Journal of the Neurological Sciences 352 (2015) 29–33
Contents lists available at ScienceDirect
Journal of the Neurological Sciences journal homepage: www.elsevier.com/locate/jns
Exome sequencing identifies a novel intronic mutation in ENG that causes recurrence of pulmonary arteriovenous malformations Naoki Saji a,⁎,1,2, Toshitaka Kawarai b,⁎⁎,1,2, Ryosuke Miyamoto b,1, Takahiro Sato a, Hiroyuki Morino c, Antonio Orlacchio d,e, Ryosuke Oki b, Kazumi Kimura a, Ryuji Kaji b a
Department of Stroke Medicine, Kawasaki Medical School, 577 Matsushima, Kurashiki, Okayama 701-0192, Japan Department of Clinical Neuroscience, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima 770-8503, Japan Department of Epidemiology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima, Japan. d Laboratorio di Neurogenetica, CERC-IRCCS Santa Lucia, Rome, Italy e Dipartimento di Medicina dei Sistemi, Università di Roma “Tor Vergata”, Rome, Italy b c
a r t i c l e
i n f o
Article history: Received 2 November 2014 Received in revised form 16 January 2015 Accepted 3 February 2015 Available online 2 April 2015 Keywords: Hereditary hemorrhagic telangiectasia Endoglin Arteriovenous malformation Whole exome sequencing Intronic mutation Aberrant transcript
a b s t r a c t Hereditary hemorrhagic telangiectasia (HHT) occasionally can be discovered in patients with cerebrovascular disease. Pulmonary arteriovenous malformation (PAVM) is one of the complications in HHT and occasionally is causative for life-threatening embolic stroke. Several genetic defects have been reported in patients with HHT. The broad spectrum of phenotype and intrafamilial phenotype variations, including age-at-onset of vascular events, often make an early diagnosis difficult. We present here a Japanese family with a novel intronic heterozygous mutation of ENG, which was identified using whole exome sequencing (WES). The intronic mutation, IVS3 + 4delAGTG, results in in-frame deletion of exon 3 and would produce a shorter ENG protein lacking the extracellular forty-seven amino acid sequences, which is located within the orphan domain. Our findings highlight the importance of the domain for the downstream signaling pathway of transforming growth factor-beta and bone morphogenesis protein superfamily receptors. Considering the phenotype variations and the available treatment for vascular complications, an early diagnosis using genetic testing, including WES, should be considered for individuals at risk of HHT. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Hereditary hemorrhagic telangiectasia (HHT), also known as Osler– Weber–Rendu disease, is an autosomal dominantly inherited vascular disease. Salient clinical features include the presence of mucocutaneous
Abbreviations: AD, autosomal dominant; AVM, arteriovenous malformation; CT, computed tomography; ENG, endoglin; ESS, epistaxis severity score; HHT, hereditary hemorrhagic telangiectasia; MRI, magnetic resonance imaging; NGS, next-generation sequencing; PAVM, pulmonary arteriovenous malformation; WES, Whole exome sequencing. ⁎ Correspondence to: N. Saji, Department of Stroke Medicine, Kawasaki Medical School, 577 Matsushima, Kurashiki City, Okayama, 701-0192, Japan. Tel.: +81 86 462 1111; fax; +81 86 462 1199. ⁎⁎ Correspondence to: T. Kawarai, Department of Clinical Neuroscience, Institute of Health Biosciences, Graduate School of Medicine, University of Tokushima, 3-18-15, Kuramotocho, Tokushima City, 770-0042, Japan. Tel.: +81 88 633 7207; fax: +81 88 633 7208. E-mail addresses: [email protected] (N. Saji), [email protected] (T. Kawarai), [email protected] (R. Miyamoto), [email protected] (T. Sato), [email protected] (H. Morino), [email protected] (A. Orlacchio), [email protected] (R. Oki), [email protected] (K. Kimura), [email protected] (R. Kaji). 1 The first three authors contributed equally to this work. 2 The two corresponding authors contributed equally to this work.
http://dx.doi.org/10.1016/j.jns.2015.02.007 0022-510X/© 2015 Elsevier B.V. All rights reserved.
telangiectasia and arteriovenous malformations (AVM) in visceral organs, primarily the lungs, brain and liver. Early treatment of pulmonary arteriovenous malformations (PAVM), through which paradoxical brain embolism mediates, would ameliorate prognosis for a patient with HHT. Combination of accurate diagnosis by genetic testing, early treatment of PAVM, and careful clinical follow-up is crucial for HHT patients. However, even with early treatment, recurrence of pulmonary arteriovenous malformations has been reported after successful surgical removal or embolization treatment [1]. To date, four genes have been identified, ENG (HHT1), ACVRL1 (HHT2), SMAD4 and GDF2 (HHT5). It has been demonstrated that HHT is caused by either mutations in these genes or other genes that modulate transforming growth factor-beta (TGF-β) signaling (e.g., bone morphogenetic proteins). Two loci have been reported by linkage studies in familial cases, HHT3 and HHT4 on the region of 5q31.3-q32 and 7p14, respectively, in which the causative genes remain unknown [2]. Elucidation of genetic mechanisms would further contribute to better understanding the molecular pathogenicity of vasculopathy and developing new therapeutic strategies in HHT. We present here a family with a novel intronic mutation in ENG, and demonstrate further evidence of allelic heterogeneity in ENG-HHT1.
30
N. Saji et al. / Journal of the Neurological Sciences 352 (2015) 29–33
2. Material and methods
2.3. Genetic analyses
2.1. Ethics statement
After informed consent was given, genomic DNA was extracted from peripheral blood leukocytes from the family members. Whole exome sequencing (WES) was performed in the proband III-2 (Fig. 1-A), as previously described [4]. Variants were filtered against public databases, including Single Nucleotide Polymorphism Database (dbSNP 135), the 1000 genomes, and the NHLBI Exome Sequencing project (ESP5400). In addition, variants were also filtered against our in-house variant database, including 106 control exome experiments. Variations identified in the candidate genes were further subjected to filtering steps in the specific databases (Supplementary data). Genomic DNA was amplified by polymerase chain reaction (PCR) with sets of specific primers in order to investigate coding sequences and intron/exon boundaries of ENG, as previously described [5,6]. PCRdirect sequencing was performed and each sequence read was aligned to the genome reference sequences using the program Sequencher version 5.2 (Gene Codes, Ann Arbor, MI, USA). The sequence analysis was further expanded, including 400 Japanese control chromosomes. Secondary structure and minimal free energy of wild and mutant RNA were predicted by the RNAFOLD program, as described elsewhere [7,8]. Total RNA was prepared from lymphocytes using RNAiso Plus reagent (TaKaRa Bio, Inc., Kyoto, Japan) according to the manufacturer's instructions. RNA samples were treated with RNase-free DNase I (Ambion) and RNA concentrations were determined using a NanoDrop
The study was approved by the ethical review boards of the Kawasaki Medical Hospital and Tokushima University Hospital. All of the participants in this study gave written informed consent.
