ACVRL1 variation-induced hereditary hemorrhagic telangiectasia presenting with pulmonary arterial hypertension: clinical and genetic analyses of three case studies

Abstract

Background

Hereditary hemorrhagic telangiectasia (HHT) is a hereditary vascular disease, and the ACVRL1 gene plays an important role in this disease, especially when associated with pulmonary arterial hypertension (PAH). In this study, we reported the clinical and genetic characteristics of 3 cases of HHT associated PAH caused by novel ACVRL1 mutations.

Method

A retrospective analysis was conducted on the clinical and genetic data of patients diagnosed with HHT and PAH at Shanxi Cardiovascular Hospital Affiliated Shanxi Medical University from January 2023 to June 2024.

Result

These three patients were initially diagnosed with PAH. They were all females, aged between 35 and 38 years old (with an average age of 36.6 years old). One patient had epistaxis, two patients had arteriovenous fistulas, and one patient had anemia. Genetic testing showed that all three patients had ACVRL1 gene mutations. Among them, two patients had frameshift mutations and one person had missense mutations. The mutation sites were c.687del (p.lle230Serfs * 28), c.145dupG (p.Ala49GlyfsTer120), and c.680 C > A (p.Ala227Asp). According to the American College of Medical Genetics and Genomics (ACMG) guidelines, two loci are classified as likely pathogenic variations, and one locus is a pathogenic variation. After targeted drug therapy to reduce pulmonary artery pressure, the condition of all three patients was well controlled.

Conclusion

The novel discovered ACVRL1 variants in this study may play a potential role in the pathogenesis of HHT and PAH, suggesting that the coordinated application of genetic testing and targeted drug therapy for PAH can play a positive role in clarifying disease diagnosis and controlling patients’ conditions.

Background

Hereditary hemorrhagic telangiectasia (HHT), also known as Osler–Weber–Rendu syndrome, is an autosomal dominant vascular malformation disease. The main clinical symptoms are capillary dilatation of mucous membranes and skin, epistaxis, gastrointestinal bleeding, and arteriovenous malformation (AVM) of internal organs [1]. Pulmonary arteriovenous malformations (PAVM) in some HHT patients can increase right-to-left pulmonary shunt, reduce pulmonary circulation blood volume, cause pulmonary vascular remodeling and elevated pulmonary artery pressure, which is closely related to pulmonary arterial hypertension (PAH). PAH is increasingly recognized as a severe complication of HHT [2]. HHT patients with PAH have a worse prognosis compared to those without PAH [3]. PAH is a serious pulmonary vascular disease that manifests as constriction, obstruction, and remodeling of small pulmonary arteries, leading to increased pulmonary artery resistance, increased right heart loading, and ultimately right heart failure. There are few cases of HHT-associated PAH (HHT-PAH) reported at present, and its treatment-related experience is even more rarely reported. In this study, we used whole exome sequencing (WES) and Sanger sequencing technology to analyze the clinical and genetic characteristic of 3 cases of HHT-PAH caused by novel variants of ACVRL1, which provides more bases for the clinical diagnosis and treatment of HHT-PAH.

Method

Study population

We retrospectively analyzed the clinical data of three patients with HHT who were admitted to Shanxi Cardiovascular Hospital Affiliated Shanxi Medical University between January 2023 and July 2024, presenting with PAH as the initial manifestation. The study was conducted in compliance with the principles of the Declaration of Helsinki and was approved by the Ethics Committee of Shanxi Cardiovascular Hospital Affiliated Shanxi Medical University (No.2024wjw101).Written informed consent was obtained from all patients involved in the study. Diagnostic criteria for PAH: mean pulmonary artery pressure (mPAP) > 20 mmHg (1 mmHg = 0.133 kPa) measured by right heart catheterization (RHC) at rest and at sea level, and pulmonary artery wedge pressure (PAWP) ≤ 15 mmHg, and elevated pulmonary vascular resistance (PVR) > 2 Wood units as measured by RHC. Diagnostic criteria for HHT are in accordance with the International Guidelines for the Diagnosis and Treatment of Hereditary Hemorrhagic Telangiectasia (2nd Edition) [4].

Clinical data collection

We comprehensively gathered the general clinical data of the patients, including demographic characteristics, age of onset, and various clinical symptoms. Additionally, we also collected the outcomes of laboratory examinations, echocardiography, RHC, computed tomography (CT) as well as the treatment regimens.

