Clinical and genetic characteristics of pulmonary arterial hypertension in Lebanon

Clinical and genetic characteristics of pulmonary arterial hypertension in Lebanon Background: Pulmonary arterial hypertension (PAH) is a rare disease with an incidence rate of 2–6 cases per million per year. Our knowledge of the disease in the Middle East and North Africa (MENA) region is limited by the small number of clinical studies and the complete absence of genetic studies. Methods: Our aim was to shed light on the clinical and genetic characteristics of PAH in Lebanon and the region by using exome sequencing on PAH patients referred to the American University of Beirut Medical Center (AUBMC). Twenty-one idiopathic, hereditary and Congenital Heart Disease (CHD) PAH patients were prospectively recruited, their clinical data summarized, and sequencing performed. Results: The mean age at diagnosis was 33 years with a female preponderance of 70%. The mean pulmonary artery pressure at the time of diagnosis was 55. Genetic testing showed that 5 out of 19 idiopathic and Congenital Heart Disease PAH patients had Bone Morphogenetic Protein Receptor 2 (BMPR2) mutations at 25% prevalence, with 2 of these patients exhibiting a novel mutation. It also showed the presence of 1 BMPR2 mutation with 100% penetrance in a heritable PAH family. In the remaining cases, the lack of a complete genotype/phenotype correlation entailed a multigenic inheritance; suspected interactions involved previously associated genes T-box transcription factor 4 (TBX4), Bone Morphogenic Protein 10 (BMP10) and Growth Differentiation Factor 2 (GDF2). Conclusions: This is the first study that looks into the genetic causes of PAH, including known and new BMPR2 mutations, in the MENA region. It is also the first study to characterize the clinical features of the disease in Lebanon. Keywords: Pulmonary hypertension, BMPR2, Mutation Background Early PAH registries in the eighties provided valuable Pulmonary Arterial Hypertension (OMIM #178600) is a information on the baseline characteristics and out- devastating disorder of the small pulmonary arteries. It comes of the disease and helped with the understanding is characterized by progressive neo-intimal prolifera- of the natural progression of PAH [4]. Patients in early tion, smooth muscle hypertrophy and occlusive vascu- registries were younger with a mean age of 36 and with lar lesions leading to increased vascular resistance and female preponderance. Data from more recent European pressure [1]. It is a rare disease with prevalence esti- and North American registries suggest that PAH pa- mated to be between 15 and 60 cases per million [2]. tients are becoming older and have a better survival [5, Males are at less risk than females with a 2–3:1 female: 6]. A longitudinal observational study on PAH incidence male ratio [2, 3]. in the United Kingdom and Ireland spaning a 9-year-period noticed an increase in the age of diagnosis of PAH patients from 45 to 52 years over the same * Correspondence: gn08@aub.edu.lb; ib08@aub.edu.lb period. These changes in demographics may reflect not Ossama K. Abou Hassan and Wiam Haidar contributed equally to this work. Department of Biochemistry and Molecular Genetics, Faculty of Medicine, only an improvement in survival but also better referral American University of Beirut, P.O.Box: 11-0236, Beirut, Lebanon practices in these countries [7]. The incidence, preva- Department of Internal Medicine, Faculty of Medicine, American University lence and burden of disease in the MENA region have of Beirut, P.O.Box: 11-0236, Beirut, Lebanon Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abou Hassan et al. BMC Medical Genetics (2018) 19:89 Page 2 of 9 never been reported before. Clinical and demographic (prevalence case) and patients who were referred to our characteristics of PAH patients have only been docu- center were approached for enrollement in the study. mented in a single center registry in Saudi Arabia, and They were eligible for enrolment if they had idiopathic in an online Iranian registry. Both sources show a mean PAH, heritable PAH or PAH associated with congenital age of 36 years and a 2:1 female preponderance [8, 9]. systemic to pulmonary shunt. All of these conditions are Despite our growing knowledge of the disease and its classified under Group 1 PAH, based on the World progression, PAH patients have significantly reduced sur- Health Organization’s (WHO) 2013 guidelines for diag- vival [10]. Most of drugs used (endothelin receptor antag- nosis of pulmonary hypertension [2]. Minimal inclusion onists, guanylate cyclase stimulators, prostacyclin criteria also included a Right Heart Catheterization analogues and prostacyclin receptor agonists) are prohibi- (RHC) measuring a mean pulmonary arterial pressure tively expensive and not readily available in middle to (mPAP) > 25 mmHg at rest and a pulmonary artery low-income countries such as Lebanon. This may affect wedge pressure (PAWP) < 15mmHG. Following the pa- disease severity and prevalence in these countries, making tient’s consent, clinical data was collected and reviewed, the care of PAH patients even more challenging. including medical history, physical exam, family history In the year 2000, the BMPR2 gene was first reported to of PAH, and previously performed tests. These tests in- be linked to the progression of the disease [11]. Around clude pulmonary function test (PFT), chest computer- 80% of hereditary PAH patients and 25% of idiopathic dis- ized tomography (CT), CT pulmonary angiography, ease patients have BMPR2 mutations [12, 13]. Twenty-one ventilation perfusion scan and pro-BNP. For those pa- other genes have also been implicated in the development tients not diagnosed and treated at AUBMC, specialist PAH, with the most frequent mutations occurring in physicians along with our investigators reviewed all ALK1, ENG, SMAD9, CAV1, KCNK3, MADH9 and medical records for accuracy of diagnosis. Treatment of EIF2AK4 [14]. The penetrance of BMPR2 mutations PAH was also noted. After collection of clinical data, a ranges between 27 and 50% in females and 14–43% in blood sample was collected from the patients for genetic males [3], depending on the mutation site within the gene analysis by whole exome sequencing. Twenty-one PAH [15]. BMRR2 mutations have also been linked to PAH with patients in total, belonging to 20 families were included congenital heart disease [16]. Trying to understand the in the study. In the case of patients found to have a mu- low penetrance of BMPR2 and the yet unidentified genetic tation, non-affected family members were approached causes of the remaining 20% of the hereditary form of for inclusion in the study as controls and blood was col- PAH, new research strategies have shifted towards using lected for sanger analysis of the mutated protein. The next-generation sequencing (NGS) in the search for add- study was approved by the institutional review board itional mutations or compound interactions of known (IRB) at the American University of Beirut. Genetic ana- genes [17]. There is a a growing acceptance of a double hit lyses and return of genetic data were performed in ac- phenomenon – a major gene with an additional modifier cordance with protocols approved by the Partners gene- supporting the lower penetrance of mutations in Human Research Committee. Data collection and gen- genes associated with PAH. As such NGS is poised to be etic analysis were performed in accordance with the pro- the best technology to unravel this genetic interaction and tocols approved by the IRB at AUBMC. accounts of all genotype-phenotype discrepancies [18, 19]. This study has many objectives. One objective is to de- Genetic studies scribe the demographic and clinical characteristics of PAH DNA was extracted from peripheral blood. Whole ex- patients in Lebanon. These findings will shed light on the ome libraries were compiled with DNA samples from all burden of disease and the state of referral and care of pa- affected individuals recruited in this study. The exome tients with PAH in