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RESEARCH REPORT |
1 Department of Oral Medicine, Peking University School of Stomatology, 22 South Zhong Guan Cun Street, Beijing 100081, People’s Republic of China;
2 Molecular Laboratory for Gene Therapy, School of Stomatology, Capital Medical University, 4 Tian Tan Xi Li, Chongwen District, Beijing 100050, People’s Republic of China; and
3 Chinese National Human Genome Center, 707 North Yongchang Road, BDA, Beijing 100176, People’s Republic of China
* corresponding authors, xuyy{at}mail.nsfc.gov.cn and songlinwang{at}dentist.org.cn
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: hereditary hemorrhagic telangiectasia (HHT) • thrombomodulin • mutation • ALK-1
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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The disease has two known variants, HHT1 and HHT2 (Johnson et al., 1996; Kjeldsen et al., 2001). A higher frequency of pulmonary arteriovenous malformations (PAVMs) has been reported for HHT1, while HHT2 is thought to be associated with a lower penetrance and milder disease manifestations (Shovlin et al., 2000). Studies have confirmed that the pathogenic genes are the endoglin gene (ENG) and the activin receptor-like kinase-1 gene (ALK-1) for HHT1 and HHT2, respectively. Several mutations of the two genes have been identified in HHT patients in many countries (Johnson et al., 1996; Shovlin et al., 1997; Kjeldsen et al., 2001); however, there is little reported for the Chinese population (Zhang et al., 2004). ENG and ALK-1 are type III and type I transforming growth factor beta (TGF-ß) family receptors, respectively (Lux et al., 1999). Animal models have shown that these receptors are important not only for maintaining vascular integrity, but also for angiogenesis during embryonic development and tumor growth (van den Driesche et al., 2003). The precise mechanisms of vascular abnormalities elicited by these mutations observed in HHT patients are still unclear, although analysis of the mechanism of intracellular signal transduction has been performed, and a change in targeted gene expression was found with the use of mutant recombinant endoglin or ALK-1 proteins (Azuma, 2000).
Thrombomodulin (TM) is a glycoprotein, present on intact endothelial cell surfaces, that plays a major role in activation of the protein C anticoagulant system (Esmon, 1995). ALK-1 is a type I TGF-ß receptor and is exclusively expressed on vascular endothelial cells (Lux et al., 1999). TGF-ß1 has been shown to down-regulate thrombomodulin mRNA expression in cultured human endothelial cells (Ohji et al., 1995). Although many studies have suggested that the TGF-ß signaling family may play a key role in HHT (Bourdeau et al., 1999), the precise mechanism giving rise to the vascular abnormalities is unclear. There have been few reports on the transcriptional changes and thrombomodulin levels in HHT. In the present study, we performed mutation screening on the ENG and ALK-1 genes in one Chinese family with HHT2, who visited our clinic with recurrent tongue bleeding. We measured ALK-1 mRNA and thrombomodulin to investigate the possible mechanisms of formation of the vascular abnormalities in this HHT family.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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). The diagnosis of HHT2 was based on family history and the presence of cutaneous or mucocutaneous arteriovenous malformations (Shovlin et al., 2000). The study was conducted with the consent of all family members and was approved by the Ethics Committee of the Peking University Health Science Center. Physical examinations were performed for all 13 family members.
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Sequence Analysis
We performed mutation screening on the ENG and ALK-1 genes on all 13 family members, with 110 normal, unrelated individuals (220 chromosomes) as controls. Intronic primers flanking the exons were used for amplifying and sequencing PCR products. Primers were designed using Primer 3 software (ABI, Inc.). PCR amplification was performed with 15 µL of a PCR reaction mix containing 50 ng of genomic DNA, 10 mM Tris (pH 8.4), 50 mM KCl, 3.0 mM MgCl2, 200 µM of each dNTP, 0.6 units HotStataqTMfinal extension was performed at 72°C for 10 min. The amplification products were purified with the use of a MultiScreen-PCR plate (Millipore, Billerica, MA, USA). Cycle sequencing was conducted with the use of the Big Dye Deoxy Terminator Cycle Sequencing kit (Perkin-Elmer) according to the manufacturer’s instructions. All sequencing was carried out on an ABI Prism 3700 DNA Analyzer. The results were analyzed with BioEdit software and compared with sequences in the NCBI databases.
