- Email: [email protected]
Mutational screening in genes related with porto-pulmonary hypertension: An analysis of 6 cases
Med Clin (Barc). 2017;148(7):310–313
www.elsevier.es/medicinaclinica
Clinical report
Mutational screening in genes related with porto-pulmonary hypertension: An analysis of 6 cases夽 Guillermo Pousada a,b,c , Adolfo Baloira d , Diana Valverde a,b,∗ a
Departamento de Bioquímica, Genética e Inmunología, Facultad de Biología, Universidad de Vigo, Vigo, Pontevedra, Spain Instituto de Investigación Sanitaria Galicia Sur (IIS-Galicia Sur), Pontevedra, Spain Centro de Investigaciones Biomédicas (CINBIO), Pontevedra, Spain d Servicio de Neumología, Complexo Hospitalario Universitario de Pontevedra, Pontevedra, Spain b c
a r t i c l e
i n f o
Article history: Received 28 October 2016 Accepted 12 January 2017 Available online 24 April 2017 Keywords: Pulmonary arterial hypertension Porto-pulmonary hypertension Mutations Genetic analysis Genetic modifiers
a b s t r a c t Introduction: Portopulmonary hypertension (PPH) is a rare disease with a low incidence and without a clearly-identified genetic component. The aim of this work was to check genes and genetic modifiers related to pulmonary arterial hypertension in patients with PPH in order to clarify the molecular basis of the pathology. Patients: We selected a total of 6 patients with PPH and amplified the exonic regions and intronic flanking regions of the relevant genes and regions of interest of the genetic modifiers. Results: Six patients diagnosed with PPH were analyzed and compared to 55 healthy individuals. Potentially-pathogenic mutations were identified in the analyzed genes of 5 patients. None of these mutations, which are highly conserved throughout evolution, were detected in the control patients nor different databases analyzed (1000 Genomes, ExAC and DECIPHER). After analyzing for genetic modifiers, we found different variations that could favor the onset of the disease. Conclusions: The genetic analysis carried out in this small cohort of patients with PPH revealed a large number of mutations, with the ENG gene showing the greatest mutational frequency. ˜ S.L.U. All rights reserved. © 2017 Elsevier Espana,
Cribado mutacional en genes relacionados con la hipertensión portopulmonar: análisis de 6 casos r e s u m e n Palabras clave: Hipertensión arterial pulmonar Hipertensión portopulmonar Mutaciones Estudio genético Modificadores genéticos
Introducción: La hipertensión portopulmonar (HPP) es una enfermedad rara de baja incidencia y sin una alteración genética claramente identificada. El principal objetivo de este estudio fue analizar los genes y modificadores genéticos relacionados con la hipertensión arterial pulmonar en pacientes con HPP. Pacientes: Se seleccionaron 6 pacientes diagnosticados de HPP y se amplificaron las regiones exónicas y sus límites intrónicos de los genes y la región de interés en los modificadores genéticos. Resultados: Se analizaron 6 pacientes diagnosticados de HPP y se compararon con 55 individuos sanos. Se identificaron mutaciones potencialmente patogénicas en 5 pacientes en alguno de los genes analizados. Ninguna de estas mutaciones, que se encuentran altamente conservadas a lo largo de la evolución, fue detectada en los controles analizados ni en las diferentes bases de datos consultadas (1000 Genomas, ExAC y DECIPHER). Tras el análisis de los modificadores genéticos encontramos diferentes variaciones que podrían favorecer el desarrollo de la enfermedad. ˜ serie de pacientes con HPP ha mostrado un elevado Conclusiones: El análisis genético en esta pequena número de mutaciones, siendo el gen ENG el que muestra una mayor frecuencia mutacional. ˜ S.L.U. Todos los derechos reservados. © 2017 Elsevier Espana,
夽 Please cite this article as: Pousada G, Baloira A, Valverde D. Cribado mutacional en genes relacionados con la hipertensión portopulmonar: análisis de 6 casos. Med Clin (Barc). 2017;148:310–313. ∗ Corresponding author. E-mail address: [email protected] (D. Valverde). ˜ S.L.U. All rights reserved. 2387-0206/© 2017 Elsevier Espana,
G. Pousada et al. / Med Clin (Barc). 2017;148(7):310–313
Introduction Portopulmonary hypertension (PPH, ORPHA 275813) is considered a clinical entity in itself and is defined as pulmonary arterial hypertension (PAH; OMIM #178600; ORPHA 422) in the presence of portal hypertension from any cause, with or without liver cirrhosis. It is a rare type of PAH, representing just over 5% of PAH,1 according to the REVEAL registry. For its diagnosis, there must be evidence of portal hypertension and hemodynamic values compatible with PAH. The exact incidence and prevalence are not known. Percentages between 6% and 8% have been observed in studies of patients referred for liver transplant evaluation.2 The values obtained in the Spanish registry (REHAP) show a percentage close to 6% for PPH on total PAHs, similar to other registries.3 There are some factors that seem to be associated with the presence of PPH, such as being female or an autoimmune etiology of liver disease, while it is less frequent in cases of hepatitis C virus.4 PPH pathogenesis is not fully clarified and histological lesions in pulmonary arteries are indistinguishable from those observed in idiopathic PAH.2,5 It is possible that stress on the vascular wall caused by increased pulmonary blood flow associated with portal hypertension, in the presence of a favorable genetic load, could cause an alteration in the balance between the various vasoactive substances of the endothelium, promoting the development of the lesions previously described.6 Data on the genes involved in this disease are very limited.6 It has been speculated that the gene encoding the bone morphogenetic protein receptor type ii (BMPR2) and the gene of kinase-like activin receptor type ii (ALK1/ACVRL1) could be involved, both related to the pathogenesis of PAH. The BMPR2 gene is altered in about 80% of patients with heritable PAH and in up to 40% of patients with idiopathic PAH.7 However, cases of PAH associated with PPH with mutations in these genes have not yet been reported. The primary objective of this study was the analysis of key genes and different genetic modifiers associated with PAH in patients with PPH, in order to delve into the molecular basis of this disease.