the country. The second objective is to was captured using the V5 SureSelect kits from Agilent assess for the first time the prevalence and types of genetic as per manufacturer’s protocol, and run on the Illumina mutations in PAH patients in Lebanon and the MENA re- HiSeq2500. The tests were performed by Macrogen, gion. This may help guide future genetic testing, counsel- South Korea. Exome sequences were aligned to hg19 ing and research in an area that shares common genetic/ using Novoalign, and variants were called using Genome ethnic background. The final objective is to explore novel Analysis Toolkit (GATK). Once variant(s) predicted to genetic mutations or interactions and their implications be damaging with a minor allele frequency (< 3%) were on the phenotypic manifestations of PAH. identified in a patient, Sanger sequencing was performed to confirm thier occurence, and all family members were Methods screened for those particular variant(s). Primers were Subject recruitment speficially designed to amplify the region(s) of interest Patients diagnosed with PAH at AUBMC between 2015 that include those variants, and amplified fragments and 2017 either newly (incidence case) or previously were analyzed by dideoxy sequencing (ABI technology) Abou Hassan et al. BMC Medical Genetics (2018) 19:89 Page 3 of 9 using the ABI3500 at the molecular core facility at the country (Table 1). The age of diagnosis as well as faculty of medicine at AUB as previously described [20]. additional phenotypes were included, and as previously The results were analyzed at the Congenital Heart mentioned, outpatients referred to our center were clinic- Disease Genetics Program (CHDGP) laboratory at the ally re-evaluated for confirmation of the phenotype. The American University of Beirut Medical Center AUBMC. cohort includes one family with 2 patients having each hereditary pulmonary arterial hypertension (PAH), 14 pa- Genetic panel selection tients with idiopathic PAH, and 5 with both PAH and con- All generated variant call files (vcf) were analyzed accord- genital heart disease (CHD). Fifteen out of 21 patients ing to an extensive virtual gene panel that was chosen to (71%) are females. The idiopathic and hereditary groups screen for the disease in Lebanon and the region and was had a mean age of 33 years at the time of diagnosis and a adopted from recent published methodology for choosing mean pulmonary artery pressure of 56 mmHg. Seventeen culprit genes in PAH [21]. Non-synonymous exon coding patients were on dual therapy (sildenafil, and bosentan or variants for BMPR2 were analyzed and a search for com- masitentan), and 6 patients on sildenafil alone. pound heterozygous variants associated with the BMPR2 mutations was performed accordingly using the Illumina BMPR2 variants variant studio. Non-synonymous missense variants, inser- For all patients, we conducted a primary analysis, whereby tion/deletions variants in the coding regions, and splicing only BMPR2 variants were included. The filtering strin- variants with allele frequencies less than 3% were filtered gency put a cutoff on all synonymous variants, inframe in according to their evolutionary conservation, location insertions/deletions, and non-coding ones. Out of the 20 within domains and prediction software (SIFT and families, we identified six with monoallelic BMPR2 Polyphen2), while additional variants with no supportive variants, thus constituting a prevalence of 30%. The roles were filtered out (Fig. 1). phenotypes of the index patients are summarized in Additional file 1: Table S1. Patients with and without Results BMPR2 mutations had a similar age of 33 years at diagno- Clinical and demographic profiles of families and patients sis, and a similar mean pulmonary arterial pressure (PAP) A total of 21 patients belonging to 20 different families of 57 mmHg. The PAH with CHD group had a mean age were recruited in the study from different parts of the of 5.4 years at diagnosis and a mean PAP of 44 mmHg. Fig. 1 Stepwise strategy for filtering variants to reach 10–15 highly likely disease-causing variants per patient screened. UTR = Untranslated Region; AF = allele frequency Abou Hassan et al. BMC Medical Genetics (2018) 19:89 Page 4 of 9 Table 1 BMPR2 variants in pulmonary hypertension patients (PHT), with their corresponding global allele frequencies (AF), and predictive protein effects (Polyphen2). ASD (Atrial Septal Defect), VSD (Ventricular Septal Defect), ExAC (Exome Aggregation Consortium), 1000G (1000 Genome from the International Genome Sample Resource) Family Sex Age at Phenotype mPAP Gene Mutation familial/ Zygosity ExAC AF 1000 G AF SIFT Polyphen2 Diagnosis (mmHg) sporadic A F 5 Large ASD and PAH 45 BMPR2 p.Q6* s Heterozygous N/A N/A (de novo) B F 38 PAH 50 BMPR2 p.N126S f Heterozygous Damaging Probably Damaging C F 25 PAH 50 BMPR2 p.R491W f Heterozygous Damaging Damaging F 25 PAH 94 BMPR2 p.R491W f Heterozygous Damaging Damaging D F 11 VSD and PAH 88 BMPR2 p.S775 N s Heterozygous 0.02516 0.00998403 Tolerated Benign E F 6 ASD and PAH 36 BMPR2 p.S775 N s Heterozygous 0.02516 0.00998403 Tolerated Benign F M 46 PAH 50 BMPR2 p.S775 N s Heterozygous 0.02516 0.00998403 Tolerated Benign G M 4 VSD and PAH 43 s H M 11 PAH 77 s I F 1 PAH VSD/ASD 33 s J M 22 PAH 56 s K F 20 PAH 50 s L F 31 PAH 41 s M F 45 PAH 58 s N F 26 PAH 62 s O F 30 PAH 63 s P F 57 PAH 46 s Q F 7 PAH 55 s R F 6 PAH 40 s S M 78 PAH 49 s T M 34 PAH 40 s Patient AII.3 (Fig. 2), a young girl who had an ASD re- screened family members. The father (BII.2), who is pair with fenestration at age one, presented at age 5 with clinically healthy, refused to participate in the study dyspnea. She was found to have PAH, with a mean PAP and may be the source of inheritance (Fig. 2). Interest- of 45 mmHg. Exome sequencing revealed a heterozygous ingly, two of the three children of the indexed patient nonsense mutation within exon 1 of BMPR2 that leads (namely individuals BIV.1 and BIV.2) also carry the to a premature stop codon (p.Q6*). Sanger sequencing variant (Fig. 2). Although they exhibit no apparent confirmed the mutation in the affected patient; however, clinical signs or symptoms of PAH, they are still young the mutation was not detected in any family members (< 10 years) compared to their mother (BIII.1) who was and none of them had pulmonary hypertension on diagnosed with PAH at age 38. follow-up, suggesting it is a de novo nonsense mutation. Patients CII.2 and CII.3 are 24-year-old female identi- Patient BIII.1 (Fig. 2), a 38-year-old-woman with no cal twins. Patient CII.2 presented with dyspnea on exer- apparent relevant family history, presented with progres- tion and syncope. Right heart catheterization (RHC) sive dyspnea. She was diagnosed with idiopathic PAH showed a mean pulmonary artery pressure (PAP) of after excluding other possible causes of PAH, with a 94 mmHg and a pulmonary vascular resistance (PVR) of mean PAP of 50 mmHg. She is being treated with sil- 25 woods units. Acute pulmonary vaso-reactivity testing denafil and macitentan. Exome sequencing showed a with inhaled nitric oxide (NO) at a concentration of novel missense mutation (p.N126S) within the trans- 40 ppm for 10 min was positive (mean PAP dropped to membrane domain of the BMPR2 receptor. The muta- 21 mmHg). She was NYHA Class III. Her mother had tion was reported as probably damaging on the died of idiopathic PAH at age 30 and her younger Polyphen-2 and SIFT prediction tools (Table 1). Sanger brother died of PAH at age 8. She was diagnosed with sequencing confirmed the mutation in the affected indi- ventricular septal defect (VSD) as a child, which was vidual, but revealed the absence of the mutation in the corrected at age 6. The patient was diagnosed with Abou Hassan et al. BMC Medical Genetics (2018) 19:89 Page 5 of 9 Fig. 2 Pedigrees for families showing the different BMPR2 mutations. The −/− symbol is for normal genes, −/+ is for heterozygous mutations and the +/+ is for homozygous mutations. Square, Male; Circle, female; open symbol, unaffected; filled symbol, affected; symbol with diagonal