Mutations In Activin Receptor-like Kinase 1 Research Articles

Shear Stress and Sub-Femtomolar Levels of Ligand Synergize to Activate ALK1 Signaling in Endothelial Cells.

Endothelial cells (ECs) respond to concurrent stimulation by biochemical factors and wall shear stress (SS) exerted by blood flow. Disruptions in flow-induced responses can result in remodeling issues and cardiovascular diseases, but the detailed mechanisms linking flow-mechanical cues and biochemical signaling remain unclear. Activin receptor-like kinase 1 (ALK1) integrates SS and ALK1-ligand cues in ECs; ALK1 mutations cause hereditary hemorrhagic telangiectasia (HHT), marked by arteriovenous malformation (AVM) development. However, the mechanistic underpinnings of ALK1 signaling modulation by fluid flow and the link to AVMs remain uncertain. We recorded EC responses under varying SS magnitudes and ALK1 ligand concentrations by assaying pSMAD1/5/9 nuclear localization using a custom multi-SS microfluidic device and a custom image analysis pipeline. We extended the previously reported synergy between SS and BMP9 to include BMP10 and BMP9/10. Moreover, we demonstrated that this synergy is effective even at extremely low SS magnitudes (0.4 dyn/cm2) and ALK1 ligand range (femtogram/mL). The synergistic response to ALK1 ligands and SS requires the kinase activity of ALK1. Moreover, ALK1's basal activity and response to minimal ligand levels depend on endocytosis, distinct from cell-cell junctions, cytoskeleton-mediated mechanosensing, or cholesterol-enriched microdomains. However, an in-depth analysis of ALK1 receptor trafficking's molecular mechanisms requires further investigation.

Impact of heterozygous ALK1 mutations on the transcriptomic response to BMP9 and BMP10 in endothelial cells from hereditary hemorrhagic telangiectasia and pulmonary arterial hypertension donors

Heterozygous activin receptor-like kinase 1 (ALK1) mutations are associated with two vascular diseases: hereditary hemorrhagic telangiectasia (HHT) and more rarely pulmonary arterial hypertension (PAH). Here, we aimed to understand the impact of ALK1 mutations on BMP9 and BMP10 transcriptomic responses in endothelial cells. Endothelial colony-forming cells (ECFCs) and microvascular endothelial cells (HMVECs) carrying loss of function ALK1 mutations were isolated from newborn HHT and adult PAH donors, respectively. RNA-sequencing was performed on each type of cells compared to controls following an 18 h stimulation with BMP9 or BMP10. In control ECFCs, BMP9 and BMP10 stimulations induced similar transcriptomic responses with around 800 differentially expressed genes (DEGs). ALK1-mutated ECFCs unexpectedly revealed highly similar transcriptomic profiles to controls, both at the baseline and upon stimulation, and normal activation of Smad1/5 that could not be explained by a compensation in cell-surface ALK1 level. Conversely, PAH HMVECs revealed strong transcriptional dysregulations compared to controls with > 1200 DEGs at the baseline. Consequently, because our study involved two variables, ALK1 genotype and BMP stimulation, we performed two-factor differential expression analysis and identified 44 BMP9-dysregulated genes in mutated HMVECs, but none in ECFCs. Yet, the impaired regulation of at least one hit, namely lunatic fringe (LFNG), was validated by RT-qPCR in three different ALK1-mutated endothelial models. In conclusion, ALK1 heterozygosity only modified the BMP9/BMP10 regulation of few genes, including LFNG involved in NOTCH signaling. Future studies will uncover whether dysregulations in such hits are enough to promote HHT/PAH pathogenesis, making them potential therapeutic targets, or if second hits are necessary.