311
pulmonary arterial pressure ≥ 25 mmHg and an arterial wedge pressure ≤ 15 mmHg without specific treatment, following the protocol in accordance with the recommendations of the European Respiratory Society/European Society of Cardiology. Patients were stable at the time of catheterization. As in previously published studies, samples from 55 healthy individuals who had no known relatives affected by PAH were used as controls.7 All patients and controls signed an informed consent, in accordance with the ethical principles for medical research involving human subjects set forth in the Declaration of Helsinki and endorsed by the World Medical Association. The local ethics committee (Autonomic Research Ethics Committee of Galicia) gave its approval for the study. Of the 6 patients, the etiology of liver disease was autoimmune in one case and alcohol-related in the other 5. At the time of diagnosis 2 patients were in functional class (FC) II, 3 patients in FC III and one in class IV (Fig. 1). The clinical and hemodynamic characteristics of patients are shown in Table 1.
Results Mutational analysis
Patient 1
Female (FC II) mutation in ENG gene
Patient 2
Female (FC III) mutation in ACVRL1 gene
After a thorough in silico analysis, potentially pathogenic mutations located in the intronic region, or synonymous mutations that can affect mRNA processing, or missense mutations, or frameshift mutations were identified. Through genetic characterization of genes BMPR2, ACVRL1, endoglin (ENG) and K+ voltage-gated channels, member 5 (KCNA5), some potentially pathogenic mutation was identified in 5 of 6patients analyzed (Table 2). None of the mutations classified as pathogenic were detected in the 110-chromosome control panel. These were searched in different databases (1000 Genomes, ExAC and DECIPHER). However, only the mutation c.1633G > A (p.G545S) of the gene ENG has been previously described in the ExAC database, being classified as pathogenic. The recommendations of the American College of Medical Genetics and Genomics were followed to classify these variants as pathogenic.8 Also, numerous changes classified as polymorphisms have been identified in these patients, and, in one patient, a variation c.572G > A (p.G191N) in the gene ENG was identified, and although the in silico analysis classifies it as pathogenic, other studies classify it as polymorphism. For this reason, until no other functional studies are conducted, it should be classified as a variant of uncertain significance. All missense mutations identified in this study are conserved throughout evolution. The exact point of the wild sequence where the study mutation was found was compared to 10 different species.
Patient 3
Male (FC III) mutation in KCNA5 gene
Analysis of genetic modifiers
Patient 4
Male (FC IV) mutation in genes BMPR2 and ENG
Patient 5
Female (FC III) Mutations were not identified
Patient 6
Male (FC II) mutation in genes ACVRL1 and ENG
Patient presentation
PPH
6 patients were included in this study, and the diagnosis was made based on medical record, imaging tests, liver biopsy and hemodynamic studies. A right heart catheterization was performed in all cases. PAH was considered if there was a mean
Fig. 1. Characteristics of patients with portopulmonary hypertension included in this study. FC: functional class; PPH: portopulmonary hypertension.