line, dead individual heritable PAH and she was started on a calcium channel PAH with no cardiac defects, and his RHC showed a blocker and sildenafil. A repeat RHC, 3 months later, mean PAP of 50. showed a decrease in mean PAP to 54 mmHg and loss of vaso-reactivity response to NO. She improved clinic- Other variants ally to NYHA II and bosentan was added to her treat- In the rest of the families (Additional file 2: Figure S1), ment. Her twin sister CII.3 was asymptomatic and was we adopted the same stringent filtering mentioned for screened for PAH. She was found to have PAH with a BMPR2 on a list of 431 genes curated from the literature mean PAP of 50 mmHg. There were no signs of VSD. and online databases (Table 1). For each patient, a list of Family C has a previously reported mutation (p.R491W) variants was extracted (Additional file 1: Table S1). None [22]; it occurs in a highly conserved kinase domain would explain the phenotype on its own, since there encoded by exon 3 of the gene. Records from the de- were no homozygous variants in these genes (data not ceased mother and brother showed that the mutation shown). Examples for potential variants are the p.R55W was found in all affected family members CI.2, CII.1, in BMP4 in family M, and p.M416I variant in BMP10 in CII.2, and CII.3. The father CI.1 who is phenotypically Family L (Additional file 1: Table S1). In contrast, in normal did not participate in the study. The mutation families with predicted benign and/or low penetrance was labeled as “damaging” on Polyphen-2 and SIFT BMPR2 mutations (families D, E, and F, Table 1), a (Table 1, and Fig. 2). search for probable second hit mutations was attempted. Patients DII.1, EII.3, and FII.1 have the same variant in The results suggest a potential digenic model for the dis- BMPR2, a missense mutation (p.S775 N) previously re- ease in families D and E (Fig. 3) supported by Sanger se- ported as being disease-causing with very low pene- quencing of the involved members in each case. In trance [23]. This mutation occurs in the C-terminal Family D, biallelic variants in GDF2 (p.P85L, and domain of the protein within exon 12. It is labeled as be- p.D218N), and a monoallelic variant in TBX4 (p.A246T) nign based on the Polyphen-2 and SIFT prediction tools are novel and are predicted as disease-causing. Those (Table 1). Patient DII.1 had a small PFO and he devel- mutations in combination with the “low penetrant” oped PAH at age 11. RHC showed a mean PAP of BMPR2 variant (p.S775 N) could explain the phenotype 68 mmHg. He has been stable on sildenafil-bosentan for in the only affected member of the family who is the the last 5 years. Patient EII.3 had an ASD repair at age 1 only one harboring all these variants (Fig. 3, Additional and developed PAH at age 6 with a mean PAP of file 1: Table S1). The same applies to Family E, albeit a 36 mmHg. Patient FII.1 presented at age 46 with dys- weak correlation does exist with the p.A1324G variant pnea on exertion. He was diagnosed with idiopathic in KDR (Fig. 2, Additional file 1: Table S1). Abou Hassan et al. BMC Medical Genetics (2018) 19:89 Page 6 of 9 Fig. 3 Pedigrees for the p.S775 BMPR2 mutation in three families with the second hit mutations shown in the table. Square, Male; Circle, female; open symbol, unaffected; filled symbol, affected; symbol with diagonal line, dead individual; the −/− symbol is for normal genes, −/+ is for heterozygous mutations and the +/+ is for homozygous mutations. N/A stands for non-applicable because DNA is not available Discussion than 300 different mutations have been identified, with With our 21 patients, and a worldwide PAH prevalence 80% of familial cases caused by mutations in this gene rate of few dozen per million, our study nearly accounts [16, 25]. Our results show a predominant BMPR2 vari- for all of the recently diagnosed cases in Lebanon, a ants rate of 30% that would perfectly align with small country with 4 million inhabitants. The mean age previous findings, and reinforces the need to look for of diagnosis of our cohort of PAH patients is 33 years additional genetic causes, taking into account the impli- which is similar to the mean age of diagnosis in other cation of environmental factors or compound MENA region registries [8, 9], but is significantly lower mutations on the onset of the disease in genetically than the mean age reported in recent European and “pre-disposed” individuals. north American registries [5, 6]. This difference in Growing evidence implicates the BMPR2 pathway in mean age at diagnosis may be due to differences in sur- structural heart defects. Roberts et al. report BMPR2 vival of PAH patients in our region or due to mutations in 6% of patients with diagnosed PAH and differences in referral and diagnostic practices. Older congenital defects [26]. Interestingly, three of our pro- patients with pulmonary hypertension have more co- bands presented with atrial or ventricular septal de- morbidities, and are not recognized early enough, if at fects. A septal defect was also identified in the sister of all,as having PAH. These findings highlight the need for patient CII.2 with the same mutation (Fig. 2). Mice more education in Lebanon and the MENA region to models of BMPR2 knock-outs show cardiac anomalies recognize that PAH is no longer a disease affecting only analogous to human septal defects and AV canal con- young patients. BMPR2 mutations are known to be the genital heart defects [27–29]. As such, both pulmonary main genetic causes of familial PAH [24]. To date, more vasculature involvement and hemodynamic stress from Abou Hassan et al. BMC Medical Genetics (2018) 19:89 Page 7 of 9 increased flow contribute to the pulmonary hyperten- patients of this small registry confirms the findings of sion in those patients. the literature but raises questions regarding its recurrent significance, especially that in all cases it is inherited High penetrance mutations: The BMPR2 variants from either one of the healthy parents with no ancestry Deleterious mutations usually lead to a tangible pheno- history of PAH. Moreover, the level of pulmonary artery type earlier in the patients’ lives. Patient AII.3 with the pressures measured in both patients was clinically milder BMPR2 p.Q6* mutation was followed at an early stage in relation to other BMPR2 mutations. However, it is im- after being diagnosed with ventricular septal defect and portant to note here that both patients D and E pre- pulmonary hypertension. His pulmonary artery pressures sented with the disease at a young age implicating a were higher than would be expected from shunting significant commitment of the mutation by environmen- alone. After surgical correction of the ventricular defect, tal or genetic interactions. The affected member in the pulmonary pressures failed to revert and an under- family B harbors a monoallelic inherited missense muta- lying cause of the pulmonary hypertension was worked tion p.N126S within the extracellular domain of the up with likelihood of idiopathic disease. Truncating and/ BMPR2 protein. Previous studies on mutations within or missense mutations affecting the N-terminal part of this domain have shown intracellular retention of the re- the protein have been hypothesized to result in more ex- ceptors and a dampening in SMAD activation [16, 31]. tensive pulmonary vascular remodeling and more rapid Such functional alteration is critical to the signaling progression of the disease process. This hypothesis is pathway, which might explain the relatively elevated sys- supported by the observation that the worse prognosis tolic pulmonary artery pressure reading on follow-up associated with a BMPR2 mutation in patients diagnosed echocardiography of the affected individual (data not before the age of 30 years is not completely attenuated shown). The fact that in both cases we do not have a after adjustment for pulmonary vascular resistance, car- clear answer about the pathogenicity of the mutation diac index, and vasoreactivity [30]. The early age at pres- prompted us to look at other “modifiers” in the