Pulmonary arterial hypertension in hereditary hemorrhagic telangiectasia associated with ACVRL1 mutation: a case report

IntroductionHereditary hemorrhagic telangiectasia is an autosomal dominant condition with an estimated prevalence of 1 in 5000. It is characterized by the presence of abnormalities of vascular structures, and may affect many organ systems, including the lungs, brain, spinal cord, gastrointestinal tract, and liver. A causative mutation is identified in approximately 97% of patients with definite hereditary hemorrhagic telangiectasia in one of three genes including a mutation in endoglin, a mutation in a locus mapped to chromosome 5, and an activin receptor-like kinase-1 (ACVRL1) mutation that is associated with an increased incidence of primary pulmonary hypertension. Pulmonary arterial hypertension is a rare (15–25 cases per million people) but severe vascular disorder. Heritable pulmonary arterial hypertension is associated with several gene mutations, with 75% having a mutation in the bone morphogenetic protein receptor 2 (BMPR2). However, the remaining 25% of patients have other associated genetic mutations including ACVLR1, which is also associated with hereditary hemorrhagic telangiectasia. Pulmonary arterial hypertension is a rare complication in patients with hereditary hemorrhagic telangiectasia (< 1% of the hereditary hemorrhagic telangiectasia population). We describe a case report with this rare occurrence.Case presentationA 70-year-old white/caucasian Irish male presented for screening for hereditary hemorrhagic telangiectasia due to a history of recurrent epistaxis (once/week) and a family history suggestive of pulmonary hypertension. Genetic testing confirmed an ACVRL1 mutation, while an echocardiogram and right heart catheterization confirmed pulmonary arterial hypertension. On examination, he had several mucocutaneous telangiectasia across his face. He was commenced on tadalafil and macitentan. However, this led to increased iron deficiency anemia and pedal edema. Selexipag was also added to his drug regime. He continues to require intermittent admissions for diuresis and blood transfusions.ConclusionThe association of hereditary hemorrhagic telangiectasia and pulmonary arterial hypertension is rare (< 1%). Here we describe a case of hereditary hemorrhagic telangiectasia complicated with pulmonary arterial hypertension as a result of an ACVRL1 mutation. We also describe the clinical challenges of treating these two conditions together, as treatment options for pulmonary arterial hypertension tend to worsen hereditary hemorrhagic telangiectasia symptoms.

Clinical characteristics and prognosis analysis of idiopathic and hereditary pulmonary hypertension patients with ACVRL1 gene mutations.

Pulmonary arterial hypertension is a kind of heart and lung vascular disease with low incidence and poor prognosis. Genetic variants are the important factors of pulmonary arterial hypertension. The mutations of activin receptor-like kinase-1 (ACVRL1) could cause pulmonary arteriole obstruction and occlusion in pulmonary arterial hypertension patients. The ACVRL1 gene mutation and clinical characteristics of Chinese idiopathic or hereditary pulmonary hypertension (IPAH/HPAH) patients are still unclear. This study aimed to retrospectively study the mutation characteristics of ACVRL1 gene in Chinese IPAH/HPAH patients and its effect on clinical prognosis. We analyzed the clinical, functional, hemodynamic and mutation characteristics of 12 IPAH/HPAH patients with ACVRL1 mutations and compared with 94 IPAH/HPAH patients (27 patients carried bone morphogenetic protein receptor type 2 (BMPR2) mutations and 67 without mutations). All ACVRL1 mutations of 12 patients were single nucleotide missense mutations. The ratio of male to female in 12 patients was 1:1. The diagnosis age of ACVRL1 mutation patients was younger than that of BMPR2 mutation patients (13.6 ± 11.3 years vs. 16.0 ± 12.9 years) but higher than that of patients without mutations (13.6 ± 11.3 years vs. 8.8 ± 8.5 years, p = 0.006). IPAH/HPAH patients with ACVRL1 mutation have rapid disease progresses, high overall mortality rate (approximately 50%) and no response to the acute pulmonary vasodilation test. In conclusion, this is the first study to analyze the ACVRL1 gene mutation and clinical characteristics of Chinese IPAH/HPAH patients. It is beneficial to screen ACVRL1 gene mutation for IPAH/HPAH patients to facilitate genetic counseling and early prevention and treatment.

