial hypertension: a systematic review. HIV Med. 2010;11:620–34. 14. Cicalini S, Almodovar S, Grilli E, Flores S. Pulmonary hypertension and human immunodeficiency virus infection: epidemiology, pathogenesis, and clinical approach. Clin Microbiol Infect. 2011;17:25–33. 15. Austin ED, Cogan JD, West JD, Hedges LK, Hamid R, Dawson EP, et al. Alterations in oestrogen metabolism: Implications for higher penetrance of familial pulmonary arterial hypertension in females. Eur Respir J. 2009;34:1093–9. 16. Almodovar S, Cicalini S, Petrosillo N, Flores SC. Pulmonary hypertension associated with HIV infection: pulmonary vascular disease: the global perspective. Chest. 2010;137 6 Suppl.:6S–12S. 17. Elliot CG, Glissmeyer EW, Havlena GT, Carlquist J, McKinney JT, Rich S, et al. Relationship of BMPR2 mutations to vasoreactivity in pulmonary arterial hypertension. Circulation. 2006;113:2509–15. 18. Liu D, Wu WH, Mao YM, Yuan P, Zhang R, Ju FL, et al. BMPR2 mutations influence phenotype more obviously in male patients with pulmonary arterial hypertension. Circ Cardiovasc Genet. 2012;5:511–8. 19. Mallet C, Lamribet K, Giraud S, Dupuis-Girod S, Feige JJ, Bailly S, et al. Functional analysis of endoglin mutations from hereditary hemorrhagic telangiectasia type 1 patients reveals different mechanisms for endoglin loss of function. Hum Mol Genet. 2015;144:261–4. 20. Förg T, Hafner M, Lux A. Investigation of endoglin wild-type and missense mutant protein heterodimerisation using fluorescence microscopy based IF, BiFC and FRET analyses. PLOS ONE. 2014;9:e102998.
www.elsevier.es/medicinaclinica
Clinical report
Pulmonary arterial hypertension associated with human immunodeficiency virus infection: study of 4 cases夽 ˜ d , Diana Valverde a,b,∗ Guillermo Pousada a,b , Adolfo Baloira c , Olalla Castro-Anón a
Departamento de Bioquímica, Genética e Inmunología, Facultad de Biología, Universidad de Vigo, Vigo, Spain Instituto de Investigación Biomédica de Ourense-Pontevedra-Vigo, Pontevedra, Spain Servicio de Neumología, Complexo Hospitalario Universitario de Pontevedra, Pontevedra, Spain d Servicio de Neumología y Unidad de Desórdenes del Sue˜ no, Hospital Universitario Lucus Augusti, Lugo, Spain b c
a r t i c l e
i n f o
Article history: Received 6 November 2015 Accepted 17 December 2015 Available online 24 June 2016 Keywords: Pulmonary arterial hypertension Human immunodeficiency virus Ambrisentan Mutational analysis
a b s t r a c t Background and objective: Pulmonary arterial hypertension (PAH) is a rare and progressive disease that can be inherited as autosomal dominant form. The BMPR2, ACVRL1 and ENG genes are main genes involved in the pathology. PAH associated to human immunodeficiency virus (HIV) is another rare disease with a low incidence, prevalence and survival. The main objective of this analysis was to study the clinical and molecular characteristics of PAH associated to HIV patients. Patients: We present 4 cases of HIV patients who developed PAH and have been treated with ambrisentan. Results: Pathogenic mutations have been identify in analyzed genes in 3 of the four analyzed patients. In addition, these patients present other changes classified as benign after a thorough in silico analysis. We identified some changes in genetic modifiers that predispose to these patients to more severe phenotype. Conclusions: The clinical analysis can help to define monitoring for these patients and the administration of appropriate treatment. These patients also have shown several pathogenic mutations. ˜ S.L.U. All rights reserved. © 2016 Elsevier Espana,
Hipertensión arterial pulmonar asociada a infección por el virus de la inmunodeficiencia humana: análisis de 4 casos r e s u m e n Palabras clave: Hipertensión arterial pulmonar Virus de la inmunodeficiencia humana Ambrisentan Análisis mutacional