WES

Collect 2 ml of venous blood from each patient, DNA was isolated using TIANGEN Blood Genomic DNA Extraction Kit (TIANGEN, Beijing, China Cat No. DP318-03). WES sequencing was performed at iGeneTech Biotech (Beijing) Co., LTD. The capture kit was Agilent SureSelect XT Human All Exon V6 kit (Agilent, USA) with Illumina NovaSeq sequencing system for high-throughput sequencing with a read length of 2 × 150 bp. The original FASTQ format data was filtered for low-quality reads and adapter sequences using Trimmomatic, and then the quality was evaluated with FastQC. An appropriate reference genome was selected, and the filtered reads were aligned using BWA. The alignment files were processed with SAMtools. SNVs and InDels were detected using GATK and structural variations were detected using Lumpy. Functional, frequency and disease association annotations were performed using ANNOVAR.

Sanger sequencing

The primers (forward primer sequence: 5’-TTTCAGAGTGTCAGCTGCTCTG-3’, reverse primer sequence: 5’-AGCAGTAGTGGTTGACGAACTC-3’) and primers (forward primer sequence: 5’-AAAAGGCCGCTATGGCGAA-3’, reverse primer sequence: 5’-TCCAGTGAGATTGCACCACT-3’) were designed with reference to the gene sequence of ACVRL1 (NM_000020.3) in the NCBI database, synthesized by Shanghai Sangong Biotech Co. The PCR reaction system was as follows: genomic DNA (20ng/ul) 2ul, 2 ×Mix solution 15ul, primer (10umol/ul) 1ul each, supplemented with double steaming water to 30ul. The reaction conditions were as follows: pre-denaturation at 94℃ for 3 min; There were 35 cycles of denaturation at 94℃ for 30s, annealing at 65℃ for 30s, and extension at 72℃ for 30s. Finally extended for 5 min at 72℃. The PCR products were detected by 1% agarose gel electrophoresis, and the expected PCR products were analyzed by Sanger sequencing.

Result

Basic clinical information of the patient

Case 1, a 37-year-old female patient began to experience shortness of breath after activity 1 year ago, accompanied by intermittent pitting edema in both lower extremities. In the past two months, her activity tolerance gradually decreased, and shortness of breath was obvious when walking for 50 m. Physical examination revealed that the second heart sound in the pulmonary valve auscultation area was accentuated. There was mild edema in both lower extremities. The N-terminal pro-B-type natriuretic peptide (NT-proBNP) was 2170 ng/L, and the World Health Organization Functional Classification (WHO-FC) was graded as class III. Ultrasonic cardiogram (UCG) showed that the right atrium and right ventricle were enlarged, the left ventricle was compressed into a D-shaped change, and pulmonary artery systolic pressure (PASP) was 75 mmHg. The results of RHC showed that the mPAP was 56 mmHg, the PAWP was 8 mmHg, and the PVR was 19.62 Wood units. The patient was found to carry a mutation in the ACVRL1 gene through genetic testing. As the patient was adopted, the genetic mutation status of their biological parents cannot be determined. However, the patient’s son and daughter also carry the same ACVRL1 gene mutation and have experienced nosebleeds, so she was diagnosed with HHT. The patient was given a combination of macitentan, selexipag, and tadalafil as targeted drugs to reduce pulmonary artery pressure, along with comprehensive treatments to improve cardiac function. The patient was discharged in good condition without any special discomfort (Table 1).

Table 1 Comparison of the 3 patients before and after treatment

Case 2, a 38-year-old female patient developed shortness of breath in the late pregnancy and was admitted to the hospital. UCG showed that the PASP reached 100 mmHg. Treprostinil was administered to reduce the pulmonary artery pressure. After cesarean section, the patient was transferred to the department of cardiology for further treatment. Physical examination showed that systolic murmur of grade 2/6 could be heard in the auscultation area of pulmonary valve. The electrocardiogram (ECG) showed sinus rhythm, right axis deviation, and ST-T changes. The UCG showed thickening of the right ventricular wall and increased pulmonary artery pressure. The results of RHC showed that the mPAP was 55 mmHg, PAWP was 12 mmHg, and the PVR was 11.8 Wood units. Echocardiography of the right heart and CT showed a right-to-left shunt with a PAVM (Fig. 1). The 6 min walk distance (6WMD) was 275 m. According to the symptoms of the case, genetic testing was conducted, and the results showed that the patient carried a mutation in the ACVRL1 gene. Based on the above results, the patient was diagnosed with HHT, PAH and mild anemia. Due to the unsatisfactory effect of using treprostinil alone, macitentan and tadalafil were added along with comprehensive treatments to improve cardiac function. At the time of discharge, she did not report any special discomfort.