The c.1-1853 1897del44 gene polymorphism in the serotonin transporter (SLC6A4/SERT1) is present in 4 patients analyzed. For the endothelin-1 (EDN1) gene, c.5665G > T polymorphism only appears in 2 patients analyzed. The 3 polymorphisms studied, associated with the transient receptor potential channel C, isoform 6 (TRPC6) gene, are present in some patients. The variation c.1-361A > T appears in 4 patients, and variations c.1-254C > G and c.1-218C > T are present in 3 patients, one of each. The c.1166A > C polymorphism of angiotensin receptor type i(AGTR1) gene was identified in 3 patients. Finally, in 5 of the cases analyzed, the CCTTT repeat polymorphism of inducible nitric oxide synthase (NOS2) gene has at least
312
G. Pousada et al. / Med Clin (Barc). 2017;148(7):310–313
Table 1 Clinical and hemodynamic characteristics in patients with portopulmonary hypertension. Patients with portopulmonary hypertension
Clinical and hemodynamic characteristics Sex Liver disease Age at diagnosis (years) Functional class MPAP (mmHg) SPAP (mmHg) PVR (mmHg/l−1 /m−1 ) CI (l/m−1 m−2 ) T6M (m) Death
Patient 1
Patient 2
Patient 3
Patient 4
Patient 5
Patient 6
Female AC 51 II 36 60 9.2 2.25 440 No
Female Autoimmune 55 III 50 85 8.3 2.53 480 No
Male AC 61 III 54 97 4.33 3.36 504 Yes
Male AC 65 IV 60 85 10.2 3.03 120 Yes
Female AC 65 III 60 28 6.2 2,2 389 No
Male AC 47 II 47 68 8.6 1.36 505 No
CA: alcoholic cirrhosis; CI: cardiac index; MPAP: mean pulmonary artery pressure; SPAP: systolic pulmonary arterial pressure; PVR: pulmonary vascular resistance; 6MWT: 6-minute walk test. Table 2 Molecular characterization of key genes in patients with portopulmonary hypertension. Patients with portopulmonary hypertension
Genes
BMPR2 ACVRL1 ENG KCNA5
Patient 1
Patient 2
Patient 3
Patient 4
Patient 5
Patient 6
– – c.1272 + 6A > T –
– c.478delT (p.S160Pfs*5) – –
– – – c.551G > C (p.R184P)
c.1021G > A (p.V341M) – c.498G > A (p.Q166Q) –
– – – –
– c.694T > A (p.S232T) c.1633G > A (p.G545S) –
Table 3 Molecular characterization of genetic modifiers in portopulmonary hypertension patients. Patients with portopulmonary hypertension
Genetic modifiers
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
Patient 1
Patient 2
Patient 3
Patient 4
Patient 5
Patient 6
Short/long G/G A/A C/G C/C A/C 12/10
Short/long G/G A/A C/C C/C A/A 10/8
Short/short G/G A/T C/G C/T A/C 10/8
Short/long G/G A/T C/G C/T A/A 13/12
Long/long G/T A/T C/C C/C A/A 13/10
Short/short G/T A/T C/C C/T A/C 13/10
A: adenine; C: cytosine; G: guanine; T: thymine. a The 3 TRPC6 gene polymorphisms are associated with a worse prognosis, with the carriers of the 3 polymorphisms being more susceptible to a more severe disease. b It has been shown that patients with several CCTTT repeats in the gene NOS2 ≤ 12 have a reduced vasodilator capacity compared to those with at least one allele with more than 12 repeats.
one allele with less than 12 reps, which means less synthesis capacity for this nitric oxide synthase. These results are shown in Table 3. Discussion 6 patients diagnosed with PPH are described in this study, conducting a study of the key genes and genetic modifiers associated with the development of PAH. To date, there have been published series of patients with PPH who present mutations in key genes that affect PAH, although this is the first study describing mutations in genes BMPR2, ACVRL1, ENG and KCNA57 in this group of patients. In this study, we have identified potentially pathogenic mutations in 5 out of 6 patients analyzed, with ENG gene being the most frequently affected, while only one patient was carrying mutations in the BMPR2 gene. Given the small number of patients, it is difficult to make comparisons with idiopathic PAH, where the most commonly affected gene is BMPR2. Larger series will be necessary to confirm these findings. The characterization of the mutations was performed through an in silico analysis, following the recommendations of the American College of Medical Genetics and Genomics. Patients with amino acid change mutations or mutations that produce a premature stop codon can produce alterations in mRNA processing, leading to smaller proteins. Consequently, their correct folding can be altered, as well as the subcellular localization
of the protein, or they may be susceptible to degradation.9 These mutations may cause small changes in receptor structure, interfering with downstream signaling in the BMP pathway.10 One of the patients analyzed had a localized mutation in the intronic region and another had a synonymous mutation, both in the ENG gene. These mutations were classified as potentially pathogenic after the splicing analysis, being able to produce an aberrant protein and prevent proper anchoring of the protein to the plasma membrane, affecting TGF/ALK1 signaling and favoring the onset of disease.10,11 Two of the patients included in this study had more than one potentially pathogenic mutation. This fact, called second hit, was identified in different studies.11 The second hit theory gives evidence that patients with several pathogenic mutations account for more than one alteration in one or more cellular pathways and have a greater predisposition to the development of PAH.11 All patients studied were carriers of several polymorphisms in the genetic modifiers analyzed. The relative effect of each modifier is difficult to assess and, in many cases, a part of the phenotypic variability can be associated with the nature of mutations in the major gene. However, it is known that these favor the development of PAH at an earlier age and have been associated with a more severe phenotype due to its potential vasoconstrictor and proliferative effect.12