Fundamentos y objetivos: La hipertensión arterial pulmonar (HAP) es una enfermedad rara y progresiva que se puede heredar de forma autosómica dominante. Los genes BMPR2, ACVRL1 y ENG son los principales relacionados con la enfermedad. La HAP asociada al virus de la inmunodeficiencia humana (VIH) es otra enfermedad rara con una incidencia, prevalencia y supervivencia muy bajas. El principal objetivo de este trabajo fue analizar las características clínicas y moleculares de pacientes con HAP asociada al VIH. Pacientes: Presentamos 4 casos de pacientes con VIH que han desarrollado HAP y han sido tratados con ambrisentan. Resultados: Se han identificado mutaciones patogénicas en los genes analizados en 3 de los 4 pacientes estudiados. Asimismo, estos pacientes presentan otros cambios clasificados como benignos tras un exhaustivo análisis in silico. Tras el análisis de los modificadores genéticos se han identificado cambios que predisponen a los pacientes a padecer un fenotipo más grave. Conclusiones: El análisis clínico nos ayudará a definir un seguimiento para estos pacientes y a la administración de un tratamiento adecuado. Asimismo, estos pacientes han mostrado un elevado número de mutaciones patogénicas. ˜ S.L.U. Todos los derechos reservados. © 2016 Elsevier Espana,
夽 Please cite this article as: Pousada G, Baloira A, Castro-Anón ˜ O, Valverde D. Hipertensión arterial pulmonar asociada a infección por el virus de la inmunodeficiencia humana: análisis de 4 casos. Med Clin (Barc). 2016;146:350–353. ∗ Corresponding author. E-mail address: [email protected] (D. Valverde). ˜ S.L.U. All rights reserved. 2387-0206/© 2016 Elsevier Espana,
G. Pousada et al. / Med Clin (Barc). 2016;146(8):350–353
Introduction Pulmonary arterial hypertension (PAH) (PAH; OMIM # 178600, ORPHA 422) is a rare, progressive disease with reduced incidence and prevalence in the Spanish population, but with poor prognosis in terms of quality of life, morbidity and mortality.1 Its aetiology is diverse, producing a large variability, both clinically and genetically, and hindering the management of these patients, as well as a systematic diagnostic study.2,3 The first case of PAH associated with human immunodeficiency virus (HIV PAH), was described in 1987, with more cases being reported later. Today, HIV-PAH is considered a rare disease whose prevalence is estimated at up to 0.5%.4 These patients present with plexiform lesions similar to those of other patients with associated PAH.4–6 Diastolic dysfunction characteristic of HIV patients could contribute to increased intracardiac pressure, exacerbating the PAH phenotype.5 Viral antigens have been shown to stimulate abnormal pulmonary endothelial cell growth and proliferation, resulting in an increase in apoptotic markers and secretion of endothelin1, creating an imbalance between apoptosis and proliferation.5 Since the onset of the first symptoms until patients with HIV-PAH are diagnosed, an average of 6 months elapsed, while in patients with idiopathic PAH, the time involved is an average of 2½ years, approximately.6 The HIV-PAH patients have a 3-year survival of 84%, which is reduced to 28% in patients with severe PAH.6 59% of cases of HIV-PAH patients are using drugs intravenously.6,7 As for the genetic basis of PAH, the main gene involved is the Bone Morphogenetic Protein Receptor type II (BMPR2) located on chromosome 2q33.8 Since then, more genes have been described, such as Activin A Receptor Type II-Like 1 (ACVRL1) or Endoglin (ENG).8,9 Genetic modifiers that interact with the genes involved in the pathogenesis of PAH have also been associated, modifying the phenotype to produce vasoconstriction and proliferation (TRPC6, AGTR1, EDN1, . . .) or vasodilation (NOS2).10,11 In this paper we describe the clinical characteristics and genetic alterations of key genes and genetic modifiers of 4 patients with HIV-PAH. Clinical observation Three men and 1 woman with a mean age at diagnosis of 44 years (42–47) are included. Hepatitis C virus infection coexisted in 2 patients. The onset of symptoms of PAH occurred after an average of 6 years (3–15) following the first positive serology for HIV. In one case it was a chance discovery when performing an echocardiogram after suspected pulmonary embolism, as the patient did not report dyspnoea. In other cases, the average time elapsed since the patient experienced any symptoms until diagnosis of PAH was 1 month. Three of the patients had been addicted to intravenous drugs. All were under antiretroviral treatment. Pulmonary embolism
351