Pulmonary arterial hypertension in families with hereditary hemorrhagic telangiectasia: a 13-year follow-up study

Abstract Background Pulmonary arterial hypertension (PAH) can occur in families with hereditary hemorrhagic telangiectasia (HHT), but it has not been well-characterized. Purpose This study sought to characterize the genetic defects, clinical and hemodynamic features, and outcomes of PAH patients in HHT families. Methods All HHT families with at least one case of PAH investigated in our center from January 2006 to December 2018 were enrolled in the study. We did whole-exome sequencing (WES) or whole-genome sequencing (WGS) to every proband and available family members. The clinical data, hemodynamic features and outcomes of PAH patients were reviewed. Results A total of 64 PAH patients in 57 families were enrolled. Only 7 (12.3%) families had more than one case of PAH. Activin-receptor-like kinase 1 (ALK-1) mutation and Endoglin (ENG) mutation were confirmed in 49 (86.0%) and 3 (5.3%) families and other 5 (8.8%) had no known mutation associated with HHT or PAH. The median age when PAH diagnosis was 22 [9–33] years and female was dominant (70.3%). Most patients (84.4%) had signs of HHT and anemia was recorded in 14 (21.9%) patients. Remarkably, these patients showed severely compromised hemodynamics with elevated mean pulmonary artery pressure (62 [51, 77] mm Hg) and pulmonary vascular resistance index (17.0 [11.2, 22.8] Wood units*m2). Also, impaired exercise capacity was recorded at diagnosis with decreased six-minute walking distance (410 [342–485] meters) and over half (54.7%) were in WHO functional class III or IV. The 1-, 3-, 5- and 10-year transplantation-free survival for the overall PAH patients was 95.0%, 75.9%, 67.1% and 36.4%, respectively. Anemia (HR: 4.24 [1.32–13.65], p=0.016) and CI &amp;lt;2.5 l/min/m2 (HR: 4.39 [1.20–16.09], p=0.026) were independent risk factors for mortality. Conclusions PAH in HHT families is a devastating condition characterized by a young age at PAH diagnosis, poor clinical status and outcomes mainly underlying ALK-1 mutation, which emphasizes the importance to pay attention to this group of patients. Transplantation-free survival Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): The Beijing Natural Science Foundation (7181009), the National Key Research and Development Program of China (2016YFC0901502)

Role of activin receptor-like kinase 1 in vascular development and cerebrovascular diseases.

Activin receptor-like kinase 1 (ALK1) is a transmembrane serine/threonine receptor kinase of the transforming growth factor beta (TGFβ) receptor superfamily. ALK1 is specifically expressed in vascular endothelial cells, and its dynamic changes are closely related to the proliferation of endothelial cells, the recruitment of pericytes to blood vessels, and functional differentiation during embryonic vascular development. The pathophysiology of many cerebrovascular diseases is today understood as a disorder of endothelial cell function and an imbalance in the proportion of vascular cells. Indeed, mutations in ALK1 and its co-receptor endoglin are major genetic risk factors for vascular arteriovenous malformation. Many studies have shown that ALK1 is closely related to the development of cerebral aneurysms, arteriovenous malformations, and cerebral atherosclerosis. In this review, we describe the various roles of ALK1 in the regulation of angiogenesis and in the maintenance of cerebral vascular homeostasis, and we discuss its relationship to functional dysregulation in cerebrovascular diseases. This review should provide new perspectives for basic research on cerebrovascular diseases and offer more effective targets and strategies for clinical diagnosis, treatment, and prevention.