pathway entation reflects the drastic functional role of the leading to PAH involving BMPR2. Compound mutations mutation. Such a drastic mutation is probably linked to within the BMPR2 pathway have been hypothesized and haploinsufficiency with a complete loss of one allele due recently predicted in the pathogenesis of the disease to mRNA decay rather than a protein with hampered [32]. This is highlighted more in mutations that are cellular localization and/or function. otherwise identified as benign mutations, rendering the Moreover, such deleterious mutations usually occur at analysis of genetic variants trickier. An interesting candi- functionally important domains within the large 3D struc- date mutation in patient DII.1 is the TBX4 variant ture of the receptor protein. Apart from an early trunca- (p.A246T): this gene has been implicated in small patella tion as in patient A.II3, the mutation p.R491W in family C syndrome and associated childhood onset pulmonary ar- (Fig. 2) occurs at a highly conserved site within the TGF-β terial hypertension [33]. GDF2, KDR and BMP10 muta- superfamily kinase domain of the receptor. Alteration of tions modifier moutations are also evident in families D, the Serine/Threonine kinase function hinders the down- E and F respectively (Fig. 3). Failure to detect segregating stream activation cascade within the BMPR2 pathway. mutations in other families may hint to copy number This mutation segregates within the family members de- variants, unknown gene mutations, as well as the possi- fining a high penetrance familial disease. bility of environmental stressors. One possible link between the genetic basis of pene- Low penetrance and the “second-hit mutation”: trance and stressors is wild-type BMPR2 transcript levels Besides the de novo mutation in BMPR2, only two mis- themselves. Hamid et al. demonstrated that the level of sense monoallelic mutations in BMPR2 were accounted production of BMPR2 transcript and protein by the for in 4 families: p.S775 N and p.N126S. The p.S775 N wild-type allele was associated with disease penetrance mutation identified in families D, E, and F has been re- [34]. HPAH patients with BMPR2 mutations had lower ported before and is of questionable clinical relevance transcript levels compared to unaffected mutation car- [23]. Clinical correlation to this mutation has failed to riers [17]. Another possibility is the role of highlight consistent phenotype, as this mutation is com- hemodynamic stressors imposed by the associated con- mon (Minor allele frequency (MAF) on ExAC 0.02516), genital disease that hasten or activate disease progres- and when found in patients with pulmonary hyperten- sion in otherwise low penetrance mutations. This is sion it does not associate with age of presentation and evident in patients D and E. The role of hemodynamic vaso-reactivity [23]. As such, the p.S775 N mutation is stressors is not very well understood; however, the role seen as a possible cause of disease with very low pene- of external triggers is well documented in the disease. A trance, perhaps requiring additional genetic or environ- trend of earlier presentation of the disease is evident in mental stimuli. The occurrence of the mutation in three patients on appetite suppressants [35]. Although such Abou Hassan et al. BMC Medical Genetics (2018) 19:89 Page 8 of 9 environmental stressors are not central to our cohort, and return of genetic data were performed in accordance with protocols approved by the Partners Human Research Committee. hemodynamic stressors may play a greater role in the failure to sustain sufficient BMPR2 transcript levels Consent for publication against a continuous stressor. The parents gave consent for information about themselves/their children or to be published in scientific journals. Conclusions Competing interests The authors declare that they have no competing interests. In conclusion, this is the first genetic study by exome se- quencing of PAH in the MENA region. The study un- Publisher’sNote veiled novel mutations in BMPR2, which may be unique Springer Nature remains neutral with regard to jurisdictional claims in published to the MENA region. This shows that more mutations maps and institutional affiliations. may be found if genetic studies are expanded to other Author details regions and ethnic groups. Our study also hinted at mul- Department of Internal Medicine, Faculty of Medicine, American University tiple gene interactions being responsible for the develop- of Beirut, P.O.Box: 11-0236, Beirut, Lebanon. Department of Biochemistry ment of PAH, which calls for further investigation of the and Molecular Genetics, Faculty of Medicine, American University of Beirut, P.O.Box: 11-0236, Beirut, Lebanon. Univeriste St Jospeh, Beirut, Lebanon. double-hit theory in larger genetic databases. Finally, the study shed light on the limitations of using standardized Received: 10 January 2018 Accepted: 18 May 2018 genetic testing based on North American and European findings in other regions of the world, emphasizing the References need for conducting whole exome sequencing (WES) for 1. Hoeper MM, Bogaard HJ, Condliffe R, et al. Definitions and diagnosis of genetic testing of PAH. pulmonary hypertension. J Am Coll Cardiol. 2013;62(25 Suppl):D42–50. https://doi.org/10.1016/j.jacc.2013.10.032. 2. Galiè N, Humbert M, Vachiery J-L, et al. 2015 ESC/ERS guidelines for the Additional files diagnosis and treatment of pulmonary hypertension. Rev Esp Cardiol (Engl Ed). 2016;69(2):177. https://doi.org/10.1016/j.rec.2016.01.002. 3. Larkin EK, Newman JH, Austin ED, et al. Longitudinal analysis casts doubt on Additional file 1: Table S1. 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Hemodynamic and clinical onset in patients with hereditary pulmonary arterial hypertension and BMPR2 Ethics approval and consent to participate mutations. Respir Res. 2011;12:99. https://doi.org/10.1186/1465-9921-12-99. The study was approved by the institutional review board (IRB) at the American 14. Garcia-Rivas G, Jerjes-Sánchez C, Rodriguez D, Garcia-Pelaez J, Trevino V. A University of Beirut (protocol number: Med.IB.01). All subjects gave written systematic review of genetic mutations in pulmonary arterial hypertension. informed consent in accordance with the Declaration of Helsinki. Children BMC Med Genet. 2017;18(1):82. https://doi.org/10.1186/s12881-017-0440-5. and adolescents under age of 16 signed an assent form, and got their 15. Newman JH, Wheeler L, Lane KB, et al. Mutation in the gene for bone parents’s written consents to be included in the study. Genetic analyses morphogenetic protein receptor II as a cause of primary pulmonary Abou Hassan et al. 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Shibbani K, Fahed A, Al-Shaar L, et al. Primary carnitine deficiency: novel mutations and insights into the cardiac phenotype. Clin Genet. 2013; https://doi.org/10.1111/cge.12112. 21. Zhao M, Austin ED, Hemnes AR, Loyd JE, Zhao Z. An evidence-based knowledgebase of pulmonary arterial hypertension to identify genes and pathways relevant to pathogenesis. Mol BioSyst. 2014;10(4):732–40. https:// doi.org/10.1039/c3mb70496c. 22. Deng Z, Morse JH, Slager SL, et al. Familial primary pulmonary hypertension (gene PPH1) is caused by mutations in the bone morphogenetic protein receptor-II gene. Am J Hum Genet. 2000;67(3):737–44. https://doi.org/10. 1086/303059. 23. Elliott CG, Glissmeyer EW, Havlena GT, et al. Relationship of BMPR2 mutations to vasoreactivity in pulmonary arterial hypertension. Circulation. 2006;113(21):2509–15. https://doi.org/10.1161/CIRCULATIONAHA.105.601930. 24. Newman JH, Trembath RC, Morse JA, et al. Genetic basis of pulmonary arterial hypertension: current understanding and future directions. J Am Coll Cardiol. 2004;43(12 Suppl S):33S–9S. https://doi.org/10.1016/j.jacc.2004.02.028. 25. Soubrier F, Chung WK, Machado R, et al. Genetics and genomics of pulmonary arterial hypertension. J Am Coll Cardiol. 2013;62(25 Suppl):D13– 21. https://doi.org/10.1016/j.jacc.2013.10.035. 