was excluded in the 4 cases. Dyspnoea was the main symptom. Two patients were in functional class (FC) II and the other 2 in FC III. The echocardiogram showed severe dilation of right cavities with deviated septum to the left in all cases. The mean pulmonary arterial pressure measured by catheterization was 50 mmHg (35–57), cardiac index 2.43 l/m/m2 (1.71–3.2), portal blood pressure 11 mmHg (10–12) and pulmonary vascular resistance 780 ds/cm−5 /m2 (400–1120). The vasodilator test was negative in all patients. The average distance covered in the 6-min walk test was 350 m (225–556). In one patient, treatment was initiated with sildenafil. This had to be withdrawn due to interactions with antiretroviral drugs and was substituted with ambrisentan. Ambrisentan was chosen for the remaining patients, at a dose of 5 mg/d. A favourable response was observed in 3 patients, being necessary to add nebulized iloprost in one of them. After 3, 4 and 6 years of follow up, they continue alive in FC II. The other case responded well initially, staying in FC II for a year but showing progressive worsening of the underlying disease and died from sepsis within 20 months of PAH diagnosis. These results are shown in Table 1. Genetic study The study was approved by the Galician Regional Research Ethics Committee, in harmony with the ethical and clinical practices of the Government of Spain and the Declaration of Helsinki. All patients signed an informed consent for genetic testing. After the genetic analysis of these 4 patients, mutations were detected in genes BMPR2, ACVRL1 and ENG. Two of the patients have a pathogenic mutation in the BMPR2 gene, 2 patients have mutations in the ENG gene and only one patient is carrying a pathogenic mutation in the ACVRL1 gene. Case 1 presented a mutation in BMPR2 (p.H688Q) and ENG (p.R554C) genes, both classified as pathogenic after the in silico analysis of the changes. Case 2 showed two pathogenic mutations, one in the ACVRL1 (P.S232T) gene and another in the ENG (P.F474Y) gene. Case 3 presented a pathogenic mutation in the BMPR2 (P.V341M) gene. Finally, case 4 revealed a mutation in the ENG (P.G191N) gene, however, we cannot say whether this mutation is pathogenic, since although the in silico analysis classified it as pathogenic other studies classified it as polymorphism (Table 2). Except the p.R554C of the ENG gene, all other detected mutations are preserved throughout evolution, as these are not altered after having compared the wild-type sequence of the altered point with 10 different species, showing a high homology with other species. Therefore, the changes observed are rare variants. On the contrary, the point where the p.R554C mutation is located presents no homologies between different species (Fig. 1). Regarding the genetic modifiers, 3 of the 4 cases studied show the c.1-1853 1897del44 polymorphism in the SLC6A4 gene and the
Table 1 Clinical and haemodynamic characteristics in patients with HIV-PAH included in this analysis. Clinical and haemodynamic characteristics
Sex Age at diagnosis (years) MPAP (mmHg) SPAP (mmHg) PVR (mmHg l−1 m−1 ) CI (lm−1 m−2 ) 6MWT (m) Exitus Treatment
Patients with PAH associated with HIV Patient 1
Patient 2
Patient 3
Patient 4
Female 44 56 88 10 1.82 260 No Ambrisentan
Male 45 57 90 14 1.71 270 Yes Ambrisentan
Male 47 52 35 9 3.21 235 No Ambrisentan
Male 42 75 50 8 1.93 556 No Ambrisentan
HIV-PAH, pulmonary arterial hypertension associated with HIV; CI, cardiac index; MPAP, mean pulmonary artery pressure; SPAP, systolic pulmonary arterial pressure; PVR, pulmonary vascular resistance; 6MWT, 6-min walk test.
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Table 2 Molecular characterization of key genes and genetic modifiers of patients with HIV-PAH included in this analysis. Genes and genetic modifiers
BMPR2 ACVRL1 ENG c.1-1853 1897del44-SLC6A4 c.5665G>T-EDN1 c.1-361A>T-TRPC6a c.1-254C>G-TRPC6a c.1-218C>T-TRPC6a c.1166A>C-AGTR1 CCTTT-NOS2b
Patients with PAH associated with HIV Patient 1
Patient 2
Patient 3
Patient 4
(p.H688Q) – (p.R554C) S/L G/T A/T C/G C/T A/C 9/16
– (p.S232T) (p.F474Y) S/S G/T A/A C/C C/C A/C 12/10
(p.V341M) – – L/L G/T A/T C/G C/T A/C 9/11
– – (p.G191N) L/L G/T A/A C/G C/C A/A 8/12
HIV-PAH, pulmonary arterial hypertension associated with human immunodeficiency virus. a The 3 polymorphisms of the TRPC6 gene are associated with a worse prognosis, being the carriers of the 3 polymorphisms more susceptible to severe disease. b It has been shown that patients with a number of CCTTT repeats in the NOS2 gene ≤12 have their vasodilator capacity reduced compared to patients who have at least one allele with more than 12 repetitions.