Treatment with low-dose tacrolimus inhibits bleeding complications in a patient with hereditary hemorrhagic telangiectasia and pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) can be found in patients suffering from a loss-of-function mutation of the gene encoding for the activin receptor-like kinase 1 (ALK-1), a bone morphogenetic protein (BMP) type 1 receptor. Interestingly, ALK-1 mutations also lead to hereditary hemorrhagic telangiectasia (HHT), an autosomal dominant disease characterized by arteriovenous malformations (AVMs) leading to potentially life-threatening bleeding complications such as epistaxis. Current therapeutic options for both diseases are limited and often only temporary or accompanied by severe side effects. Here, we report of a patient with a mutation of the ALK-1 gene suffering from both HHT and PAH. Recently, it was shown that tacrolimus increased ALK-1 signaling and had beneficial effects in selected end-stage PAH patients. We thus hypothesized that treatment with tacrolimus may prevent disease progression in this patient. Surprisingly, treatment with low-dose tacrolimus dramatically improved his HHT-associated epistaxis but did not attenuate progression of PAH.

ALK1 Loss Results in Vascular Hyperplasia in Mice and Humans Through PI3K Activation.

ALK1 (activin-receptor like kinase 1) is an endothelial cell-restricted receptor with high affinity for BMP (bone morphogenetic protein) 9 TGF-β (transforming growth factor-β) family member. Loss-of-function mutations in ALK1 cause a subtype of hereditary hemorrhagic telangiectasia-a rare disease characterized by vasculature malformations. Therapeutic strategies are aimed at reducing potential complications because of vascular malformations, but currently, there is no curative treatment for hereditary hemorrhagic telangiectasia. In this work, we report that a reduction in ALK1 gene dosage (heterozygous ALK1+/- mice) results in enhanced retinal endothelial cell proliferation and vascular hyperplasia at the sprouting front. We found that BMP9/ALK1 represses VEGF (vascular endothelial growth factor)-mediated PI3K (phosphatidylinositol 3-kinase) by promoting the activity of the PTEN (phosphatase and tensin homolog). Consequently, loss of ALK1 function in endothelial cells results in increased activity of the PI3K pathway. These results were confirmed in cutaneous telangiectasia biopsies of patients with hereditary hemorrhagic telangiectasia 2, in which we also detected an increase in endothelial cell proliferation linked to an increase on the PI3K pathway. In mice, genetic and pharmacological inhibition of PI3K is sufficient to abolish the vascular hyperplasia of ALK1+/- retinas and in turn normalize the vasculature. Overall, our results indicate that the BMP9/ALK1 hub critically mediates vascular quiescence by limiting PI3K signaling and suggest that PI3K inhibitors could be used as novel therapeutic agents to treat hereditary hemorrhagic telangiectasia.

Gene Analysis of A Chinese Family with Hereditary Hemorrhagic Telangiectasia and its Curative Effect of Thalidomide

To analyze a hereditary hemorrhagic telangiectasia(HHT) family Activin receptor-like kinase 1(ACVRL1), Endoglin (ENG) and Mothers against decapentaplegic homolog 4 (MADH4, SMAD4) gene mutation, meanwhile, to observe the curative effect of thalidomide in treatment of HHT patients. The clinical feature of the HHT family was analyzed, the polymerase chain reaction (PCR) combined with Sanger sequencing of ACVRL1, ENG and SMAD4 were used to investigate the proband. The suspicious mutations were further detected in the other 7 family members. Proband was treated with thalidomide (100 mg/day) for 6 months, and the frequency and quantity of bleeding and blood transfusion frequency were assayed to evaluate the curative efficacy. One ACVRL1 mutation (c.1231C>T) was identified in proband(II-1) and the other 4 family members(II-2, III-1, III-2, III-3), which was reported as a pathogenic gene and revealed cosegregation with HHT clinical phenotype. One ENG mutation (c.1096G>C) was previously reported as gene polymorphism, which was identified in some family members(II-1, II-3, III-1, III-2) without cosegregation with clinical phenotype. No gene mutation was found in SMAD4. After thalidomide treatment, the frequency and quantity of bleeding and blood transfusion frequncy in the proband were reduced, hemoglobin concentration and serum iron level were increased. There is phenotypic heterogeneity in hereditary hemorrhagic telangiectasia and the features are age-dependent, the pathogenic gene of this pedigree is ACVRL1 mutation (c.1231C>T;p. R 411 W). Thalidomide is effective for the treatment of hemorrhage in hereditary hemorrhagic telangiectasia.