G. Pousada et al. / Med Clin (Barc). 2017;148(7):310–313
Patients showed a mixed progression during the follow-up period. Two of them had a satisfactory response, in the case of 2 other patients it was necessary to combine 3 drugs, and 2 died 15 months and 5 years after diagnosis, the first of them, carrying 2 mutations and various polymorphisms in genetic modifiers. The main limitation of this study was the small number of patients analyzed, which has not allowed us to make a genotype–phenotype correlation and draw conclusions about the impact on the therapeutic response. However, the low incidence of PPH is a limiting factor regarding including more patients in the analysis. Another potential limitation is not having conducted a similar genetic study in patients with portal hypertension without PAH. However, the mutations described are extremely rare in healthy population, are associated with the development of PAH and have never been described in patients with liver disease, which makes it very unlikely to have a higher incidence in patients with liver disease without PAH. In conclusion, we report a small series of patients with PPH, with most of them carrying mutations in the genes related to PAH. To date, this is the first study where potentially pathogenic mutations have been identified in this group of patients. Funding This study has been funded through the projects CO-0118-2012 of Actelion Pharmaceuticals and INBIOMED 2009-063 of Xunta de Galicia. Authorship GP has been involved in the study design, genetic analysis, data analysis, correlations and manuscript preparation. AB has been responsible for the collection of samples and clinical data, data analysis and manuscript preparation. DV has been involved in the study design and coordination, data analysis and manuscript preparation. All authors have read and approved the final manuscript. Conflict of interests Adolfo Baloira has received research grants from Actelion and has acted as a consultant for Actelion, GSK, Pfizer, Lilly, Bayer and Ferrer. The other authors declare no conflicts of interest.
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Acknowledgements We wish to thank all the patients with PAH-PPH who participated in this study after signing an informed consent, according ˜ and to current law. We also thank pulmonologists Carlos Vilarino Jose Manuel Cifrián for their contribution regarding patient inclusion in this series. Finally, we wish to thank the Spanish Association of Pulmonary Hypertension and Biomedical Capabilities Support Program (BIOCAPS) FP7-REGPOT316265 for their collaboration. References 1. Benza RL, Miller DP, Gomberg-Maitland M, Frantz RP, Foreman AJ, Coffey CS, et al. Predicting survival in pulmonary arterial hypertension: insights from the Registry to Evaluate Early and Long-Term Pulmonary Arterial Hypertension Disease Management (REVEAL). Circulation. 2010;122:164–72. 2. Saleemi S. Portopulmonary hypertension. Ann Thorac Med. 2010;5:5–9. 3. Escribano-Subias P, Blanco I, López-Meseguer M, Lopez-Guarch CJ, Roman A, Morales P, et al. Survival in pulmonary hypertension in Spain: insights from the Spanish registry. Eur Respir J. 2012;40:596–603. 4. Kawut SM, Krowka MJ, Trotter JF, Roberts KE, Benza RL, Badesch DB, et al. Clinical risk factors for portopulmonary hypertension. Hepatology. 2008;48: 196–203. 5. Medarov BI, Chopra A, Judson MA. Clinical aspects of portopulmonary hypertension. Respir Med. 2014;108:943–54. 6. Liberal R, Grant CR, Baptista R, Macedo G. Porto-pulmonary hypertension: a comprehensive review. Clin Res Hepatol Gastroenterol. 2015;39: 157–67. ˜ C, Cifrian JM, Valverde D. Novel mutations in 7. Pousada G, Baloira A, Vilarino BMPR2ACVRL1 and KCNA5 genes and hemodynamic parameters in patients with pulmonary arterial hypertension. PLoS One. 2014;9:e100261. 8. Richards S, Aziz N, Bale S, Bick D, Das S, Gastier-Foster J, et al. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med. 2015;17: 405–24. 9. 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. 10. Chida A, Shintani M, Nakayama T, Furutani Y, Hayama E, Inai K, et al. Missense mutations of the BMPR1B (ALK6) gene in childhood idiopathic pulmonary arterial hypertension. Circ J. 2012;76:1804–11. 11. Pousada G, Baloira A, Valverde D. Complex inheritance in pulmonary arterial hypertension patients with several mutations. Sci Rep. 2016;6:33570. 12. 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.