26. Roberts KE, McElroy JJ, Wong WPK, et al. BMPR2 mutations in pulmonary arterial hypertension with congenital heart disease. Eur Respir J. 2004;24(3): 371–4. https://doi.org/10.1183/09031936.04.00018604. 27. Délot EC, Bahamonde ME, Zhao M, Lyons KM. BMP signaling is required for septation of the outflow tract of the mammalian heart. Development. 2003; 130(1):209–20. http://www.ncbi.nlm.nih.gov/pubmed/12441304 28. Jiao K, Kulessa H, Tompkins K, et al. An essential role of Bmp4 in the atrioventricular septation of the mouse heart. Genes Dev. 2003;17(19):2362– 7. https://doi.org/10.1101/gad.1124803. 29. Beppu H, Malhotra R, Beppu Y, Lepore JJ, Parmacek MS, Bloch KD. BMP type II receptor regulates positioning of outflow tract and remodeling of atrioventricular cushion during cardiogenesis. Dev Biol. 2009;331(2):167–75. https://doi.org/10.1016/j.ydbio.2009.04.032. 30. Evans JDW, Girerd B, Montani D, et al. BMPR2 mutations and survival in pulmonary arterial hypertension: an individual participant data meta- analysis. Lancet Respir Med. 2016;4(2):129–37. https://doi.org/10.1016/S2213- 2600(15)00544-5. 31. Rudarakanchana N, Flanagan JA, Chen H, et al. Functional analysis of bone morphogenetic protein type II receptor mutations underlying primary pulmonary hypertension. Hum Mol Genet. 2002;11(13):1517–25. http://www. ncbi.nlm.nih.gov/pubmed/12045205 32. Eichstaedt CA, Song J, Benjamin N, et al. EIF2AK4 mutation as “second hit” in hereditary pulmonary arterial hypertension. Respir Res. 2016;17(1):141. https://doi.org/10.1186/s12931-016-0457-x. 33. Kerstjens-Frederikse WS, Bongers EMHF, Roofthooft MTR, et al. TBX4 mutations (small patella syndrome) are associated with childhood-onset pulmonary arterial hypertension. J Med Genet. 2013;50(8):500–6. https://doi. org/10.1136/jmedgenet-2012-101152. 34. Hamid R, Cogan JD, Hedges LK, et al. Penetrance of pulmonary arterial hypertension is modulated by the expression of normal BMPR2 allele. Hum Mutat. 2009;30(4):649–54. https://doi.org/10.1002/humu.20922. 35. Humbert M, Deng Z, Simonneau G, et al. BMPR2 germline mutations in pulmonary hypertension associated with fenfluramine derivatives. Eur Respir J. 2002;20(3):518–23. http://www.ncbi.nlm.nih.gov/pubmed/12358323 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png BMC Medical Genetics Springer Journals

Clinical and genetic characteristics of pulmonary arterial hypertension in Lebanon

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Abstract

Background: Pulmonary arterial hypertension (PAH) is a rare disease with an incidence rate of 2–6 cases per million per year. Our knowledge of the disease in the Middle East and North Africa (MENA) region is limited by the small number of clinical studies and the complete absence of genetic studies. Methods: Our aim was to shed light on the clinical and genetic characteristics of PAH in Lebanon and the region by using exome sequencing on PAH patients referred to the American University of Beirut Medical Center (AUBMC). Twenty-one idiopathic, hereditary and Congenital Heart Disease (CHD) PAH patients were prospectively recruited, their clinical data summarized, and sequencing performed. Results: The mean age at diagnosis was 33 years with a female preponderance of 70%. The mean pulmonary artery pressure at the time of diagnosis was 55. Genetic testing showed that 5 out of 19 idiopathic and Congenital Heart Disease PAH patients had Bone Morphogenetic Protein Receptor 2 (BMPR2) mutations at 25% prevalence, with 2 of these patients exhibiting a novel mutation. It also showed the presence of 1 BMPR2 mutation with 100% penetrance in a heritable PAH family. In the remaining cases, the lack of a complete genotype/phenotype correlation entailed a multigenic inheritance; suspected interactions involved previously associated genes T-box transcription factor 4 (TBX4), Bone Morphogenic Protein 10 (BMP10) and Growth Differentiation Factor 2 (GDF2). Conclusions: This is the first study that looks into the genetic causes of PAH, including known and new BMPR2 mutations, in the MENA region. It is also the first study to characterize the clinical features of the disease in Lebanon. Keywords: Pulmonary hypertension, BMPR2, Mutation Background Early PAH registries in the eighties provided valuable Pulmonary Arterial Hypertension (OMIM #178600) is a information on the baseline characteristics and out- devastating disorder of the small pulmonary arteries. It comes of the disease and helped with the understanding is characterized by progressive neo-intimal prolifera- of the natural progression of PAH [4]. Patients in early tion, smooth muscle hypertrophy and occlusive vascu- registries were younger with a mean age of 36 and with lar lesions leading to increased vascular resistance and female preponderance. Data from more recent European pressure [1]. It is a rare disease with prevalence esti- and North American registries suggest that PAH pa- mated to be between 15 and 60 cases per million [2]. tients are becoming older and have a better survival [5, Males are at less risk than females with a 2–3:1 female: 6]. A longitudinal observational study on PAH incidence male ratio [2, 3]. in the United Kingdom and Ireland spaning a 9-year-period noticed an increase in the age of diagnosis of PAH patients from 45 to 52 years over the same * Correspondence: gn08@aub.edu.lb; ib08@aub.edu.lb period. These changes in demographics may reflect not Ossama K. Abou Hassan and Wiam Haidar contributed equally to this work. Department of Biochemistry and Molecular Genetics, Faculty of Medicine, only an improvement in survival but also better referral American University of Beirut, P.O.Box: 11-0236, Beirut, Lebanon practices in these countries [7]. The incidence, preva- Department of Internal Medicine, Faculty of Medicine, American University lence and burden of disease in the MENA region have of Beirut, P.O.Box: 11-0236, Beirut, Lebanon Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abou Hassan et al. BMC Medical Genetics (2018) 19:89 Page 2 of 9 never been reported before. Clinical and demographic (prevalence case) and patients who were referred to our characteristics of PAH patients have only been docu- center were approached for enrollement in the study. mented in a single center registry in Saudi Arabia, and They were eligible for enrolment if they had idiopathic in an online Iranian registry. Both sources show a mean PAH, heritable PAH or PAH associated with congenital age of 36 years and a 2:1 female preponderance [8, 9]. systemic to pulmonary shunt. All of these conditions are Despite our growing knowledge of the disease and its classified under Group 1 PAH, based on the World progression, PAH patients have significantly reduced sur- Health Organization’s (WHO) 2013 guidelines for diag- vival [10]. Most of drugs used (endothelin receptor antag- nosis of pulmonary hypertension [2]. Minimal inclusion onists, guanylate cyclase stimulators, prostacyclin criteria also included a Right Heart Catheterization analogues and prostacyclin receptor agonists) are prohibi- (RHC) measuring a mean pulmonary arterial pressure tively expensive and not readily available in middle to (mPAP) > 25 mmHg at rest and a pulmonary artery low-income countries such as Lebanon. This may affect wedge pressure (PAWP) < 15mmHG. Following the pa- disease severity and prevalence in these countries, making tient’s consent, clinical data was collected and reviewed, the care of PAH patients even more challenging. including medical history, physical exam, family history In the year 2000, the BMPR2 gene was first reported to of PAH, and previously performed tests. These tests in- be linked to the progression of the disease [11]. Around clude pulmonary function test (PFT), chest computer- 80% of hereditary PAH patients and 25% of idiopathic dis- ized tomography (CT), CT pulmonary angiography, ease patients have BMPR2 mutations [12, 13]. Twenty-one ventilation perfusion scan and pro-BNP. For those pa- other genes have also been implicated in the development tients not diagnosed and treated at AUBMC, specialist PAH, with the most frequent mutations occurring in