Identification of multiple ACVRL1 mutations in patients with pulmonary arterial hypertension by targeted exome capture.

Pulmonary artery hypertension (PAH) is characterized as sustained elevation of pressure in the pulmonary vascular system that is attributable to a variety of causes. More than a dozen genes have previously been proposed as being associated with PAH. To examine potential mutations of these genes in patients with PAH, we developed a targeted exome kit containing 22 PAH-associated genes for genetic screens of 80 unrelated patients with PAH. As a result, we identified 16 different mutations in the BMPR2 gene and four different mutations in ACVRL1, the gene for activin receptor-like kinase-1 (ACVRL1). However, no deleterious mutations were found in the remaining 20 genes. In the present study, we provided detailed characterization of the ACVRL1 mutations in four pedigrees, including two novel missense mutations (c.676G>A, p.V226M; c.955G>C, p.G319R) and two recurrent mutations (c.1231C>T, p.R411W; c.1450C>T, p.R484W). Furthermore, we showed that markedly reduced Smad1/5 phosphorylation levels and reduced activities of luciferase reporters in each of the four ACVRL1 mutant-transfected NIH-3T3 cells. Therefore, our findings demonstrated that missense mutations of ACVRL1 identified in the present study significantly affected the bone morphogenetic protein 9 (BMP-9) pathway, implicating PAH pathogenesis. Detailed genotype-phenotype correlation analysis revealed initial symptoms of hereditary haemorrhagic telangiectasia (HHT) in some of the patients, suggesting the importance of sequencing molecular markers for early identification and intervention of individuals at risk for PAH and potential HHT. We developed a customized exome sequencing system to identify mutations in these PAH-associated genes, and found two novel missense mutations and two recurrent mutations in the ACVRL1 gene in four unrelated Chinese families; we also determined hypomorphicalleles using functional studies.

Mucocutaneous Telangiectasia as a Diagnostic Clue of Hereditary Hemorrhagic Telangiectasia: An Activin Receptor-Like Kinase-1 Mutation in a Korean Patient.

Dear Editor: Hereditary hemorrhagic telangiectasia (HHT), also known as Osler-Weber-Rendu syndrome, is an autosomal dominant vascular disorder manifested by variable-sized arteriovenous malformations (AVMs) of the skin, central nervous system, and respiratory, gastrointestinal, and urogenital tracts1,2. A 43-year-old woman visited our dermatology clinic with a 3-year history of increasing numbers of purpuric, punctate, and tiny macules of the lip, oral cavity, and fingers (Fig. 1A~C). The patient had no history of recurrent epistaxis; however, her mother had recurrent epistaxis and brain AVMs. No other abnormalities were detected in laboratory workups, particularly to detect visceral AVMs, including computed tomography scans of the chest and abdomen, brain magnetic resonance imaging, and gastrointestinal endoscopy. Under clinical suspicion, we performed a mutation analysis for HHT in the patient and her mother. The mutation c.940C>T (p.His314Tyr) in activin receptor-like kinase-1 (ACVRL1) was confirmed in both the patient and her mother, confirming the diagnosis of HHT (Fig. 1D). Interestingly, this genetic alteration, previously reported in a Western pedigree3, is a novel point mutation in a Korean family. The cutaneous lesions were well improved by pulsed dye laser (Fig. 2). Fig. 1 (A~C) Typical cutaneous telangiectasias at characteristic sites: lip, oral cavity, and fingers. (D) Presence of the mutation c.940C>T (p.His314Tyr) in activin receptor-like kinase-1. Fig. 2 Skin lesions on the tongue showed excellent response after a single treatment using pulsed dye laser (Vbeam Perfecta; Candela Laser Corporation, Wayland, MA, USA): (A) before treatment and (B) after treatment. Clinically, HHT is diagnosed by the Curacao criteria, which include spontaneous and recurrent epistaxis, mucocutaneous telangiectasias, visceral AVMs, and family history of HHT in a first-degree relative1,3. HHT is definitively diagnosed when three of the criteria are satisfied, whereas a suspected diagnosis is given when two of the criteria are met. In molecular diagnosis, mutations are found in approximately 85% of HHT families; most of these cases involve mutations in the endoglin (HHT1) or ACVRL1 (HHT2) genes4. Mutations of the endoglin gene on chromosome 9, which cause HHT1, were not detected in this family. The prevalence of specific vascular abnormalities varies according to genotype; patients with HHT1 have a higher prevalence of pulmonary and cerebral AVMs, while hepatic AVMs occur more commonly in HHT2 patients4. In our case, although the patient and her mother did not fulfill the criteria for a definitive diagnosis, HHT was strongly suspected because of the patient's typical mucocutaneous lesions. Therefore, targeted genetic testing for the endoglin and ACVRL1 genes should be performed to establish an early diagnosis if there are characteristic cutaneous features and/or a family history of HHT. The cutaneous stigmata, which are characterized by an accumulation of small-caliber and superficial vessels, can cause cosmetic concerns and a risk for bleeding1,2,5. In our case, treatment with pulsed dye laser resulted in dramatic improvement of the skin lesions, similar to results reported by Halachmi et al.2. Multiple treatments and follow-up visits can be necessary because of the lower response of HHT than non-HHT telangiectasia and the possible accumulation of new vascular lesions2. In conclusion, to initially rule out an HHT diagnosis, genetic study is an available option. Because of the superficial nature of vascular lesions, shorter wavelength vascular lasers such as the pulsed dye laser can be considered an effective and safe treatment option for HHT.