www.elsevier.es/medicinaclinica
Clinical report
Mutational screening in genes related with porto-pulmonary hypertension: An analysis of 6 cases夽 Guillermo Pousada a,b,c , Adolfo Baloira d , Diana Valverde a,b,∗ a
Departamento de Bioquímica, Genética e Inmunología, Facultad de Biología, Universidad de Vigo, Vigo, Pontevedra, Spain Instituto de Investigación Sanitaria Galicia Sur (IIS-Galicia Sur), Pontevedra, Spain Centro de Investigaciones Biomédicas (CINBIO), Pontevedra, Spain d Servicio de Neumología, Complexo Hospitalario Universitario de Pontevedra, Pontevedra, Spain b c
a r t i c l e
i n f o
Article history: Received 28 October 2016 Accepted 12 January 2017 Available online 24 April 2017 Keywords: Pulmonary arterial hypertension Porto-pulmonary hypertension Mutations Genetic analysis Genetic modifiers
a b s t r a c t Introduction: Portopulmonary hypertension (PPH) is a rare disease with a low incidence and without a clearly-identified genetic component. The aim of this work was to check genes and genetic modifiers related to pulmonary arterial hypertension in patients with PPH in order to clarify the molecular basis of the pathology. Patients: We selected a total of 6 patients with PPH and amplified the exonic regions and intronic flanking regions of the relevant genes and regions of interest of the genetic modifiers. Results: Six patients diagnosed with PPH were analyzed and compared to 55 healthy individuals. Potentially-pathogenic mutations were identified in the analyzed genes of 5 patients. None of these mutations, which are highly conserved throughout evolution, were detected in the control patients nor different databases analyzed (1000 Genomes, ExAC and DECIPHER). After analyzing for genetic modifiers, we found different variations that could favor the onset of the disease. Conclusions: The genetic analysis carried out in this small cohort of patients with PPH revealed a large number of mutations, with the ENG gene showing the greatest mutational frequency. ˜ S.L.U. All rights reserved. © 2017 Elsevier Espana,
Cribado mutacional en genes relacionados con la hipertensión portopulmonar: análisis de 6 casos r e s u m e n Palabras clave: Hipertensión arterial pulmonar Hipertensión portopulmonar Mutaciones Estudio genético Modificadores genéticos
Introducción: La hipertensión portopulmonar (HPP) es una enfermedad rara de baja incidencia y sin una alteración genética claramente identificada. El principal objetivo de este estudio fue analizar los genes y modificadores genéticos relacionados con la hipertensión arterial pulmonar en pacientes con HPP. Pacientes: Se seleccionaron 6 pacientes diagnosticados de HPP y se amplificaron las regiones exónicas y sus límites intrónicos de los genes y la región de interés en los modificadores genéticos. Resultados: Se analizaron 6 pacientes diagnosticados de HPP y se compararon con 55 individuos sanos. Se identificaron mutaciones potencialmente patogénicas en 5 pacientes en alguno de los genes analizados. Ninguna de estas mutaciones, que se encuentran altamente conservadas a lo largo de la evolución, fue detectada en los controles analizados ni en las diferentes bases de datos consultadas (1000 Genomas, ExAC y DECIPHER). Tras el análisis de los modificadores genéticos encontramos diferentes variaciones que podrían favorecer el desarrollo de la enfermedad. ˜ serie de pacientes con HPP ha mostrado un elevado Conclusiones: El análisis genético en esta pequena número de mutaciones, siendo el gen ENG el que muestra una mayor frecuencia mutacional. ˜ S.L.U. Todos los derechos reservados. © 2017 Elsevier Espana,
夽 Please cite this article as: Pousada G, Baloira A, Valverde D. Cribado mutacional en genes relacionados con la hipertensión portopulmonar: análisis de 6 casos. Med Clin (Barc). 2017;148:310–313. ∗ Corresponding author. E-mail address: [email protected] (D. Valverde). ˜ S.L.U. All rights reserved. 2387-0206/© 2017 Elsevier Espana,
G. Pousada et al. / Med Clin (Barc). 2017;148(7):310–313
Introduction Portopulmonary hypertension (PPH, ORPHA 275813) is considered a clinical entity in itself and is defined as pulmonary arterial hypertension (PAH; OMIM #178600; ORPHA 422) in the presence of portal hypertension from any cause, with or without liver cirrhosis. It is a rare type of PAH, representing just over 5% of PAH,1 according to the REVEAL registry. For its diagnosis, there must be evidence of portal hypertension and hemodynamic values compatible with PAH. The exact incidence and prevalence are not known. Percentages between 6% and 8% have been observed in studies of patients referred for liver transplant evaluation.2 The values obtained in the Spanish registry (REHAP) show a percentage close to 6% for PPH on total PAHs, similar to other registries.3 There are some factors that seem to be associated with the presence of PPH, such as being female or an autoimmune etiology of liver disease, while it is less frequent in cases of hepatitis C virus.4 PPH pathogenesis is not fully clarified and histological lesions in pulmonary arteries are indistinguishable from those observed in idiopathic PAH.2,5 It is possible that stress on the vascular wall caused by increased pulmonary blood flow associated with portal hypertension, in the presence of a favorable genetic load, could cause an alteration in the balance between the various vasoactive substances of the endothelium, promoting the development of the lesions previously described.6 Data on the genes involved in this disease are very limited.6 It has been speculated that the gene encoding the bone morphogenetic protein receptor type ii (BMPR2) and the gene of kinase-like activin receptor type ii (ALK1/ACVRL1) could be involved, both related to the pathogenesis of PAH. The BMPR2 gene is altered in about 80% of patients with heritable PAH and in up to 40% of patients with idiopathic PAH.7 However, cases of PAH associated with PPH with mutations in these genes have not yet been reported. The primary objective of this study was the analysis of key genes and different genetic modifiers associated with PAH in patients with PPH, in order to delve into the molecular basis of this disease.