2.2. Subjects The pedigree chart is illustrated in Fig. 1. There were three family members with a history of spontaneous and repeated epistaxis including the proband (Fig. 1: II-1, III-1 and III-2). Other members in this family presented as normal. All the living family members were examined by three experienced neurologists (Drs. N.S., T.S. and K.K.) and underwent neuroimaging and genetic examinations. The Curaçao criteria for HHT was applied in the diagnosis [3]. Epistaxis severity score for hereditary hemorrhagic telangiectasia (ESS-HHT) was adopted, in which scores are assigned to each severity level: severe = 7 to 10, moderate = 4 to 7, mild = 1 to 4 and none = 0 to 1 [3]. The score is calculated automatically at the website, http://www2. drexelmed.edu/hht-ess/. Detailed history and neurological features in the deceased patient (Fig. 1; III-1) were assumed from medical records or interviews with living family members.
A
I
1
1*
II
D
3
B
?
4
5
C
6
? 1
III IV
2
2
?
2*
P 1
2
3
4
5
E
F
Fig. 1. A) Pedigree chart. Solid symbols, affected individuals; open symbols, unaffected individuals or family members showing no abnormal finding in chest roentgenogram; question mark, phenotype unknown; circles, females; squares, males; slashes, deceased; arrow, proband. *Indicates members carrying the mutation, g.IVS3 + 4delAGTG. B) Multiple red spots on the proband's tongue. The subcutaneous lesions reflect characteristic telangiectasia. C) Spontaneous recurrent nose bleeds from telangiectasia of the nasal mucosa in the proband. Examination of the nasal cavity using a flexible fiberscope demonstrates active bleeding from the mucosal telangiectasia. D) Embolic stroke occurred in the proband. Diffusion-weighted imaging shows a high intensity at the left temporal lobe (upper panel). Magnetic resonance angiography reveals obstruction of superior cortical branches of the middle cerebral artery (white arrow in lower panel). E) Pulmonary arteriovenous malformation. Unenhanced thoracic computed tomography demonstrates a thick and rope-like density within the left lower lung field in the proband (red arrow). F) Abnormal connection between the venous and arterial system is revealed by axial and volume-rendered computed tomography (red arrow head).
N. Saji et al. / Journal of the Neurological Sciences 352 (2015) 29–33
31
Table 1 Clinical data of the four affected individuals. Family member ID
Age at examination (years)
Age at onset of repeated epistaxis
ESS-HHT at examination (or deceased age)
Telangiectases of hands, face, and oral cavity
Pulmonary AVMs
Hepatic AVMs
Central nervous system AVMs
II-1 III-1 III-2 IV-5
90 65 (age at death) 61 31
Late twenties Early twenties Late twenties Asymptomatic
1.41 3.22 1.92 0.91
None Not described Hands, face and oral cavity None
Positive None Positive (recurrence) None
None Not examined None None
None Not examined None None
(ND-1000) spectrophotometer. 10 mg RNA were reverse transcribed using random primers and the PrimeScript II 1st Strand cDNA Synthesis Kit (TaKaRa Bio, Inc., Kyoto, Japan). Oligonucleotide primers ENG-82F (5′-GTCCATTGTGACCTTCAGCCTGTG-3′) and ENG-616R (5′-ACGCCTTC CAAGTGGCAGCC-3′) that span exons 2–5 of endoglin cDNA were used for amplification of cDNA. PCR products were fractionated by agarose gel electrophoresis, excised and purified by RBC HiField Gel/PCR DNA Fragments Extraction Kit (SciTrobe, Tokyo, Japan), and subjected to cycle sequencing, as previously described [9]. The three dimensional protein structures of ENG were predicted by SWISS-MODEL (http://swissmodel.expasy.org) [10]. 3. Results 3.1. Clinical features The proband, a 61-year-old man, suddenly developed behavioral disorders and referred to the emergency unit in Kawasaki Medical Hospital. Physical examination demonstrated multiple telangiectasias on his lips, tongue, fingers, and earlobes (Fig. 1-B). Epistaxis from telangiectasias of the nasal mucosa was confirmed by flexible fiberscopic
A
Exon 3
examination (Fig. 1-C). He had a history of repeated nasal bleeding since his late twenties. ESS-HTT of 1.92, categorized as mild, was given at admission. Neurological examination revealed left inferior quadrantanopsia, left hemiparesis, and unilateral spatial neglect. Laboratory findings showed iron deficiency anemia. Diffusion weighted magnetic resonance imaging scans of the brain showed a high signal intensity area in the right occipito-temporal lobe. Magnetic resonance angiography showed a blunt-ended occlusion with insufficient collaterals at the right middle cerebral artery, indicating acute embolic stroke (Fig. 1-D). A transesophageal echocardiography revealed no evidence of patent foramen ovale and intracardiac thrombus. A transcranial doppler ultrasound revealed a finding of continuous right-to-left shunt. The patient had a history of recurrent nasal hemorrhage and PAVM, which was incidentally found, and resection of the lower lobe was performed at the age of 28. Recurrence of PAVM at the left lower lobe was confirmed by thoracic computed tomography (Fig. 1-E and F). Coil embolization for pulmonary arteriovenous malformation was performed. Clinical examination and review of medical record revealed that the other two members, II-1 and III-1, were diagnosed with HHT. The asymptomatic member, IV-5, was genetically diagnosed with HHT. The clinical summary is described in Table 1.
Intron 3
control
proband T T G G C C T AC GT G A G T GT G T T C C C TCC A A C C T T G G C C T AC GT G T G T T C C C T C C A ACC B C G
C UA G U U C A C
C G +1
U +2 G +3 A +4 G +5 U +6 GUGUUCCCUCCA
+7 +8 +9
Wild-type
C U A G C U G +1 U +2 U C G +3 A U +8 C GUUCCCUCCAACCC
C G
+9
Mutant-type
Fig. 2. A) Fluorescent chromatographs of the mutation found in the ENG gene. The sequence around the heterozygous substitution is shown, with the redline rectangle pointing to the g.IVS3 + 4delAGTG mutation. B) Effect of the mutation in the ENG gene on the RNA secondary structure. The left and right figures display the wild-type and mutant RNA sequences respectively. The mutation site is emphasized in red. The natural splice site in the wild-type transcript is marked with an arrow. Positions are numbered according to the first nucleotide of intron 3 starting with +1.