Context-specific interactions between Notch and ALK1 cannot explain ALK1-associated arteriovenous malformations.

Notch and activin receptor-like kinase 1 (ALK1) have been implicated in arterial specification, angiogenic tip/stalk cell differentiation, and development of arteriovenous malformations (AVMs), and ALK1 can cooperate with Notch to up-regulate expression of Notch target genes in cultured endothelial cells. These findings suggest that Notch and ALK1 might collaboratively program arterial identity and prevent AVMs. We therefore sought to investigate the interaction between Notch and Alk1 signalling in the developing vertebrate vasculature. We modulated Notch and Alk1 activities in zebrafish embryos and examined effects on Notch target gene expression and vascular morphology. Although Alk1 is not necessary for expression of Notch target genes in arterial endothelium, loss of Notch signalling unmasks a role for Alk1 in supporting hey2 and ephrinb2a expression in the dorsal aorta. In contrast, Notch and Alk1 play opposing roles in hey2 expression in cranial arteries and dll4 expression in all arterial endothelium, with Notch inducing and Alk1 repressing these genes. Although alk1 loss increases expression of dll4, AVMs in alk1 mutants could neither be phenocopied by Notch activation nor rescued by Dll4/Notch inhibition. Control of Notch targets in arterial endothelium is context-dependent, with gene-specific and region-specific requirements for Notch and Alk1. Alk1 is not required for arterial identity, and perturbations in Notch signalling cannot account for alk1 mutant-associated AVMs. These data suggest that AVMs associated with ALK1 mutation are not caused by defective arterial specification or altered Notch signalling.

Clinical and genetic characteristics of Chinese patients with hereditary haemorrhagic telangiectasia–associated pulmonary hypertension