311
pulmonary arterial pressure ≥ 25 mmHg and an arterial wedge pressure ≤ 15 mmHg without specific treatment, following the protocol in accordance with the recommendations of the European Respiratory Society/European Society of Cardiology. Patients were stable at the time of catheterization. As in previously published studies, samples from 55 healthy individuals who had no known relatives affected by PAH were used as controls.7 All patients and controls signed an informed consent, in accordance with the ethical principles for medical research involving human subjects set forth in the Declaration of Helsinki and endorsed by the World Medical Association. The local ethics committee (Autonomic Research Ethics Committee of Galicia) gave its approval for the study. Of the 6 patients, the etiology of liver disease was autoimmune in one case and alcohol-related in the other 5. At the time of diagnosis 2 patients were in functional class (FC) II, 3 patients in FC III and one in class IV (Fig. 1). The clinical and hemodynamic characteristics of patients are shown in Table 1.
Results Mutational analysis
Patient 1
Female (FC II) mutation in ENG gene
Patient 2
Female (FC III) mutation in ACVRL1 gene
After a thorough in silico analysis, potentially pathogenic mutations located in the intronic region, or synonymous mutations that can affect mRNA processing, or missense mutations, or frameshift mutations were identified. Through genetic characterization of genes BMPR2, ACVRL1, endoglin (ENG) and K+ voltage-gated channels, member 5 (KCNA5), some potentially pathogenic mutation was identified in 5 of 6patients analyzed (Table 2). None of the mutations classified as pathogenic were detected in the 110-chromosome control panel. These were searched in different databases (1000 Genomes, ExAC and DECIPHER). However, only the mutation c.1633G > A (p.G545S) of the gene ENG has been previously described in the ExAC database, being classified as pathogenic. The recommendations of the American College of Medical Genetics and Genomics were followed to classify these variants as pathogenic.8 Also, numerous changes classified as polymorphisms have been identified in these patients, and, in one patient, a variation c.572G > A (p.G191N) in the gene ENG was identified, and although the in silico analysis classifies it as pathogenic, other studies classify it as polymorphism. For this reason, until no other functional studies are conducted, it should be classified as a variant of uncertain significance. All missense mutations identified in this study are conserved throughout evolution. The exact point of the wild sequence where the study mutation was found was compared to 10 different species.
Patient 3
Male (FC III) mutation in KCNA5 gene
Analysis of genetic modifiers
Patient 4
Male (FC IV) mutation in genes BMPR2 and ENG
Patient 5
Female (FC III) Mutations were not identified
Patient 6
Male (FC II) mutation in genes ACVRL1 and ENG
Patient presentation
PPH
6 patients were included in this study, and the diagnosis was made based on medical record, imaging tests, liver biopsy and hemodynamic studies. A right heart catheterization was performed in all cases. PAH was considered if there was a mean
Fig. 1. Characteristics of patients with portopulmonary hypertension included in this study. FC: functional class; PPH: portopulmonary hypertension.