32
N. Saji et al. / Journal of the Neurological Sciences 352 (2015) 29–33
protein lacking the forty-seven amino acid sequences (Supplementary data).
3.2. Genetic analyses WES generated a good coverage depth, which was sufficiently covered to pass our thresholds for calling SNPs and indels. After the exome sequencing and variant calling, 64,746 variants were identified. Data quality and quantity are described in the Supplementary data. Combination of filtering sequence variants and bioinformatic analyses demonstrated no compelling variant mapped on the candidate regions for HHT3 and HHT4. Manual review of intronic variants revealed a 5′ splice site deletion, g.IVS3 + 4delAGTG, at intron 3 in ENG. Validation of the intronic variant and segregation with the phenotype in the family was confirmed by Sanger sequencing (Fig. 2-A). The deletion variant was absent in 400 control chromosomes. Analysis by the RNAFOLD program demonstrated an altered predicted secondary structure of the mutant RNA (Fig. 2-B and Supplementary data). The secondary structure of the wild-type RNA sequence contains a stem–loop structure; the four residues, AGTG at the position + 4 to + 7, are located within the stem (δ G − 3.70 kcal/mol). The mutation, g.IVS3 + 4delAGTG, created a smaller stem–loop structure (δ G −2.60 kcal/mol). Reverse transcriptase PCR (RT-PCR) assay demonstrated aberrant transcripts in the proband (Fig. 3-A). Sanger sequencing of the short PCR fragments showed ENG transcripts lacking exon 3 (Fig. 3-B), which lead to in-frame deletion. The normal ENG protein is 658 amino acid residues in length, however, mutant protein having a short length of 611 amino acids would be produced in this case. No significant structural alteration was predicted by in silico analysis. Formation of twisted U-shaped construction seemed unchanged in the mutant ENG
4. Discussion To date, more than 27 ENG mutations have been reported in hereditary hemorrhagic telangiectasia (HHT), including nonsense, missense, frame shift, deletion and splicing mutations [3,5,6]. Haploinsufficiency is presumably a cardinal pathomechanism in HHT caused by mutant ENG. Missense mutant ENG proteins dimerize with themselves, as well as with wild-type ENG proteins, and localize in the rough endoplasmic reticulum (rER) or in the plasma membrane of the cells. When the mutant ENG proteins are retained in the rER, the amount of endogenous wild-type endoglin on the plasma membrane is reduced through interception in the rER [11]. A total of four 5′ splice site mutations in intron 3 have been published, c.360 + 1GNA (IVS3 + 1GNA), c.360 + 1GNC (IVS3 + 1GNC), c.360 + 4ANG (IVS3 + 4ANG) and c.360 + 5GNC (IVS3 + 5GNC) (Supplementary data). In-frame deletion of exon 3 was demonstrated in the two mutations, c.360 + 1GNA (IVS3 + 1GNA) and c.360 + 1GNC (IVS3 + 1GNC) (Supplementary data). A total of six 5′ splice site mutations in intron 3 have been listed in the HHT Mutation Database (http://www.hhtmutation.org/index.php) including the c.360 + 4delAGTG (IVS3 + 4delAGTG), however, the biological effect by the mutation is not assessed. The prediction of secondary pre-mRNA structure would indicate an alteration of splice site selection. When an alternative structure is too different from the lowest free-energy structure (ground state), the sequence is not recognized by the splicing machinery
(2) Impairment of heterodimer complex formaon?
A
C
554 bp 413 bp
Extracellular part
Plasma membrane (1) Insufficient transport to the cell surface
TGF-β receptor type I and II
B Exon 2
Exon 4
Decreased signaling from TGF-β/BMP superfamily in nucleus 74-120
1
Altered gene expression in angiogenesis
Phe
Pro
71
72
Thr 73
Asn
Ser
Ser
121
122
123
ENG protein synthesis 658
Fig. 3. A) Results of the RT-PCR assay. PCR product obtained from the proband's cDNA shows two bands — 554 bp and 413 bp — revealing that aberrant RNA splicing occurred by the intronic deletion mutation. B) Sequence analysis of the shorter PCR fragment shows the ENG cDNA sequence with in-flame deletion of exon 3. Amino acid numbering is based on the longest protein isoform (NCBI accession no. NM_001114753.2). C) Bar diagram of ENG with the domains indicated and highlighted in different colors. SP, signal peptide in red, TM, transmembrane region in black, ZP, zona pellucida in dark blue and OD, orphan domain in orange. The position of an in-frame deleted part is indicated with a two-way arrow. Numbers indicate the amino acid of endoglin (starting at the N terminus). Two possible biological effects by the deletion mutation are commented in the figure.
N. Saji et al. / Journal of the Neurological Sciences 352 (2015) 29–33
[12]. As a consequence, the corresponding exon is prone to skipping. The effect by the reported mutations at 5′ splice site mutations in intron 3 were evaluated, however, no distinctive difference was demonstrated under the model of minimum free energy. The RNA structural stability of the sequence containing the mutation of IVS3 + 1GNA is similar to that in the wild type sequence, but exon 3 skipping was biologically confirmed (Supplementary data). Exon 3 splicing may be regulated by a more complicated structural alteration or splicing regulatory elements. Deletion of the four nucleotides at the 5′ splice site in intron 3 influences the RNA structure with reduced free energy, leading to the inframe skipping of an exon 3. The mutant protein, lacking amino acids encoded by the exon 3, was detected as an intracellular homodimer, while the normal protein was expressed at the cell surface. The amount of biologically active ENG protein was decreased to 50% of control levels in umbilical vein endothelial cells from a patient carrying the heterozygous mutation [13]. The in-frame deletion of exon 3 would not affect the protein synthesis and would result in the production of shorter length protein, lacking the forty-seven amino acid sequences. The region deleted by the mutation contains two asparagine residues, 88Asn and 102Asn, which are subject to glycosylation after protein synthesis (post-translational). The absence of the two glycosylated asparagine residues or the forty-seven amino acid sequences per se might lead to insufficient transporting of ENG protein to the cell surface or impairment of heterodimer complex formation with TGF-β receptors (Fig. 3-C). The proband (III-2) undertook a surgical removal of PAVM more than 30 years before the current embolic stroke. Several explanations for the recurrence of PAVM have been postulated, including persistent small PAVMs which would grow due to the altered blood flow after the surgery [14,15]. In addition, a continuous defect in the signaling pathway of the TGF-β/BMP superfamily might lead to angiodysgenesis acquired after the lobectomy. Intrafamilial phenotype variation was demonstrated in the family, including age-at-onset, severity of epistaxis and telangiectasias on skin and mucosa. It has been reported that the age-at-onset of recurrent epistaxis is adolescence or later. The age-at-onset may be influenced by genotype. Later onset of symptoms in HHT2 is reported in comparison with HHT1. In the study of genetically confirmed HHT cases in the Japanese population, the mean age of HHT2 is 48.6 years old while that of HHT1 is 35.1 years old [5]. Genetic testing is indispensable for family members with unlikely diagnosis for HHT, thus positive family history only is reported, in order to exclude gene mutation in currently-known HHT genes. Unlike other genetic neurological disorders, prophylaxis for vascular complications could be provided for HHT patients, including surgical operation, endovascular treatment and antithrombotic/anticoagulant therapy [16,17]. The role of genetic analysis in the diagnosis of HHT is of importance. In conclusion, our findings demonstrate further allelic heterogeneity in ENG-HHT1 and indicate that the extracellular domain encoded by exon 3 might be crucial for the signaling pathway of the TGF-beta/ BMP superfamily. It is also demonstrated that the utility of WES facilitates the diagnosis of genetically highly heterogeneous diseases. The natural history of HHT can be variable, however, the incidence of embolic stroke obviously confers poor prognosis and occasionally causes unexpected sudden death. Prognosis of HTT would become more favorable by the combination of early diagnosis, using WES, treatment of PAVM and therapeutic anticoagulation. Author contribution Naoki Saji and Takahiro Sato: patient review and medical writing for content. Toshitaka Kawarai: genetic testing, sequencing of mutation, and manuscript preparation. Ryosuke Miyamoto, Hiroyuki Morino and Ryosuke Oki: genetic testing and bioinformatic analysis. Antonio Orlacchio, Kazumi Kimura and Ryuji Kaji: study supervision and coordination.