Mutations in activin receptor-like kinase-1 (ACVRL-1) or endoglin (ENG) are mostly identified in patients with hereditary haemorrhagic telangiectasia (HHT) associated with pulmonary hypertension (PH), but have not yet been studied in Chinese patients. In this study, we investigated the clinical and molecular genetic features of Chinese patients with HHT-associated PH and analysed genotype/phenotype correlations in 14 probands and their relatives. Mutation analyses in ACVRL-1, bone morphogenetic protein receptor type 2 (BMPR2) and ENG were performed in 14 Chinese Han patients with HHT-associated PH. The overall mutation rate was 71·4%, including 8 ACVRL-1 mutations and 2 ENG mutations, 6 of which were novel. Six patients were identified with arteriovenous malformations (AVMs), including four patients with pulmonary AVMs and two patients with liver AVMs. Five of the patients with AVMs were identified with mutations. Most patients received targeted therapy for PH. Our findings have revealed the clinical phenotype and molecular genetic features of HHT-associated PH in Chinese Han patients and indicate that mutations of ACVRL-1 and ENG are genetic predisposing factors in Chinese patients. Our data further addressed clinical management and have provided limited experience in treating this group of disorders.

Missense Mutations of the &lt;i&gt;BMPR1B&lt;/i&gt; (&lt;i&gt;ALK6&lt;/i&gt;) Gene in Childhood Idiopathic Pulmonary Arterial Hypertension

Mutations in the bone morphogenetic protein receptor type 2 (BMPR2) gene, the activin receptor-like kinase 1 (ALK1) gene, and SMAD8 gene have been reported in heritable pulmonary arterial hypertension (HPAH) and in idiopathic pulmonary arterial hypertension (IPAH). However, almost 30% of HPAH cases and 60-90% of IPAH cases have no mutations in those genes. This suggests that there remain unidentified genes associated with HPAH and IPAH. This study screened for mutations in endoglin, SMAD1, SMAD2, SMAD3, SMAD4, SMAD5, SMAD6, SMAD7, bone morphogenetic protein receptor type 1A (BMPR1A) and bone morphogenetic protein receptor type 1B (BMPR1B) genes in 43 IPAH patients who had no mutations in BMPR2, ALK1 and SMAD8. Two missense mutations (c.479 G>A S160N, c.1176 C>A F392L) in BMPR1B were each identified in 2 IPAH patients. Immunoblot analysis revealed that the BMPR1B F392L protein promoted SMAD8 phosphorylation. The response to BMP was analyzed using promoter-reporter activities. The transcriptional activation of the BMPR1B F392L protein with SMAD8 increased above that of wild-type BMPR1B with SMAD8, and those of BMPR1B S160N and F392L with SMAD8 and SMAD4 were each increased above those of the wild-type BMPR1B with SMAD8 and SMAD4. We identified 2 novel mutations in BMPR1B in 2 patients with IPAH. Our study suggests that BMPR1B mutations are associated with the pathogenesis of IPAH.

Mutation analysis of "Endoglin" and "Activin receptor-like kinase" genes in German patients with hereditary hemorrhagic telangiectasia and the value of rapid genotyping using an allele-specific PCR-technique

BackgroundHereditary hemorrhagic telangiectasia (HHT), also known as Rendu-Osler-Weber syndrome, is an autosomal dominant disorder which is clinically characterised by recurrent epistaxis, mucocutaneous telangiectasia and visceral arteriovenous malformations. Genetic linkage studies identified two genes primarily related to HHT: endoglin (ENG) on chromosome 9q33-34 and activin receptor-like kinase1 (ACVRL1) on chromosome 12q13. We have screened a total of 41 unselected German patients with the suspected diagnosis of HHT. Mutation analysis for the ENG and ACVRL1 genes in all patients was performed by PCR amplification. Sequences were then compared to the HHT database http://www.hhtmutation.org sequences of the ENG mRNA (accession no. BC014271.2) and the ACVRL1 mRNA (accession no. NM000020.1).ResultsWe identified 15 different mutations in 18 cases by direct sequencing. Among these mutations, one novel ENG mutation could be detected which has not yet been described in the literature before. The genotype-phenotype correlation was consistent with a higher frequency of pulmonary arteriovenous malformations in patients with ENG mutations than in patients with ACVRL1 mutations in our collective.ConclusionFor rapid genotyping of mutations and SNPs (single nucleotide polymorphisms) in ENG and ACVRL1, allele-specific PCR methods with sequence-specific primers (PCR-SSP) were established and their value analysed.