The c.1-1853 1897del44 gene polymorphism in the serotonin transporter (SLC6A4/SERT1) is present in 4 patients analyzed. For the endothelin-1 (EDN1) gene, c.5665G > T polymorphism only appears in 2 patients analyzed. The 3 polymorphisms studied, associated with the transient receptor potential channel C, isoform 6 (TRPC6) gene, are present in some patients. The variation c.1-361A > T appears in 4 patients, and variations c.1-254C > G and c.1-218C > T are present in 3 patients, one of each. The c.1166A > C polymorphism of angiotensin receptor type i(AGTR1) gene was identified in 3 patients. Finally, in 5 of the cases analyzed, the CCTTT repeat polymorphism of inducible nitric oxide synthase (NOS2) gene has at least
312
G. Pousada et al. / Med Clin (Barc). 2017;148(7):310–313
Table 1 Clinical and hemodynamic characteristics in patients with portopulmonary hypertension. Patients with portopulmonary hypertension
Clinical and hemodynamic characteristics Sex Liver disease Age at diagnosis (years) Functional class MPAP (mmHg) SPAP (mmHg) PVR (mmHg/l−1 /m−1 ) CI (l/m−1 m−2 ) T6M (m) Death
Patient 1
Patient 2
Patient 3
Patient 4
Patient 5
Patient 6
Female AC 51 II 36 60 9.2 2.25 440 No
Female Autoimmune 55 III 50 85 8.3 2.53 480 No
Male AC 61 III 54 97 4.33 3.36 504 Yes
Male AC 65 IV 60 85 10.2 3.03 120 Yes
Female AC 65 III 60 28 6.2 2,2 389 No
Male AC 47 II 47 68 8.6 1.36 505 No
CA: alcoholic cirrhosis; CI: cardiac index; MPAP: mean pulmonary artery pressure; SPAP: systolic pulmonary arterial pressure; PVR: pulmonary vascular resistance; 6MWT: 6-minute walk test. Table 2 Molecular characterization of key genes in patients with portopulmonary hypertension. Patients with portopulmonary hypertension
Genes
BMPR2 ACVRL1 ENG KCNA5
Patient 1
Patient 2
Patient 3
Patient 4
Patient 5
Patient 6
– – c.1272 + 6A > T –
– c.478delT (p.S160Pfs*5) – –
– – – c.551G > C (p.R184P)
c.1021G > A (p.V341M) – c.498G > A (p.Q166Q) –
– – – –
– c.694T > A (p.S232T) c.1633G > A (p.G545S) –
Table 3 Molecular characterization of genetic modifiers in portopulmonary hypertension patients. Patients with portopulmonary hypertension
Genetic modifiers
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
Patient 1
Patient 2
Patient 3
Patient 4
Patient 5
Patient 6
Short/long G/G A/A C/G C/C A/C 12/10
Short/long G/G A/A C/C C/C A/A 10/8
Short/short G/G A/T C/G C/T A/C 10/8
Short/long G/G A/T C/G C/T A/A 13/12
Long/long G/T A/T C/C C/C A/A 13/10
Short/short G/T A/T C/C C/T A/C 13/10
A: adenine; C: cytosine; G: guanine; T: thymine. a The 3 TRPC6 gene polymorphisms are associated with a worse prognosis, with the carriers of the 3 polymorphisms being more susceptible to a more severe disease. b It has been shown that patients with several CCTTT repeats in the gene NOS2 ≤ 12 have a reduced vasodilator capacity compared to those with at least one allele with more than 12 repeats.
one allele with less than 12 reps, which means less synthesis capacity for this nitric oxide synthase. These results are shown in Table 3. Discussion 6 patients diagnosed with PPH are described in this study, conducting a study of the key genes and genetic modifiers associated with the development of PAH. To date, there have been published series of patients with PPH who present mutations in key genes that affect PAH, although this is the first study describing mutations in genes BMPR2, ACVRL1, ENG and KCNA57 in this group of patients. In this study, we have identified potentially pathogenic mutations in 5 out of 6 patients analyzed, with ENG gene being the most frequently affected, while only one patient was carrying mutations in the BMPR2 gene. Given the small number of patients, it is difficult to make comparisons with idiopathic PAH, where the most commonly affected gene is BMPR2. Larger series will be necessary to confirm these findings. The characterization of the mutations was performed through an in silico analysis, following the recommendations of the American College of Medical Genetics and Genomics. Patients with amino acid change mutations or mutations that produce a premature stop codon can produce alterations in mRNA processing, leading to smaller proteins. Consequently, their correct folding can be altered, as well as the subcellular localization
of the protein, or they may be susceptible to degradation.9 These mutations may cause small changes in receptor structure, interfering with downstream signaling in the BMP pathway.10 One of the patients analyzed had a localized mutation in the intronic region and another had a synonymous mutation, both in the ENG gene. These mutations were classified as potentially pathogenic after the splicing analysis, being able to produce an aberrant protein and prevent proper anchoring of the protein to the plasma membrane, affecting TGF/ALK1 signaling and favoring the onset of disease.10,11 Two of the patients included in this study had more than one potentially pathogenic mutation. This fact, called second hit, was identified in different studies.11 The second hit theory gives evidence that patients with several pathogenic mutations account for more than one alteration in one or more cellular pathways and have a greater predisposition to the development of PAH.11 All patients studied were carriers of several polymorphisms in the genetic modifiers analyzed. The relative effect of each modifier is difficult to assess and, in many cases, a part of the phenotypic variability can be associated with the nature of mutations in the major gene. However, it is known that these favor the development of PAH at an earlier age and have been associated with a more severe phenotype due to its potential vasoconstrictor and proliferative effect.12