33
Conflict of interest The authors report no conflicts of interest. Acknowledgements This work was supported by Grants-in-Aid from the Research Committee of CNS Degenerative Diseases of the Ministry of Health, Labour and Welfare of Japan (R.K.), the Japan Society for the Promotion of Science (JSPS KAKENHI Grant no. 26870765 to N.S. and no. 26461294 to T.K.), the Brain Science Foundation, Japan (Grant to T.K.), and Grantin-Aid for Research on rare and intractable diseases, the Research Committee on Establishment of Novel Treatments for Amyotrophic Lateral Sclerosis from the Ministry of Health, Labour and Welfare of Japan (Grant to T.K. and R.K.), and the Ministry of Health, Italy (Grant no. GR09.109 to A.O.). The authors thank the patient and family members involved in this study. We thank Michela Renna (MA) for her language advice and assistance, as well as the Support Center for Advanced Medical Sciences, Tokushima University School of Medicine, for the use of their facilities to prepare the manuscript. The authors also thank Ms. Akemi Takahashi, Ms. Mariko Hasegawa and Ms. Akie Tanabe for their technical support. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.jns.2015.02.007. References [1] Chao HS, Chern MS, Chen YC, Chang SC. Recurrence of pulmonary arteriovenous malformations in a female with hereditary hemorrhagic telangiectasia. Am J Med Sci 2004;327:294–8. [2] Duffau P, Lazarro E, Viallard JF. Hereditary hemorrhagic telangiectasia. Rev Med Interne 2014;35:21–7. [3] Shovlin CL, Guttmacher AE, Buscarini E, Faughnan ME, Hyland RH, Westermann CJ, et al. Diagnostic criteria for hereditary hemorrhagic telangiectasia (Rendu–Osler– Weber syndrome). Am J Med Genet 2000;91:66–7. [4] Miyamoto R, Morino H, Yoshizawa A, Miyazaki Y, Maruyama H, Murakami N, et al. Exome sequencing reveals a novel MRE11 mutation in a patient with progressive myoclonic ataxia. J Neurol Sci 2014;337:219–23. [5] Komiyama M, Ishiguro T, Yamada O, Morisaki H, Morisaki T. Hereditary hemorrhagic telangiectasia in Japanese patients. J Hum Genet 2014;59:37–41. [6] Shovlin CL, Hughes JM, Scott J, Seidman CE, Seidman JG. Characterization of endoglin and identification of novel mutations in hereditary hemorrhagic telangiectasia. Am J Hum Genet 1997;61:68–79. [7] Zuker M, Stiegler P. Optimal computer folding of large RNA sequences using thermodynamics and auxiliary information. Nucleic Acids Res 1981;9:133–48. [8] Kleiner-Fisman G, Rogaeva E, Halliday W, Houle S, Kawarai T, Sato C, et al. Benign hereditary chorea: clinical, genetic, and pathological findings. Ann Neurol 2003; 54:244–7. [9] Kawarai T, Pasco PM, Teleg RA, Kamada M, Sakai W, Shimozono K, et al. Application of long-range polymerase chain reaction in the diagnosis of X-linked dystoniaparkinsonism. Neurogenetics 2013;14:167–9. [10] Biasini M, Bienert S, Waterhouse A, Arnold K, Studer G, Schmidt T, et al. SWISSMODEL: modelling protein tertiary and quaternary structure using evolutionary information. Nucleic Acids Res 2014;42:W252–8. [11] Forg T, Hafner M, Lux A. Investigation of endoglin wild-type and missense mutant protein heterodimerisation using fluorescence microscopy based IF, BiFC and FRET analyses. PLoS One 2014;9:e102998. [12] Lopez AJ. Alternative splicing of pre-mRNA: developmental consequences and mechanisms of regulation. Annu Rev Genet 1998;32:279–305. [13] Pece N, Vera S, Cymerman U, White Jr RI, Wrana JL, Letarte M. Mutant endoglin in hereditary hemorrhagic telangiectasia type 1 is transiently expressed intracellularly and is not a dominant negative. J Clin Invest 1997;100:2568–79. [14] Robertson RJ, Robertson IR. Pulmonary arteriovenous malformations. Thorax 1995; 50:707–8. [15] Abdel Aal AK, Hamed MF, Biosca RF, Saddekni S, Raghuram K. Occlusion time for Amplatzer vascular plug in the management of pulmonary arteriovenous malformations. AJR Am J Roentgenol 2009;192:793–9. [16] Devlin HL, Hosman AE, Shovlin CL. Antiplatelet and anticoagulant agents in hereditary hemorrhagic telangiectasia. N Engl J Med 2013;368:876–8. [17] Edwards CP, Shehata N, Faughnan ME. Hereditary hemorrhagic telangiectasia patients can tolerate anticoagulation. Ann Hematol 2012;91:1959–68.