physicians along with our investigators reviewed all ALK1, ENG, SMAD9, CAV1, KCNK3, MADH9 and medical records for accuracy of diagnosis. Treatment of EIF2AK4 [14]. The penetrance of BMPR2 mutations PAH was also noted. After collection of clinical data, a ranges between 27 and 50% in females and 14–43% in blood sample was collected from the patients for genetic males [3], depending on the mutation site within the gene analysis by whole exome sequencing. Twenty-one PAH [15]. BMRR2 mutations have also been linked to PAH with patients in total, belonging to 20 families were included congenital heart disease [16]. Trying to understand the in the study. In the case of patients found to have a mu- low penetrance of BMPR2 and the yet unidentified genetic tation, non-affected family members were approached causes of the remaining 20% of the hereditary form of for inclusion in the study as controls and blood was col- PAH, new research strategies have shifted towards using lected for sanger analysis of the mutated protein. The next-generation sequencing (NGS) in the search for add- study was approved by the institutional review board itional mutations or compound interactions of known (IRB) at the American University of Beirut. Genetic ana- genes [17]. There is a a growing acceptance of a double hit lyses and return of genetic data were performed in ac- phenomenon – a major gene with an additional modifier cordance with protocols approved by the Partners gene- supporting the lower penetrance of mutations in Human Research Committee. Data collection and gen- genes associated with PAH. As such NGS is poised to be etic analysis were performed in accordance with the pro- the best technology to unravel this genetic interaction and tocols approved by the IRB at AUBMC. accounts of all genotype-phenotype discrepancies [18, 19]. This study has many objectives. One objective is to de- Genetic studies scribe the demographic and clinical characteristics of PAH DNA was extracted from peripheral blood. Whole ex- patients in Lebanon. These findings will shed light on the ome libraries were compiled with DNA samples from all burden of disease and the state of referral and care of pa- affected individuals recruited in this study. The exome tients with PAH in the country. The second objective is to was captured using the V5 SureSelect kits from Agilent assess for the first time the prevalence and types of genetic as per manufacturer’s protocol, and run on the Illumina mutations in PAH patients in Lebanon and the MENA re- HiSeq2500. The tests were performed by Macrogen, gion. This may help guide future genetic testing, counsel- South Korea. Exome sequences were aligned to hg19 ing and research in an area that shares common genetic/ using Novoalign, and variants were called using Genome ethnic background. The final objective is to explore novel Analysis Toolkit (GATK). Once variant(s) predicted to genetic mutations or interactions and their implications be damaging with a minor allele frequency (< 3%) were on the phenotypic manifestations of PAH. identified in a patient, Sanger sequencing was performed to confirm thier occurence, and all family members were Methods screened for those particular variant(s). Primers were Subject recruitment speficially designed to amplify the region(s) of interest Patients diagnosed with PAH at AUBMC between 2015 that include those variants, and amplified fragments and 2017 either newly (incidence case) or previously were analyzed by dideoxy sequencing (ABI technology) Abou Hassan et al. BMC Medical Genetics (2018) 19:89 Page 3 of 9 using the ABI3500 at the molecular core facility at the country (Table 1). The age of diagnosis as well as faculty of medicine at AUB as previously described [20]. additional phenotypes were included, and as previously The results were analyzed at the Congenital Heart mentioned, outpatients referred to our center were clinic- Disease Genetics Program (CHDGP) laboratory at the ally re-evaluated for confirmation of the phenotype. The American University of Beirut Medical Center AUBMC. cohort includes one family with 2 patients having each hereditary pulmonary arterial hypertension (PAH), 14 pa- Genetic panel selection tients with idiopathic PAH, and 5 with both PAH and con- All generated variant call files (vcf) were analyzed accord- genital heart disease (CHD). Fifteen out of 21 patients ing to an extensive virtual gene panel that was chosen to (71%) are females. The idiopathic and hereditary groups screen for the disease in Lebanon and the region and was had a mean age of 33 years at the time of diagnosis and a adopted from recent published methodology for choosing mean pulmonary artery pressure of 56 mmHg. Seventeen culprit genes in PAH [21]. Non-synonymous exon coding patients were on dual therapy (sildenafil, and bosentan or variants for BMPR2 were analyzed and a search for com- masitentan), and 6 patients on sildenafil alone. pound heterozygous variants associated with the BMPR2 mutations was performed accordingly using the Illumina BMPR2 variants variant studio. Non-synonymous missense variants, inser- For all patients, we conducted a primary analysis, whereby tion/deletions variants in the coding regions, and splicing only BMPR2 variants were included. The filtering strin- variants with allele frequencies less than 3% were filtered gency put a cutoff on all synonymous variants, inframe in according to their evolutionary conservation, location insertions/deletions, and non-coding ones. Out of the 20 within domains and prediction software (SIFT and families, we identified six with monoallelic BMPR2 Polyphen2), while additional variants with no supportive variants, thus constituting a prevalence of 30%. The roles were filtered out (Fig. 1). phenotypes of the index patients are summarized in Additional file 1: Table S1. Patients with and without Results BMPR2 mutations had a similar age of 33 years at diagno- Clinical and demographic profiles of families and patients sis, and a similar mean pulmonary arterial pressure (PAP) A total of 21 patients belonging to 20 different families of 57 mmHg. The PAH with CHD group had a mean age were recruited in the study from different parts of the of 5.4 years at diagnosis and a mean PAP of 44 mmHg. Fig. 1 Stepwise strategy for filtering variants to reach 10–15 highly likely disease-causing variants per patient screened. UTR = Untranslated Region; AF = allele frequency Abou Hassan et al. BMC Medical Genetics (2018) 19:89 Page 4 of 9 Table 1 BMPR2 variants in pulmonary hypertension patients (PHT), with their corresponding global allele frequencies (AF), and predictive protein effects (Polyphen2). ASD (Atrial Septal Defect), VSD (Ventricular Septal Defect), ExAC (Exome Aggregation Consortium), 1000G (1000 Genome from the International Genome Sample Resource) Family Sex Age at Phenotype mPAP Gene Mutation familial/ Zygosity ExAC AF 1000 G AF SIFT Polyphen2 Diagnosis (mmHg) sporadic A F 5 Large ASD and PAH 45 BMPR2 p.Q6* s Heterozygous N/A N/A (de novo) B F 38 PAH 50 BMPR2 p.N126S f Heterozygous Damaging Probably Damaging C F 25 PAH 50 BMPR2 p.R491W f Heterozygous Damaging Damaging F 25 PAH 94 BMPR2 p.R491W f Heterozygous Damaging Damaging D F 11 VSD and PAH 88 BMPR2 p.S775 N s Heterozygous 0.02516 0.00998403 Tolerated Benign E F 6 ASD and PAH 36 BMPR2 p.S775 N s Heterozygous 0.02516 0.00998403 Tolerated Benign F M 46 PAH 50 BMPR2 p.S775 N s Heterozygous 0.02516 0.00998403 Tolerated Benign G M 4 VSD and PAH 43 s H M 11 PAH 77 s I F 1 PAH VSD/ASD 33 s J M 22 PAH 56 s K F 20 PAH 50 s L F 31 PAH 41 s M F 45 PAH 58 s N F 26 PAH 62 s O F 30 PAH 63 s P F 57 PAH 46 s Q F 7 PAH 55 s R F 6 PAH 40 s S M 78 PAH 49 s T M 34 PAH 40 s Patient AII.3 (Fig. 2), a young girl who had an ASD re- screened family members. The father (BII.2), who is pair with fenestration at age one, presented at age 5 with clinically healthy, refused to participate in the study dyspnea. She was found to have PAH, with a mean PAP and may be the source of inheritance (Fig. 2). Interest- of 45 mmHg. Exome sequencing revealed a heterozygous ingly, two of the