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Patients showed a mixed progression during the follow-up period. Two of them had a satisfactory response, in the case of 2 other patients it was necessary to combine 3 drugs, and 2 died 15 months and 5 years after diagnosis, the first of them, carrying 2 mutations and various polymorphisms in genetic modifiers. The main limitation of this study was the small number of patients analyzed, which has not allowed us to make a genotype–phenotype correlation and draw conclusions about the impact on the therapeutic response. However, the low incidence of PPH is a limiting factor regarding including more patients in the analysis. Another potential limitation is not having conducted a similar genetic study in patients with portal hypertension without PAH. However, the mutations described are extremely rare in healthy population, are associated with the development of PAH and have never been described in patients with liver disease, which makes it very unlikely to have a higher incidence in patients with liver disease without PAH. In conclusion, we report a small series of patients with PPH, with most of them carrying mutations in the genes related to PAH. To date, this is the first study where potentially pathogenic mutations have been identified in this group of patients. Funding This study has been funded through the projects CO-0118-2012 of Actelion Pharmaceuticals and INBIOMED 2009-063 of Xunta de Galicia. Authorship GP has been involved in the study design, genetic analysis, data analysis, correlations and manuscript preparation. AB has been responsible for the collection of samples and clinical data, data analysis and manuscript preparation. DV has been involved in the study design and coordination, data analysis and manuscript preparation. All authors have read and approved the final manuscript. Conflict of interests Adolfo Baloira has received research grants from Actelion and has acted as a consultant for Actelion, GSK, Pfizer, Lilly, Bayer and Ferrer. The other authors declare no conflicts of interest.
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Acknowledgements We wish to thank all the patients with PAH-PPH who participated in this study after signing an informed consent, according ˜ and to current law. We also thank pulmonologists Carlos Vilarino Jose Manuel Cifrián for their contribution regarding patient inclusion in this series. Finally, we wish to thank the Spanish Association of Pulmonary Hypertension and Biomedical Capabilities Support Program (BIOCAPS) FP7-REGPOT316265 for their collaboration. References 1. Benza RL, Miller DP, Gomberg-Maitland M, Frantz RP, Foreman AJ, Coffey CS, et al. Predicting survival in pulmonary arterial hypertension: insights from the Registry to Evaluate Early and Long-Term Pulmonary Arterial Hypertension Disease Management (REVEAL). Circulation. 2010;122:164–72. 2. Saleemi S. Portopulmonary hypertension. Ann Thorac Med. 2010;5:5–9. 3. Escribano-Subias P, Blanco I, López-Meseguer M, Lopez-Guarch CJ, Roman A, Morales P, et al. Survival in pulmonary hypertension in Spain: insights from the Spanish registry. Eur Respir J. 2012;40:596–603. 4. Kawut SM, Krowka MJ, Trotter JF, Roberts KE, Benza RL, Badesch DB, et al. Clinical risk factors for portopulmonary hypertension. Hepatology. 2008;48: 196–203. 5. Medarov BI, Chopra A, Judson MA. Clinical aspects of portopulmonary hypertension. Respir Med. 2014;108:943–54. 6. Liberal R, Grant CR, Baptista R, Macedo G. Porto-pulmonary hypertension: a comprehensive review. Clin Res Hepatol Gastroenterol. 2015;39: 157–67. ˜ C, Cifrian JM, Valverde D. Novel mutations in 7. Pousada G, Baloira A, Vilarino BMPR2ACVRL1 and KCNA5 genes and hemodynamic parameters in patients with pulmonary arterial hypertension. PLoS One. 2014;9:e100261. 8. Richards S, Aziz N, Bale S, Bick D, Das S, Gastier-Foster J, et al. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med. 2015;17: 405–24. 9. 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. 10. Chida A, Shintani M, Nakayama T, Furutani Y, Hayama E, Inai K, et al. Missense mutations of the BMPR1B (ALK6) gene in childhood idiopathic pulmonary arterial hypertension. Circ J. 2012;76:1804–11. 11. Pousada G, Baloira A, Valverde D. Complex inheritance in pulmonary arterial hypertension patients with several mutations. Sci Rep. 2016;6:33570. 12. 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.