Contents lists available at ScienceDirect
Journal of the Neurological Sciences journal homepage: www.elsevier.com/locate/jns
Exome sequencing identifies a novel intronic mutation in ENG that causes recurrence of pulmonary arteriovenous malformations Naoki Saji a,⁎,1,2, Toshitaka Kawarai b,⁎⁎,1,2, Ryosuke Miyamoto b,1, Takahiro Sato a, Hiroyuki Morino c, Antonio Orlacchio d,e, Ryosuke Oki b, Kazumi Kimura a, Ryuji Kaji b a
Department of Stroke Medicine, Kawasaki Medical School, 577 Matsushima, Kurashiki, Okayama 701-0192, Japan Department of Clinical Neuroscience, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima 770-8503, Japan Department of Epidemiology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima, Japan. d Laboratorio di Neurogenetica, CERC-IRCCS Santa Lucia, Rome, Italy e Dipartimento di Medicina dei Sistemi, Università di Roma “Tor Vergata”, Rome, Italy b c
a r t i c l e
i n f o
Article history: Received 2 November 2014 Received in revised form 16 January 2015 Accepted 3 February 2015 Available online 2 April 2015 Keywords: Hereditary hemorrhagic telangiectasia Endoglin Arteriovenous malformation Whole exome sequencing Intronic mutation Aberrant transcript
a b s t r a c t Hereditary hemorrhagic telangiectasia (HHT) occasionally can be discovered in patients with cerebrovascular disease. Pulmonary arteriovenous malformation (PAVM) is one of the complications in HHT and occasionally is causative for life-threatening embolic stroke. Several genetic defects have been reported in patients with HHT. The broad spectrum of phenotype and intrafamilial phenotype variations, including age-at-onset of vascular events, often make an early diagnosis difficult. We present here a Japanese family with a novel intronic heterozygous mutation of ENG, which was identified using whole exome sequencing (WES). The intronic mutation, IVS3 + 4delAGTG, results in in-frame deletion of exon 3 and would produce a shorter ENG protein lacking the extracellular forty-seven amino acid sequences, which is located within the orphan domain. Our findings highlight the importance of the domain for the downstream signaling pathway of transforming growth factor-beta and bone morphogenesis protein superfamily receptors. Considering the phenotype variations and the available treatment for vascular complications, an early diagnosis using genetic testing, including WES, should be considered for individuals at risk of HHT. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Hereditary hemorrhagic telangiectasia (HHT), also known as Osler– Weber–Rendu disease, is an autosomal dominantly inherited vascular disease. Salient clinical features include the presence of mucocutaneous
Abbreviations: AD, autosomal dominant; AVM, arteriovenous malformation; CT, computed tomography; ENG, endoglin; ESS, epistaxis severity score; HHT, hereditary hemorrhagic telangiectasia; MRI, magnetic resonance imaging; NGS, next-generation sequencing; PAVM, pulmonary arteriovenous malformation; WES, Whole exome sequencing. ⁎ Correspondence to: N. Saji, Department of Stroke Medicine, Kawasaki Medical School, 577 Matsushima, Kurashiki City, Okayama, 701-0192, Japan. Tel.: +81 86 462 1111; fax; +81 86 462 1199. ⁎⁎ Correspondence to: T. Kawarai, Department of Clinical Neuroscience, Institute of Health Biosciences, Graduate School of Medicine, University of Tokushima, 3-18-15, Kuramotocho, Tokushima City, 770-0042, Japan. Tel.: +81 88 633 7207; fax: +81 88 633 7208. E-mail addresses: [email protected] (N. Saji), [email protected] (T. Kawarai), [email protected] (R. Miyamoto), [email protected] (T. Sato), [email protected] (H. Morino), [email protected] (A. Orlacchio), [email protected] (R. Oki), [email protected] (K. Kimura), [email protected] (R. Kaji). 1 The first three authors contributed equally to this work. 2 The two corresponding authors contributed equally to this work.
http://dx.doi.org/10.1016/j.jns.2015.02.007 0022-510X/© 2015 Elsevier B.V. All rights reserved.
telangiectasia and arteriovenous malformations (AVM) in visceral organs, primarily the lungs, brain and liver. Early treatment of pulmonary arteriovenous malformations (PAVM), through which paradoxical brain embolism mediates, would ameliorate prognosis for a patient with HHT. Combination of accurate diagnosis by genetic testing, early treatment of PAVM, and careful clinical follow-up is crucial for HHT patients. However, even with early treatment, recurrence of pulmonary arteriovenous malformations has been reported after successful surgical removal or embolization treatment [1]. To date, four genes have been identified, ENG (HHT1), ACVRL1 (HHT2), SMAD4 and GDF2 (HHT5). It has been demonstrated that HHT is caused by either mutations in these genes or other genes that modulate transforming growth factor-beta (TGF-β) signaling (e.g., bone morphogenetic proteins). Two loci have been reported by linkage studies in familial cases, HHT3 and HHT4 on the region of 5q31.3-q32 and 7p14, respectively, in which the causative genes remain unknown [2]. Elucidation of genetic mechanisms would further contribute to better understanding the molecular pathogenicity of vasculopathy and developing new therapeutic strategies in HHT. We present here a family with a novel intronic mutation in ENG, and demonstrate further evidence of allelic heterogeneity in ENG-HHT1.
30
N. Saji et al. / Journal of the Neurological Sciences 352 (2015) 29–33
2. Material and methods
2.3. Genetic analyses
2.1. Ethics statement
After informed consent was given, genomic DNA was extracted from peripheral blood leukocytes from the family members. Whole exome sequencing (WES) was performed in the proband III-2 (Fig. 1-A), as previously described [4]. Variants were filtered against public databases, including Single Nucleotide Polymorphism Database (dbSNP 135), the 1000 genomes, and the NHLBI Exome Sequencing project (ESP5400). In addition, variants were also filtered against our in-house variant database, including 106 control exome experiments. Variations identified in the candidate genes were further subjected to filtering steps in the specific databases (Supplementary data). Genomic DNA was amplified by polymerase chain reaction (PCR) with sets of specific primers in order to investigate coding sequences and intron/exon boundaries of ENG, as previously described [5,6]. PCRdirect sequencing was performed and each sequence read was aligned to the genome reference sequences using the program Sequencher version 5.2 (Gene Codes, Ann Arbor, MI, USA). The sequence analysis was further expanded, including 400 Japanese control chromosomes. Secondary structure and minimal free energy of wild and mutant RNA were predicted by the RNAFOLD program, as described elsewhere [7,8]. Total RNA was prepared from lymphocytes using RNAiso Plus reagent (TaKaRa Bio, Inc., Kyoto, Japan) according to the manufacturer's instructions. RNA samples were treated with RNase-free DNase I (Ambion) and RNA concentrations were determined using a NanoDrop
The study was approved by the ethical review boards of the Kawasaki Medical Hospital and Tokushima University Hospital. All of the participants in this study gave written informed consent.