c.1021G > A (p.V341M) – BMPR2
c.572G > A (p.G191D) ENG
c.2064C > G (p.H688Q) – BMPR2
c.694T > A (p.S232T) – ACVRL1
c.1402G > C (p.F474Y) ENG
c.1660C > A (p.R554C) ENG
Fig. 1. Electropherograms of different mutations identified in patients with HIV-PAH and their orthologues. (1) Homo sapiens (sp. | P17813 # 1); (2) Homo sapiens mutated (sp. | P17813 # 1); (3) Mus musculus (sp. | Q63961 # 1); (4) Rattus norvegicus (sp. | Q6Q3E8 # 1); (5) Macaca mulatta (sp. | F7BB68 # 1); (6) Sus scrofa (sp. | P37176 # 1); (7) Oryctolagus cuniculus (sp. | G1SSF2 # 1); (8) Canis familiaris (sp. | F1P847 # 1); (9) Bos taurus (sp. | Q1RMV1 # 1); (10) Equus (sp. | W046 F6 # 1); (11) Loxodonta africana (sp. | G3SR82 # 1); (12): Ailuropoda melanoleuca (sp. | G1 M9D6 # 1).
c.5665G>T polymorphism of the EDN1 gene appears in all cases. On the contrary, as can be seen in Table 2, polymorphisms associated with the TRPC6 gene show variability among patients. The c.1166A>C polymorphism of the AGTR1 gene appears in 3 of the 4 patients included. Finally, in all cases, the CCTTT repeat polymorphism of the NOS2 gene shows at least one allele with less than 12 repetitions.
Discussion We present 4 patients with HIV-PAH, describing their clinical features, therapeutic response and genetic study of the major genes associated with PAH and genetic modifiers. Since the first case of HIV-PAH was described, there has been speculation about its prevalence, which is variable in the different populations analyzed.4,12
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To date very few series of HIV-PAH cases have been published because of its rarity.13 Today PAH is considered as one of the most serious complications HIV patients can have. This involvement occurs in all stages of the disease and does not appear to be related to the degree of immune deficiency.14 However, HIV-PAH presents clinical features, pathological manifestations and imaging that is very similar to that of patients with idiopathic PAH, making their differentiation difficult when based on its clinical signs and symptoms.13 There is a female predominance in patients with PAH, however, there are some differences between the various types of PAH. In idiopathic PAH, the ratio of women affected with respect to men is 1.7:1 in the USA register and 1.6:1 in the National Register of France.7,15 In the case of patients with HIV-PAH, 55% are male,7 with a ratio of men to women of 3:1. This may be due to the prevalence of drug addiction in men over women. Three of our patients showed a satisfactory progression during follow-up. By contrast, the fourth patient died after a progressive worsening. All patients were treated with ambrisentan. Data on specific treatment efficacy for PAH associated with HIV are limited. To date there have been no reported studies aimed at these patients.13 In addition to the rarity of the disease, another reason for this may be that patients with HIV-PAH are excluded from clinical trials because of the possibility that therapies for PAH interfere with antiretroviral drugs and treatment compliance is not optimal. Meaningful conclusions about treatment efficacy cannot be drawn from our small series. The published mean survival after 3 years of follow up is around 21%.5,16 However, in our patients it reached 75%. One of the most relevant data of this study is the presence of several pathogenic mutations in 2 patients analyzed and a pathogenic mutation in another patient, identified in the BMPR2, ACVRL1 and ENG genes. These amino acid change mutations alter the protein sequence and may produce a protein misfolding, altering its subcellular localization, or cause alterations in mRNA processing.8,17–19 Another patient presented a p.G191N change, but we cannot confirm its involvement in the development of PAH as it has been described by some authors as polymorphism, although in silico analyses classify it as pathogenetic.19,20 All patients had a change in genetic modifiers with potential vasoconstrictor effect in the pulmonary arteries, possibly favouring a more severe PAH phenotype.10,11 In conclusion, we show a number of HIV-PAH cases treated with ambrisentan, the majority of which showed an improvement. To date this is the first Spanish study where the mutational frequency of patients with HIV-PAH was analyzed, confirming the importance of such studies. Our results highlight the importance of genetics in HIV-PAH onset, together with the HIV effects themselves. Funding This study has been funded with the projects IN-202-05 of SOGAPAR, CO-0118-2012 of Actelion Pharmaceuticals and INBIOMED 2009-063 of Xunta de Galicia.