Fig. 1

CT imaging for PAVM of case 2

Case 3, a 35-year-old female patient, complained of bilateral lower limb edema for six months, shortness of breath with nausea for one month. Upon admission, a physical examination revealed cyanosis of the lips. The second heart sound of the pulmonary artery was accentuated. ECG showed sinus rhythm, prolonged QT interval and right deviation of electrical axis. The UCG indicated: enlargement of the right atrium and right ventricle, a relatively small left ventricle, severe tricuspid insufficiency, widened pulmonary artery, and PASP was 96 mmHg. RHC showed an mPAP of 57 mmHg, a PAWP of 13 mmHg, and PVR of 7.88 Wood units. Nasal endoscopy showed nosebleeds (bilateral). Enhanced abdominal CT shows hepatic arteriovenous malformation (HAVM) (Fig. 2). The patient had been diagnosed with anemia for half a year and had previously visited the hematology department. Based on the manifestations of the case, a diagnosis of HHT was made. Genetic testing was performed on the case, and the result showed that the patient carried an ACVRL1 gene mutation. The patient was treated with ambrisentan and selexipag to reduce pulmonary artery pressure and improve cardiac function, she did not report any special discomfort after discharge.

Fig. 2

Enhanced abdominal CT shows HAVM of case 3

Genetic testing results

In case 1, the ACVRL1 gene c.687del (p.Ile230Serfs*28) is a frameshift mutation. The population frequency of this variant locus is not documented according to the ExAC database, Thousands database, and gnomAD database analysis (PM2_Supporting). The variant is located on exon 6 and may lead to nonsense-mediated mRNA degradation (NMD) (PVS1). Combined with the clinical phenotype of the subject it is consistent with the features of HHT (PAH) (PP4). No data reported in the Clinvar database. The available evidence supports a judgment of likely pathogenic according to the ACMG gene variant interpretation guidelines (PVS1 + PM2_Supporting + PP4) (Table 2).

Table 2 ACVRL1Genetic variation information of 3 patients

In case 2, the ACVRL1 gene c.145dupG (p.Ala49GlyfsTer120) is a frameshift mutation. The population frequency of this variant locus is not documented according to the ExAC database, Thousands database, and gnomAD database analysis (PM2_Supporting). The variant is located on exon 3 and may lead to nonsense-mediated mRNA degradation (NMD) (PVS1). Combined with the clinical phenotype of the subject, the situation is consistent with the features of HHT (anemia, PAVM, PAH) (PP4). There are 8 documents on the pathogenicity of this locus in the clinvar database (PS4). The available evidence supports a judgment of pathogenic (PVS1 + PS4 + PM2_Supporting + PP4) according to the ACMG guidelines.

In case 3, the ACVRL1 gene c.680 C > A (p.Ala227Asp) is a missense mutation. According to the analysis of ExAC database, Thousands database, and gnomAD database, the population frequency of this variant locus is not documented (PM2_Supporting). Bioinformatics software, such as MutPred, REVEL, and BayesDel, predicted that this variant is deleterious to the gene or gene product (PP3_strong). Combined with the clinical phenotype of the subject, it was consistent with the features of HHT (epistaxis, hepatic AVM, PAH) (PP4). The available evidence supports a judgment of likely pathogenic according to the ACMG Guidelines (PM2_Supporting + PP3_strong + PP4).

Discussion

HHT is an autosomal dominant vascular disease with a prevalence of 1/10,000 [5], and 85–95% of cases are caused by pathogenic variants of ACVRL1 and ENG [6]. ACVRL1 encodes a vascular endothelial cell growth related protein (activator receptor like kinase 1, ALK1) located in the autosomal 12q11-q14 region. Previous studies have shown that pure heterozygous ACVRL1 knockout mice exhibit embryonic lethality with severe vascular malformations. Heterozygous ACVRL mice develop spontaneous pulmonary hypertension in adulthood, with an increase in right ventricular systolic pressure, right ventricular hypertrophy, and vascular remodeling [7]. There are various types of mutations in the ACVRL1 gene, including missense mutations, frameshift mutations due to insertion or deletion, nonsense mutations, splice site mutations, etc., of which missense mutations account for the largest percentage and most of the mutations have been reported only once. In this study, all three patients presented with novel gene sites. Among them, the gene sites associated with case 1 and case 3 have not been reported worldwide, and the gene site variation of case 2 is the first discovery in the Chinese population. Of the three cases reported in this study, case 1 carried c.687del (p. lle230Serfs*28), which is a frameshift mutation in the coding region of the ACVRL1 gene caused by non triple base deletions. Case 2 carried a heterozygous mutation (p.Ala49GlyfsTer120) in the ACVRL1 gene c.145dupG, which causes the non-polar alanine at position 49 to become non-polar glycine, followed by protein frameshift expression and the appearance of a stop codon at position 119, potentially leading to nonsense mediated mRNA degradation. The c.680 C > A (p.Ala227Asp) variant carried by case 3 is a missense variant in the coding region of the ACVRL1 gene, which may result in nonsense-mediated mRNA degradation, downstream of a frameshift or a nonsense mutation.