2.2. Subjects The pedigree chart is illustrated in Fig. 1. There were three family members with a history of spontaneous and repeated epistaxis including the proband (Fig. 1: II-1, III-1 and III-2). Other members in this family presented as normal. All the living family members were examined by three experienced neurologists (Drs. N.S., T.S. and K.K.) and underwent neuroimaging and genetic examinations. The Curaçao criteria for HHT was applied in the diagnosis [3]. Epistaxis severity score for hereditary hemorrhagic telangiectasia (ESS-HHT) was adopted, in which scores are assigned to each severity level: severe = 7 to 10, moderate = 4 to 7, mild = 1 to 4 and none = 0 to 1 [3]. The score is calculated automatically at the website, http://www2. drexelmed.edu/hht-ess/. Detailed history and neurological features in the deceased patient (Fig. 1; III-1) were assumed from medical records or interviews with living family members.
A
I
1
1*
II
D
3
B
?
4
5
C
6
? 1
III IV
2
2
?
2*
P 1
2
3
4
5
E
F
Fig. 1. A) Pedigree chart. Solid symbols, affected individuals; open symbols, unaffected individuals or family members showing no abnormal finding in chest roentgenogram; question mark, phenotype unknown; circles, females; squares, males; slashes, deceased; arrow, proband. *Indicates members carrying the mutation, g.IVS3 + 4delAGTG. B) Multiple red spots on the proband's tongue. The subcutaneous lesions reflect characteristic telangiectasia. C) Spontaneous recurrent nose bleeds from telangiectasia of the nasal mucosa in the proband. Examination of the nasal cavity using a flexible fiberscope demonstrates active bleeding from the mucosal telangiectasia. D) Embolic stroke occurred in the proband. Diffusion-weighted imaging shows a high intensity at the left temporal lobe (upper panel). Magnetic resonance angiography reveals obstruction of superior cortical branches of the middle cerebral artery (white arrow in lower panel). E) Pulmonary arteriovenous malformation. Unenhanced thoracic computed tomography demonstrates a thick and rope-like density within the left lower lung field in the proband (red arrow). F) Abnormal connection between the venous and arterial system is revealed by axial and volume-rendered computed tomography (red arrow head).
N. Saji et al. / Journal of the Neurological Sciences 352 (2015) 29–33
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Table 1 Clinical data of the four affected individuals. Family member ID
Age at examination (years)
Age at onset of repeated epistaxis
ESS-HHT at examination (or deceased age)
Telangiectases of hands, face, and oral cavity
Pulmonary AVMs
Hepatic AVMs
Central nervous system AVMs
II-1 III-1 III-2 IV-5
90 65 (age at death) 61 31
Late twenties Early twenties Late twenties Asymptomatic
1.41 3.22 1.92 0.91
None Not described Hands, face and oral cavity None
Positive None Positive (recurrence) None
None Not examined None None
None Not examined None None
(ND-1000) spectrophotometer. 10 mg RNA were reverse transcribed using random primers and the PrimeScript II 1st Strand cDNA Synthesis Kit (TaKaRa Bio, Inc., Kyoto, Japan). Oligonucleotide primers ENG-82F (5′-GTCCATTGTGACCTTCAGCCTGTG-3′) and ENG-616R (5′-ACGCCTTC CAAGTGGCAGCC-3′) that span exons 2–5 of endoglin cDNA were used for amplification of cDNA. PCR products were fractionated by agarose gel electrophoresis, excised and purified by RBC HiField Gel/PCR DNA Fragments Extraction Kit (SciTrobe, Tokyo, Japan), and subjected to cycle sequencing, as previously described [9]. The three dimensional protein structures of ENG were predicted by SWISS-MODEL (http://swissmodel.expasy.org) [10]. 3. Results 3.1. Clinical features The proband, a 61-year-old man, suddenly developed behavioral disorders and referred to the emergency unit in Kawasaki Medical Hospital. Physical examination demonstrated multiple telangiectasias on his lips, tongue, fingers, and earlobes (Fig. 1-B). Epistaxis from telangiectasias of the nasal mucosa was confirmed by flexible fiberscopic
A
Exon 3
examination (Fig. 1-C). He had a history of repeated nasal bleeding since his late twenties. ESS-HTT of 1.92, categorized as mild, was given at admission. Neurological examination revealed left inferior quadrantanopsia, left hemiparesis, and unilateral spatial neglect. Laboratory findings showed iron deficiency anemia. Diffusion weighted magnetic resonance imaging scans of the brain showed a high signal intensity area in the right occipito-temporal lobe. Magnetic resonance angiography showed a blunt-ended occlusion with insufficient collaterals at the right middle cerebral artery, indicating acute embolic stroke (Fig. 1-D). A transesophageal echocardiography revealed no evidence of patent foramen ovale and intracardiac thrombus. A transcranial doppler ultrasound revealed a finding of continuous right-to-left shunt. The patient had a history of recurrent nasal hemorrhage and PAVM, which was incidentally found, and resection of the lower lobe was performed at the age of 28. Recurrence of PAVM at the left lower lobe was confirmed by thoracic computed tomography (Fig. 1-E and F). Coil embolization for pulmonary arteriovenous malformation was performed. Clinical examination and review of medical record revealed that the other two members, II-1 and III-1, were diagnosed with HHT. The asymptomatic member, IV-5, was genetically diagnosed with HHT. The clinical summary is described in Table 1.
Intron 3
control
proband T T G G C C T AC GT G A G T GT G T T C C C TCC A A C C T T G G C C T AC GT G T G T T C C C T C C A ACC B C G
C UA G U U C A C
C G +1
U +2 G +3 A +4 G +5 U +6 GUGUUCCCUCCA
+7 +8 +9
Wild-type
C U A G C U G +1 U +2 U C G +3 A U +8 C GUUCCCUCCAACCC
C G
+9
Mutant-type
Fig. 2. A) Fluorescent chromatographs of the mutation found in the ENG gene. The sequence around the heterozygous substitution is shown, with the redline rectangle pointing to the g.IVS3 + 4delAGTG mutation. B) Effect of the mutation in the ENG gene on the RNA secondary structure. The left and right figures display the wild-type and mutant RNA sequences respectively. The mutation site is emphasized in red. The natural splice site in the wild-type transcript is marked with an arrow. Positions are numbered according to the first nucleotide of intron 3 starting with +1.