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Conflict of interest The authors declare that they have no conflicts of interest. Acknowledgements We would like to thank the patients who have given their consent to participate in this study. References 1. Hoeper MM, Bogaard HJ, Condliffe R, Frantz R, Khanna D, Kurzyna M, et al. Definitions and diagnosis of pulmonary hypertension. J Am Coll Cardiol. 2013;62:25S. 2. Peacock AJ, Murphy NF, McMurray JJV, Caballero L, Stewart S. An epidemiological study of pulmonary arterial hypertension. Eur Respir J. 2007;30:104–9. 3. Simonneau G, Gatzoulis MA, Adatia I, Celermajer D, Denton C, Ghofrani A, et al. Updated clinical classification of pulmonary hypertension. J Am Coll Cardiol. 2013;62:25s. 4. Araújo I, Enjuanes-Grau C, Lopez-Guarch CJ, Narankiewicz D, Ruiz-Cano MJ, Velazquez-Martin T, et al. Pulmonary arterial hypertension related to human immunodeficiency virus infection: a case series. World J Cardiol. 2014;6:495–501. 5. Barnett CF, Hsue PY. Human immunodeficiency virus-associated pulmonary arterial hypertension. Clin Chest Med. 2013;34:283–92. 6. Correale M, Palmiotti GA, lo Storto MM, Montrone D, Foschino Barbaro MP, di Biase M, et al. HIV-associated pulmonary arterial hypertension: from bedside to the future. Eur J Clin Investig. 2015;45:515–28. 7. Sanchez O, Marié E, Lerolle U, Wermert D, Israël-Biel D, Meyer G. Pulmonary arterial hypertension in women. Rev Mal Respir. 2010;27:e79–87. ˜ C, Cifrián JM, Valverde D. Novel mutations in 8. Pousada G, Baloira A, Vilarino BMPR2, ACVRL1 and KCNA5 genes and hemodynamic parameters in patients with pulmonary arterial hypertension. PLOS ONE. 2014;9:e100261. 9. Pffar N, Szamalek-Hoegel J, Fischer C, Hinderhofer K, Nagel C, Eklken N, et al. Hemodynamic and clinical onset in patients with hereditary pulmonary arterial hypertension and BMPR2 mutations. Respir Res. 2011;12:99–109. 10. Pousada G, Baloira A, Valverde D. Molecular and clinical analysis of TRPC6 and AGTR1 genes in patients with pulmonary arterial hypertension. Orphanet J Rare Dis. 2015;10:1. ˜ ˜ 11. Baloira-Villar A, Pousada-Fernández G, Vilarino-Pombo C, Núnez-Fernández M, Cifrián-Martínez J, Valverde-Pérez D. CCTTT pentanucleotide repeats in inducible nitric oxide synthase gene expression in patients with pulmonary arterial hypertension. Arch Bronconeumol. 2014;50:141–5. 12. Tcherakian C, Rivaud E, Zucman D, Metivier AC, Couderc LJ. Curing HIVassociated pulmonary arterial hypertension. Eur Respir J. 2012;39:1045–6. 13. Janda S, Quon BS, Swiston J. HIV and pulmonary arterial hypertension: a systematic review. HIV Med. 2010;11:620–34. 14. Cicalini S, Almodovar S, Grilli E, Flores S. Pulmonary hypertension and human immunodeficiency virus infection: epidemiology, pathogenesis, and clinical approach. Clin Microbiol Infect. 2011;17:25–33. 15. Austin ED, Cogan JD, West JD, Hedges LK, Hamid R, Dawson EP, et al. Alterations in oestrogen metabolism: Implications for higher penetrance of familial pulmonary arterial hypertension in females. Eur Respir J. 2009;34:1093–9. 16. Almodovar S, Cicalini S, Petrosillo N, Flores SC. Pulmonary hypertension associated with HIV infection: pulmonary vascular disease: the global perspective. Chest. 2010;137 6 Suppl.:6S–12S. 17. Elliot CG, Glissmeyer EW, Havlena GT, Carlquist J, McKinney JT, Rich S, et al. Relationship of BMPR2 mutations to vasoreactivity in pulmonary arterial hypertension. Circulation. 2006;113:2509–15. 18. Liu D, Wu WH, Mao YM, Yuan P, Zhang R, Ju FL, et al. BMPR2 mutations influence phenotype more obviously in male patients with pulmonary arterial hypertension. Circ Cardiovasc Genet. 2012;5:511–8. 19. Mallet C, Lamribet K, Giraud S, Dupuis-Girod S, Feige JJ, Bailly S, et al. Functional analysis of endoglin mutations from hereditary hemorrhagic telangiectasia type 1 patients reveals different mechanisms for endoglin loss of function. Hum Mol Genet. 2015;144:261–4. 20. Förg T, Hafner M, Lux A. Investigation of endoglin wild-type and missense mutant protein heterodimerisation using fluorescence microscopy based IF, BiFC and FRET analyses. PLOS ONE. 2014;9:e102998.