For many years, ACVRL1 mutations have been known to cause HHT and PAH. For a small number of HHT and PAH patients, PAH is diagnosed before the clinical symptoms of HHT appear. It has been reported in the literature that some patients with ACVRL1 mutations develop symptoms of isolated PAH [8, 9]. In addition, it has also been demonstrated that ACVRL1 mutation carriers may develop severe PAH without clinical evidence of HHT. Consistent with our research findings, all three patients in this study were diagnosed with PAH as the primary diagnosis. Among them, two patients had obvious HHT-related clinical symptoms, while the other had no obvious HHT-related clinical symptoms. The International Guidelines for the Diagnosis and Treatment of Hereditary Hemorrhagic Telangiectasia (2nd Edition) state [4] that a diagnosis of HHT can be confirmed if a pathogenic mutation is detected in a known HHT-related gene (e.g., ENG, ACVRL1, SMAD4, etc.), even if the clinical symptoms do not fully match [4]. Fujiwara and colleagues identified five ACVRL1 mutations in 21 pediatric patients with PAH without any signs of HHT [10]. In addition, Harrison and colleagues report on a case of a PAH patient carrying the ACVRL1 mutation, with severe PAH diagnosed at age 51, an age at which HHT is considered almost completely explicit. The patient died at the age of 54, and no pathological features of HHT were found in the autopsy [11]. Some of the characteristic signs of HHT, especially epistaxis, are non-specific and usually not spontaneously reported by patients, so more detailed family history and clinical characteristics should be examined in such patients to prevent misdiagnosis and missed diagnosis. In addition, although PAH is a rare complication of HHT, previous studies have shown that the prognosis of such patients is poor [12, 13]. Therefore, clinical symptom screening for PAH should be performed in these patients in order to detect PAH earlier and treat it earlier. In addition, as the HHT - related clinical symptoms are not obvious or even absent in a small number of HHT patients with PAH who carry ACVRL1 mutations, a detailed family history and a careful examination of the clinical symptoms of their parents or children, especially genetic testing, may help identify these patients.

According to reports, HHT patients with PAH have lower survival rates compared to those without PAH [14]. In addition, studies have reported that carriers of ACVRL1 mutations experience faster clinical deterioration of PAH and ultimately die from PAH related causes. Compared with other types of PAH patients, carriers of ACVRL1 mutations have a faster progression of the disease [8]. Therefore, the treatment strategy for PAH in this type of patient is particularly important. Although several pulmonary vasodilators such as phosphodiesterase-5 inhibitors, endothelin receptor antagonists, and prostaglandins have been approved for the treatment of PAH, only a few case reports have described the response of pulmonary vasodilators to HHT and PAH patients carrying ACVRL1 gene mutations. Tomohisa Nakamura reported a case of a 37 year old patient treated with tadalafil. At the beginning of the treatment, there were no bleeding side effects, but 12 months later, hemodynamic deterioration and right heart failure occurred, and the patient ultimately died from right heart failure [15]. Ryo Miyake used sildenafil (60 mg/day) as a treatment for PAH in a 47 year old female patient. The patient’s hemodynamics (mPAP and PVR) and exercise ability (6WMD) were significantly improved, and the plasma BNP concentration decreased to 73 pg/mL. The patient’s epistaxis did not worsen throughout the entire treatment period [16]. In our study, all three patients achieved good therapeutic results with the application of targeted drugs for PAH, and no other related side effects occurred during treatment. A recent study demonstrated that targeted drug therapy for HHT-PAH effectively improved hemodynamics in patients and was not associated with an increased risk of side effects [17], which is consistent with our study. However, previous studies have reported that endothelin receptor antagonists (bosentan and macitentan) and soluble guanylate cyclase stimulators increase the risk of anemia [18, 19]. Phosphodiesterase-5 inhibitors are associated with increased rates of nosebleeds and prostaglandins have antiplatelet properties [20]. Therefore, patients with HHT and PAH should have a treatment plan tailored to their clinical needs and individual side effect profile.

Conclusion

In conclusion, our study reported the clinical and genetic characteristics of three patients with HHT initially diagnosed with PAH who carried new mutations in the ACVRL1 gene, providing some reference and guidance for the clinical treatment of this disease. However, this study has some limitations. Although the mutation sites of ACVRL1 gene in these three patients are different, the targeted drug therapy for PAH had achieved good therapeutic effect, and the specific mechanism needs to be further verified by animal or cell experiments.