32
N. Saji et al. / Journal of the Neurological Sciences 352 (2015) 29–33
protein lacking the forty-seven amino acid sequences (Supplementary data).
3.2. Genetic analyses WES generated a good coverage depth, which was sufficiently covered to pass our thresholds for calling SNPs and indels. After the exome sequencing and variant calling, 64,746 variants were identified. Data quality and quantity are described in the Supplementary data. Combination of filtering sequence variants and bioinformatic analyses demonstrated no compelling variant mapped on the candidate regions for HHT3 and HHT4. Manual review of intronic variants revealed a 5′ splice site deletion, g.IVS3 + 4delAGTG, at intron 3 in ENG. Validation of the intronic variant and segregation with the phenotype in the family was confirmed by Sanger sequencing (Fig. 2-A). The deletion variant was absent in 400 control chromosomes. Analysis by the RNAFOLD program demonstrated an altered predicted secondary structure of the mutant RNA (Fig. 2-B and Supplementary data). The secondary structure of the wild-type RNA sequence contains a stem–loop structure; the four residues, AGTG at the position + 4 to + 7, are located within the stem (δ G − 3.70 kcal/mol). The mutation, g.IVS3 + 4delAGTG, created a smaller stem–loop structure (δ G −2.60 kcal/mol). Reverse transcriptase PCR (RT-PCR) assay demonstrated aberrant transcripts in the proband (Fig. 3-A). Sanger sequencing of the short PCR fragments showed ENG transcripts lacking exon 3 (Fig. 3-B), which lead to in-frame deletion. The normal ENG protein is 658 amino acid residues in length, however, mutant protein having a short length of 611 amino acids would be produced in this case. No significant structural alteration was predicted by in silico analysis. Formation of twisted U-shaped construction seemed unchanged in the mutant ENG
4. Discussion To date, more than 27 ENG mutations have been reported in hereditary hemorrhagic telangiectasia (HHT), including nonsense, missense, frame shift, deletion and splicing mutations [3,5,6]. Haploinsufficiency is presumably a cardinal pathomechanism in HHT caused by mutant ENG. Missense mutant ENG proteins dimerize with themselves, as well as with wild-type ENG proteins, and localize in the rough endoplasmic reticulum (rER) or in the plasma membrane of the cells. When the mutant ENG proteins are retained in the rER, the amount of endogenous wild-type endoglin on the plasma membrane is reduced through interception in the rER [11]. A total of four 5′ splice site mutations in intron 3 have been published, c.360 + 1GNA (IVS3 + 1GNA), c.360 + 1GNC (IVS3 + 1GNC), c.360 + 4ANG (IVS3 + 4ANG) and c.360 + 5GNC (IVS3 + 5GNC) (Supplementary data). In-frame deletion of exon 3 was demonstrated in the two mutations, c.360 + 1GNA (IVS3 + 1GNA) and c.360 + 1GNC (IVS3 + 1GNC) (Supplementary data). A total of six 5′ splice site mutations in intron 3 have been listed in the HHT Mutation Database (http://www.hhtmutation.org/index.php) including the c.360 + 4delAGTG (IVS3 + 4delAGTG), however, the biological effect by the mutation is not assessed. The prediction of secondary pre-mRNA structure would indicate an alteration of splice site selection. When an alternative structure is too different from the lowest free-energy structure (ground state), the sequence is not recognized by the splicing machinery
(2) Impairment of heterodimer complex formaon?
A
C
554 bp 413 bp
Extracellular part
Plasma membrane (1) Insufficient transport to the cell surface
TGF-β receptor type I and II
B Exon 2
Exon 4
Decreased signaling from TGF-β/BMP superfamily in nucleus 74-120
1
Altered gene expression in angiogenesis
Phe
Pro
71
72
Thr 73
Asn
Ser
Ser
121
122
123
ENG protein synthesis 658
Fig. 3. A) Results of the RT-PCR assay. PCR product obtained from the proband's cDNA shows two bands — 554 bp and 413 bp — revealing that aberrant RNA splicing occurred by the intronic deletion mutation. B) Sequence analysis of the shorter PCR fragment shows the ENG cDNA sequence with in-flame deletion of exon 3. Amino acid numbering is based on the longest protein isoform (NCBI accession no. NM_001114753.2). C) Bar diagram of ENG with the domains indicated and highlighted in different colors. SP, signal peptide in red, TM, transmembrane region in black, ZP, zona pellucida in dark blue and OD, orphan domain in orange. The position of an in-frame deleted part is indicated with a two-way arrow. Numbers indicate the amino acid of endoglin (starting at the N terminus). Two possible biological effects by the deletion mutation are commented in the figure.
N. Saji et al. / Journal of the Neurological Sciences 352 (2015) 29–33
[12]. As a consequence, the corresponding exon is prone to skipping. The effect by the reported mutations at 5′ splice site mutations in intron 3 were evaluated, however, no distinctive difference was demonstrated under the model of minimum free energy. The RNA structural stability of the sequence containing the mutation of IVS3 + 1GNA is similar to that in the wild type sequence, but exon 3 skipping was biologically confirmed (Supplementary data). Exon 3 splicing may be regulated by a more complicated structural alteration or splicing regulatory elements. Deletion of the four nucleotides at the 5′ splice site in intron 3 influences the RNA structure with reduced free energy, leading to the inframe skipping of an exon 3. The mutant protein, lacking amino acids encoded by the exon 3, was detected as an intracellular homodimer, while the normal protein was expressed at the cell surface. The amount of biologically active ENG protein was decreased to 50% of control levels in umbilical vein endothelial cells from a patient carrying the heterozygous mutation [13]. The in-frame deletion of exon 3 would not affect the protein synthesis and would result in the production of shorter length protein, lacking the forty-seven amino acid sequences. The region deleted by the mutation contains two asparagine residues, 88Asn and 102Asn, which are subject to glycosylation after protein synthesis (post-translational). The absence of the two glycosylated asparagine residues or the forty-seven amino acid sequences per se might lead to insufficient transporting of ENG protein to the cell surface or impairment of heterodimer complex formation with TGF-β receptors (Fig. 3-C). The proband (III-2) undertook a surgical removal of PAVM more than 30 years before the current embolic stroke. Several explanations for the recurrence of PAVM have been postulated, including persistent small PAVMs which would grow due to the altered blood flow after the surgery [14,15]. In addition, a continuous defect in the signaling pathway of the TGF-β/BMP superfamily might lead to angiodysgenesis acquired after the lobectomy. Intrafamilial phenotype variation was demonstrated in the family, including age-at-onset, severity of epistaxis and telangiectasias on skin and mucosa. It has been reported that the age-at-onset of recurrent epistaxis is adolescence or later. The age-at-onset may be influenced by genotype. Later onset of symptoms in HHT2 is reported in comparison with HHT1. In the study of genetically confirmed HHT cases in the Japanese population, the mean age of HHT2 is 48.6 years old while that of HHT1 is 35.1 years old [5]. Genetic testing is indispensable for family members with unlikely diagnosis for HHT, thus positive family history only is reported, in order to exclude gene mutation in currently-known HHT genes. Unlike other genetic neurological disorders, prophylaxis for vascular complications could be provided for HHT patients, including surgical operation, endovascular treatment and antithrombotic/anticoagulant therapy [16,17]. The role of genetic analysis in the diagnosis of HHT is of importance. In conclusion, our findings demonstrate further allelic heterogeneity in ENG-HHT1 and indicate that the extracellular domain encoded by exon 3 might be crucial for the signaling pathway of the TGF-beta/ BMP superfamily. It is also demonstrated that the utility of WES facilitates the diagnosis of genetically highly heterogeneous diseases. The natural history of HHT can be variable, however, the incidence of embolic stroke obviously confers poor prognosis and occasionally causes unexpected sudden death. Prognosis of HTT would become more favorable by the combination of early diagnosis, using WES, treatment of PAVM and therapeutic anticoagulation. Author contribution Naoki Saji and Takahiro Sato: patient review and medical writing for content. Toshitaka Kawarai: genetic testing, sequencing of mutation, and manuscript preparation. Ryosuke Miyamoto, Hiroyuki Morino and Ryosuke Oki: genetic testing and bioinformatic analysis. Antonio Orlacchio, Kazumi Kimura and Ryuji Kaji: study supervision and coordination.