three children of the indexed patient nonsense mutation within exon 1 of BMPR2 that leads (namely individuals BIV.1 and BIV.2) also carry the to a premature stop codon (p.Q6*). Sanger sequencing variant (Fig. 2). Although they exhibit no apparent confirmed the mutation in the affected patient; however, clinical signs or symptoms of PAH, they are still young the mutation was not detected in any family members (< 10 years) compared to their mother (BIII.1) who was and none of them had pulmonary hypertension on diagnosed with PAH at age 38. follow-up, suggesting it is a de novo nonsense mutation. Patients CII.2 and CII.3 are 24-year-old female identi- Patient BIII.1 (Fig. 2), a 38-year-old-woman with no cal twins. Patient CII.2 presented with dyspnea on exer- apparent relevant family history, presented with progres- tion and syncope. Right heart catheterization (RHC) sive dyspnea. She was diagnosed with idiopathic PAH showed a mean pulmonary artery pressure (PAP) of after excluding other possible causes of PAH, with a 94 mmHg and a pulmonary vascular resistance (PVR) of mean PAP of 50 mmHg. She is being treated with sil- 25 woods units. Acute pulmonary vaso-reactivity testing denafil and macitentan. Exome sequencing showed a with inhaled nitric oxide (NO) at a concentration of novel missense mutation (p.N126S) within the trans- 40 ppm for 10 min was positive (mean PAP dropped to membrane domain of the BMPR2 receptor. The muta- 21 mmHg). She was NYHA Class III. Her mother had tion was reported as probably damaging on the died of idiopathic PAH at age 30 and her younger Polyphen-2 and SIFT prediction tools (Table 1). Sanger brother died of PAH at age 8. She was diagnosed with sequencing confirmed the mutation in the affected indi- ventricular septal defect (VSD) as a child, which was vidual, but revealed the absence of the mutation in the corrected at age 6. The patient was diagnosed with Abou Hassan et al. BMC Medical Genetics (2018) 19:89 Page 5 of 9 Fig. 2 Pedigrees for families showing the different BMPR2 mutations. The −/− symbol is for normal genes, −/+ is for heterozygous mutations and the +/+ is for homozygous mutations. Square, Male; Circle, female; open symbol, unaffected; filled symbol, affected; symbol with diagonal line, dead individual heritable PAH and she was started on a calcium channel PAH with no cardiac defects, and his RHC showed a blocker and sildenafil. A repeat RHC, 3 months later, mean PAP of 50. showed a decrease in mean PAP to 54 mmHg and loss of vaso-reactivity response to NO. She improved clinic- Other variants ally to NYHA II and bosentan was added to her treat- In the rest of the families (Additional file 2: Figure S1), ment. Her twin sister CII.3 was asymptomatic and was we adopted the same stringent filtering mentioned for screened for PAH. She was found to have PAH with a BMPR2 on a list of 431 genes curated from the literature mean PAP of 50 mmHg. There were no signs of VSD. and online databases (Table 1). For each patient, a list of Family C has a previously reported mutation (p.R491W) variants was extracted (Additional file 1: Table S1). None [22]; it occurs in a highly conserved kinase domain would explain the phenotype on its own, since there encoded by exon 3 of the gene. Records from the de- were no homozygous variants in these genes (data not ceased mother and brother showed that the mutation shown). Examples for potential variants are the p.R55W was found in all affected family members CI.2, CII.1, in BMP4 in family M, and p.M416I variant in BMP10 in CII.2, and CII.3. The father CI.1 who is phenotypically Family L (Additional file 1: Table S1). In contrast, in normal did not participate in the study. The mutation families with predicted benign and/or low penetrance was labeled as “damaging” on Polyphen-2 and SIFT BMPR2 mutations (families D, E, and F, Table 1), a (Table 1, and Fig. 2). search for probable second hit mutations was attempted. Patients DII.1, EII.3, and FII.1 have the same variant in The results suggest a potential digenic model for the dis- BMPR2, a missense mutation (p.S775 N) previously re- ease in families D and E (Fig. 3) supported by Sanger se- ported as being disease-causing with very low pene- quencing of the involved members in each case. In trance [23]. This mutation occurs in the C-terminal Family D, biallelic variants in GDF2 (p.P85L, and domain of the protein within exon 12. It is labeled as be- p.D218N), and a monoallelic variant in TBX4 (p.A246T) nign based on the Polyphen-2 and SIFT prediction tools are novel and are predicted as disease-causing. Those (Table 1). Patient DII.1 had a small PFO and he devel- mutations in combination with the “low penetrant” oped PAH at age 11. RHC showed a mean PAP of BMPR2 variant (p.S775 N) could explain the phenotype 68 mmHg. He has been stable on sildenafil-bosentan for in the only affected member of the family who is the the last 5 years. Patient EII.3 had an ASD repair at age 1 only one harboring all these variants (Fig. 3, Additional and developed PAH at age 6 with a mean PAP of file 1: Table S1). The same applies to Family E, albeit a 36 mmHg. Patient FII.1 presented at age 46 with dys- weak correlation does exist with the p.A1324G variant pnea on exertion. He was diagnosed with idiopathic in KDR (Fig. 2, Additional file 1: Table S1). Abou Hassan et al. BMC Medical Genetics (2018) 19:89 Page 6 of 9 Fig. 3 Pedigrees for the p.S775 BMPR2 mutation in three families with the second hit mutations shown in the table. Square, Male; Circle, female; open symbol, unaffected; filled symbol, affected; symbol with diagonal line, dead individual; the −/− symbol is for normal genes, −/+ is for heterozygous mutations and the +/+ is for homozygous mutations. N/A stands for non-applicable because DNA is not available Discussion than 300 different mutations have been identified, with With our 21 patients, and a worldwide PAH prevalence 80% of familial cases caused by mutations in this gene rate of few dozen per million, our study nearly accounts [16, 25]. Our results show a predominant BMPR2 vari- for all of the recently diagnosed cases in Lebanon, a ants rate of 30% that would perfectly align with small country with 4 million inhabitants. The mean age previous findings, and reinforces the need to look for of diagnosis of our cohort of PAH patients is 33 years additional genetic causes, taking into account the impli- which is similar to the mean age of diagnosis in other cation of environmental factors or compound MENA region registries [8, 9], but is significantly lower mutations on the onset of the disease in genetically than the mean age reported in recent European and “pre-disposed” individuals. north American registries [5, 6]. This difference in Growing evidence implicates the BMPR2 pathway in mean age at diagnosis may be due to differences in sur- structural heart defects. Roberts et al. report BMPR2 vival of PAH patients in our region or due to mutations in 6% of patients with diagnosed PAH and differences in referral and diagnostic practices. Older congenital defects [26]. Interestingly, three of our pro- patients with pulmonary hypertension have more co- bands presented with atrial or ventricular septal de- morbidities, and are not recognized early enough, if at fects. A septal defect was also identified in the sister of all,as having PAH. These findings highlight the need for patient CII.2 with the same mutation (Fig. 2). Mice more education in Lebanon and the MENA region to models of BMPR2 knock-outs show cardiac anomalies recognize that PAH is no longer a disease affecting only analogous to human septal defects and AV canal con- young patients. BMPR2 mutations are known to be the genital heart defects [27–29]. As such, both pulmonary main genetic causes of familial PAH [24]. To date, more vasculature involvement and hemodynamic stress from Abou Hassan et al. BMC Medical Genetics (2018) 19:89 Page 7 of 9 increased flow contribute to the pulmonary hyperten- patients of this small registry confirms the findings of sion in those patients. the literature but raises questions regarding