Hereditary hemorrhagic telangiectasia type 1 and 2 mutations in Finland

Conclusion. The finding of several new unique mutations suggests that the genes causing hereditary hemorrhagic telangiectasia (HHT), i.e. endoglin (ENG) and activin receptor-like kinase 1 (ACVRL1), have a relatively high mutation rate. As no single founder mutation was found, analysis of the whole coding sequences of ENG and ACVRL1 genes remains the first choice in genetic testing of new index patients with HHT. Objectives. Our aim was to characterize specific mutations causing HHT in our hospital in Helsinki serving a population of 1 million inhabitants. Patients and methods. HHT patients were searched from our hospital discharge records and their diagnoses were verified by review of patient records and interviews. Eight index patients who fulfilled HHT phenotypic criteria were tested. ENG and ACVRL1 mutations were identified by DNA sequencing of ENG and ACVRL1 coding regions. Results: Of the eight index patients, four had a mutation in the ENG gene, three in the ACVRL1 gene, and one had no mutations. All the mutations were different and all the four ENG mutations and one of the ACVRL1 mutations were new and had not been described previously in other populations. All the affected first-degree relatives had the same mutation as the index case.

Functional analysis of mutations in the kinase domain of the TGF-β receptor ALK1 reveals different mechanisms for induction of hereditary hemorrhagic telangiectasia

Genetic studies in mouse and zebrafish have established the importance of activin receptor-like kinase 1 (ALK1) in formation and remodeling of blood vessels. Single-allele mutations in the ALK1 gene have been linked to the human type 2 hereditary hemorrhagic telangiectasia (HHT2). However, how these ALK1 mutations contribute to this disorder remains unclear. To explore the mechanism underlying effect of the HHT-related ALK1 mutations on receptor activity, we generated 11 such mutants and investigated their signaling activities using reporter assay in mammalian cells and examined their effect on zebrafish embryogenesis. Here we show that some of the HHT2-related mutations generate a dominant-negative effect whereas the others give rise to a null phenotype via loss of protein expression or receptor activity. These data indicate that loss-of-function mutations in a single allele of the ALK1 locus are sufficient to contribute to defects in maintaining endothelial integrity.

Clinical and molecular genetic features of pulmonary hypertension in patients with hereditary hemorrhagic telangiectasia.

Most patients with familial primary pulmonary hypertension have defects in the gene for bone morphogenetic protein receptor II (BMPR2), a member of the transforming growth factor beta (TGF-beta) superfamily of receptors. Because patients with hereditary hemorrhagic telangiectasia may have lung disease that is indistinguishable from primary pulmonary hypertension, we investigated the genetic basis of lung disease in these patients. We evaluated members of five kindreds plus one individual patient with hereditary hemorrhagic telangiectasia and identified 10 cases of pulmonary hypertension. In the two largest families, we used microsatellite markers to test for linkage to genes encoding TGF-beta-receptor proteins, including endoglin and activin-receptor-like kinase 1 (ALK1), and BMPR2. In subjects with hereditary hemorrhagic telangiectasia and pulmonary hypertension, we also scanned ALK1 and BMPR2 for mutations. We identified suggestive linkage of pulmonary hypertension with hereditary hemorrhagic telangiectasia on chromosome 12q13, a region that includes ALK1. We identified amino acid changes in activin-receptor-like kinase 1 that were inherited in subjects who had a disorder with clinical and histologic features indistinguishable from those of primary pulmonary hypertension. Immunohistochemical analysis in four subjects and one control showed pulmonary vascular endothelial expression of activin-receptor-like kinase 1 in normal and diseased pulmonary arteries. Pulmonary hypertension in association with hereditary hemorrhagic telangiectasia can involve mutations in ALK1. These mutations are associated with diverse effects, including the vascular dilatation characteristic of hereditary hemorrhagic telangiectasia and the occlusion of small pulmonary arteries that is typical of primary pulmonary hypertension.