33
Conflict of interest The authors report no conflicts of interest. Acknowledgements This work was supported by Grants-in-Aid from the Research Committee of CNS Degenerative Diseases of the Ministry of Health, Labour and Welfare of Japan (R.K.), the Japan Society for the Promotion of Science (JSPS KAKENHI Grant no. 26870765 to N.S. and no. 26461294 to T.K.), the Brain Science Foundation, Japan (Grant to T.K.), and Grantin-Aid for Research on rare and intractable diseases, the Research Committee on Establishment of Novel Treatments for Amyotrophic Lateral Sclerosis from the Ministry of Health, Labour and Welfare of Japan (Grant to T.K. and R.K.), and the Ministry of Health, Italy (Grant no. GR09.109 to A.O.). The authors thank the patient and family members involved in this study. We thank Michela Renna (MA) for her language advice and assistance, as well as the Support Center for Advanced Medical Sciences, Tokushima University School of Medicine, for the use of their facilities to prepare the manuscript. The authors also thank Ms. Akemi Takahashi, Ms. Mariko Hasegawa and Ms. Akie Tanabe for their technical support. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.jns.2015.02.007. References [1] Chao HS, Chern MS, Chen YC, Chang SC. Recurrence of pulmonary arteriovenous malformations in a female with hereditary hemorrhagic telangiectasia. Am J Med Sci 2004;327:294–8. [2] Duffau P, Lazarro E, Viallard JF. Hereditary hemorrhagic telangiectasia. Rev Med Interne 2014;35:21–7. [3] Shovlin CL, Guttmacher AE, Buscarini E, Faughnan ME, Hyland RH, Westermann CJ, et al. Diagnostic criteria for hereditary hemorrhagic telangiectasia (Rendu–Osler– Weber syndrome). Am J Med Genet 2000;91:66–7. [4] Miyamoto R, Morino H, Yoshizawa A, Miyazaki Y, Maruyama H, Murakami N, et al. Exome sequencing reveals a novel MRE11 mutation in a patient with progressive myoclonic ataxia. J Neurol Sci 2014;337:219–23. [5] Komiyama M, Ishiguro T, Yamada O, Morisaki H, Morisaki T. Hereditary hemorrhagic telangiectasia in Japanese patients. J Hum Genet 2014;59:37–41. [6] Shovlin CL, Hughes JM, Scott J, Seidman CE, Seidman JG. Characterization of endoglin and identification of novel mutations in hereditary hemorrhagic telangiectasia. Am J Hum Genet 1997;61:68–79. [7] Zuker M, Stiegler P. Optimal computer folding of large RNA sequences using thermodynamics and auxiliary information. Nucleic Acids Res 1981;9:133–48. [8] Kleiner-Fisman G, Rogaeva E, Halliday W, Houle S, Kawarai T, Sato C, et al. Benign hereditary chorea: clinical, genetic, and pathological findings. Ann Neurol 2003; 54:244–7. [9] Kawarai T, Pasco PM, Teleg RA, Kamada M, Sakai W, Shimozono K, et al. Application of long-range polymerase chain reaction in the diagnosis of X-linked dystoniaparkinsonism. Neurogenetics 2013;14:167–9. [10] Biasini M, Bienert S, Waterhouse A, Arnold K, Studer G, Schmidt T, et al. SWISSMODEL: modelling protein tertiary and quaternary structure using evolutionary information. Nucleic Acids Res 2014;42:W252–8. [11] Forg T, Hafner M, Lux A. Investigation of endoglin wild-type and missense mutant protein heterodimerisation using fluorescence microscopy based IF, BiFC and FRET analyses. PLoS One 2014;9:e102998. [12] Lopez AJ. Alternative splicing of pre-mRNA: developmental consequences and mechanisms of regulation. Annu Rev Genet 1998;32:279–305. [13] Pece N, Vera S, Cymerman U, White Jr RI, Wrana JL, Letarte M. Mutant endoglin in hereditary hemorrhagic telangiectasia type 1 is transiently expressed intracellularly and is not a dominant negative. J Clin Invest 1997;100:2568–79. [14] Robertson RJ, Robertson IR. Pulmonary arteriovenous malformations. Thorax 1995; 50:707–8. [15] Abdel Aal AK, Hamed MF, Biosca RF, Saddekni S, Raghuram K. Occlusion time for Amplatzer vascular plug in the management of pulmonary arteriovenous malformations. AJR Am J Roentgenol 2009;192:793–9. [16] Devlin HL, Hosman AE, Shovlin CL. Antiplatelet and anticoagulant agents in hereditary hemorrhagic telangiectasia. N Engl J Med 2013;368:876–8. [17] Edwards CP, Shehata N, Faughnan ME. Hereditary hemorrhagic telangiectasia patients can tolerate anticoagulation. Ann Hematol 2012;91:1959–68.