its recurrent significance, especially that in all cases it is inherited High penetrance mutations: The BMPR2 variants from either one of the healthy parents with no ancestry Deleterious mutations usually lead to a tangible pheno- history of PAH. Moreover, the level of pulmonary artery type earlier in the patients’ lives. Patient AII.3 with the pressures measured in both patients was clinically milder BMPR2 p.Q6* mutation was followed at an early stage in relation to other BMPR2 mutations. However, it is im- after being diagnosed with ventricular septal defect and portant to note here that both patients D and E pre- pulmonary hypertension. His pulmonary artery pressures sented with the disease at a young age implicating a were higher than would be expected from shunting significant commitment of the mutation by environmen- alone. After surgical correction of the ventricular defect, tal or genetic interactions. The affected member in the pulmonary pressures failed to revert and an under- family B harbors a monoallelic inherited missense muta- lying cause of the pulmonary hypertension was worked tion p.N126S within the extracellular domain of the up with likelihood of idiopathic disease. Truncating and/ BMPR2 protein. Previous studies on mutations within or missense mutations affecting the N-terminal part of this domain have shown intracellular retention of the re- the protein have been hypothesized to result in more ex- ceptors and a dampening in SMAD activation [16, 31]. tensive pulmonary vascular remodeling and more rapid Such functional alteration is critical to the signaling progression of the disease process. This hypothesis is pathway, which might explain the relatively elevated sys- supported by the observation that the worse prognosis tolic pulmonary artery pressure reading on follow-up associated with a BMPR2 mutation in patients diagnosed echocardiography of the affected individual (data not before the age of 30 years is not completely attenuated shown). The fact that in both cases we do not have a after adjustment for pulmonary vascular resistance, car- clear answer about the pathogenicity of the mutation diac index, and vasoreactivity [30]. The early age at pres- prompted us to look at other “modifiers” in the pathway entation reflects the drastic functional role of the leading to PAH involving BMPR2. Compound mutations mutation. Such a drastic mutation is probably linked to within the BMPR2 pathway have been hypothesized and haploinsufficiency with a complete loss of one allele due recently predicted in the pathogenesis of the disease to mRNA decay rather than a protein with hampered [32]. This is highlighted more in mutations that are cellular localization and/or function. otherwise identified as benign mutations, rendering the Moreover, such deleterious mutations usually occur at analysis of genetic variants trickier. An interesting candi- functionally important domains within the large 3D struc- date mutation in patient DII.1 is the TBX4 variant ture of the receptor protein. Apart from an early trunca- (p.A246T): this gene has been implicated in small patella tion as in patient A.II3, the mutation p.R491W in family C syndrome and associated childhood onset pulmonary ar- (Fig. 2) occurs at a highly conserved site within the TGF-β terial hypertension [33]. GDF2, KDR and BMP10 muta- superfamily kinase domain of the receptor. Alteration of tions modifier moutations are also evident in families D, the Serine/Threonine kinase function hinders the down- E and F respectively (Fig. 3). Failure to detect segregating stream activation cascade within the BMPR2 pathway. mutations in other families may hint to copy number This mutation segregates within the family members de- variants, unknown gene mutations, as well as the possi- fining a high penetrance familial disease. bility of environmental stressors. One possible link between the genetic basis of pene- Low penetrance and the “second-hit mutation”: trance and stressors is wild-type BMPR2 transcript levels Besides the de novo mutation in BMPR2, only two mis- themselves. Hamid et al. demonstrated that the level of sense monoallelic mutations in BMPR2 were accounted production of BMPR2 transcript and protein by the for in 4 families: p.S775 N and p.N126S. The p.S775 N wild-type allele was associated with disease penetrance mutation identified in families D, E, and F has been re- [34]. HPAH patients with BMPR2 mutations had lower ported before and is of questionable clinical relevance transcript levels compared to unaffected mutation car- [23]. Clinical correlation to this mutation has failed to riers [17]. Another possibility is the role of highlight consistent phenotype, as this mutation is com- hemodynamic stressors imposed by the associated con- mon (Minor allele frequency (MAF) on ExAC 0.02516), genital disease that hasten or activate disease progres- and when found in patients with pulmonary hyperten- sion in otherwise low penetrance mutations. This is sion it does not associate with age of presentation and evident in patients D and E. The role of hemodynamic vaso-reactivity [23]. As such, the p.S775 N mutation is stressors is not very well understood; however, the role seen as a possible cause of disease with very low pene- of external triggers is well documented in the disease. A trance, perhaps requiring additional genetic or environ- trend of earlier presentation of the disease is evident in mental stimuli. The occurrence of the mutation in three patients on appetite suppressants [35]. Although such Abou Hassan et al. BMC Medical Genetics (2018) 19:89 Page 8 of 9 environmental stressors are not central to our cohort, and return of genetic data were performed in accordance with protocols approved by the Partners Human Research Committee. hemodynamic stressors may play a greater role in the failure to sustain sufficient BMPR2 transcript levels Consent for publication against a continuous stressor. The parents gave consent for information about themselves/their children or to be published in scientific journals. Conclusions Competing interests The authors declare that they have no competing interests. In conclusion, this is the first genetic study by exome se- quencing of PAH in the MENA region. The study un- Publisher’sNote veiled novel mutations in BMPR2, which may be unique Springer Nature remains neutral with regard to jurisdictional claims in published to the MENA region. This shows that more mutations maps and institutional affiliations. may be found if genetic studies are expanded to other Author details regions and ethnic groups. Our study also hinted at mul- Department of Internal Medicine, Faculty of Medicine, American University tiple gene interactions being responsible for the develop- of Beirut, P.O.Box: 11-0236, Beirut, Lebanon. Department of Biochemistry ment of PAH, which calls for further investigation of the and Molecular Genetics, Faculty of Medicine, American University of Beirut, P.O.Box: 11-0236, Beirut, Lebanon. Univeriste St Jospeh, Beirut, Lebanon. double-hit theory in larger genetic databases. Finally, the study shed light on the limitations of using standardized Received: 10 January 2018 Accepted: 18 May 2018 genetic testing based on North American and European findings in other regions of the world, emphasizing the References need for conducting whole exome sequencing (WES) for 1. Hoeper MM, Bogaard HJ, Condliffe R, et al. Definitions and diagnosis of genetic testing of PAH. pulmonary hypertension. J Am Coll Cardiol. 2013;62(25 Suppl):D42–50. https://doi.org/10.1016/j.jacc.2013.10.032. 2. Galiè N, Humbert M, Vachiery J-L, et al. 2015 ESC/ERS guidelines for the Additional files diagnosis and treatment of pulmonary hypertension. Rev Esp Cardiol (Engl Ed). 2016;69(2):177. https://doi.org/10.1016/j.rec.2016.01.002. 3. Larkin EK, Newman JH, Austin ED, et al. Longitudinal analysis casts doubt on Additional file 1: Table S1. 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BMC Medical GeneticsSpringer Journals

Published: May 30, 2018

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