Real-time Quantitative PCR
The B-lymphocytes of peripheral venous blood from all 13 family members and 50 unrelated normal controls were infected by Epstein-Barr virus, after which immortalized cell lines were established. Total RNA was extracted and reverse-transcribed into cDNA. The ALK-1 mRNA expression level, which was quantified by the cycle threshold method, was measured with the use of a QuantiTectTM SYBR® Green PCR Kit (Qiagen), according to the manufacturer’s instructions, in an ABI PRISM 7000 sequence detection system. Values were then normalized to the relative amounts of the control gene albumin (Alb, NM_000477). Primers were designed with the use of Primer 3 software, as follows: Forward and reverse primer were 5'-TCTCAGGCCTAGCTCAGATGAT-3' and5'-TAGGCTTCTCTGGACTGTTGCT-3', and the sequences were 117 bp. Alb cDNA was amplified with primers: Forward primers were 5'-AATGCCCTGTGCAGAAGACT-3', and reverse primers were 5'-CTGTGCAGCATTTGGTGACT-3', and the sequences were 101 bp. Real-time PCR was performed by three-step cycling under the following conditions: initial denaturation at 95°C for 15 min; and denaturation, annealing, and extension at 94°C for 30 sec, 59°C for 30 sec, and 72°C for 1 min (40 cycles). Fluorescence was measured at 59°C at the end of every cycle.
Cell Fractionation and Western Blot Analysis
The cell fractionations were performed as described previously from immortalized cell lines (Shimizu et al., 1997). The proteins were separated by 10% SDS-PAGE and transferred to PVDF membrane with a semi-dry transfer apparatus (Bio-Rad Laboratories, Hercules, CA, USA). The membranes were probed with monoclonal antibodies against ALK-1 (1:1000, R&D Systems, Minneapolis, MN, USA). For internal controls, the blots were stripped and reprobed with monoclonal antibodies against 
-tubulin (1: 20,000, Sigma-Aldrich, St. Louis, MO, USA). Both Western blots were probed with mouse HRP-conjugated secondary antisera (Amersham, Buckinghamshire, UK), at a dilution of 1:3000, with proteins detected with the ECL kit (Amersham).
Thrombomodulin ELISA
Peripheral venous blood samples from all 13 family members and 50 unrelated normal controls were obtained, treated with EDTA as anti-coagulant, and centrifuged at 4000 g for 20 min. The plasma was separated and stored at –70°C. Plasma was 50x diluted. Soluble plasma thrombomodulin was measured by a Thrombomodulin ELISA kit (Market Inc., San Jose, CA, USA). Each sample was assayed 3 times, according to the manufacturer’s instructions, using a spectrophotometer (Bio-Rad Model 550 Microplate Reader) at 450-nm wavelengths, and concentration was determined from a standard curve.
Statistics
All statistical calculations were performed with the use of GraphPad Prism 4 Demo statistical software; for real-time quantitative PCR, a 
2 test was used, and for ELISA, one-way ANOVA was used to test for significance. P < 0.05 was considered statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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), but without pulmonary arteriovenous malformation, so all patients were diagnosed as HHT2 on the basis of clinical symptoms. We examined all the exons of ALK-1 gene and found a C-to-T substitution mutation in exon 10 of ALK-1 (1717C>T) (GI: 4557242, NM_000020.1) (Fig. 3a). This caused the base sequence to change from codon CGA to TGA (R479X) and resulted in a substantial shortening of the ALK-1 protein in its predicted intracellular domain. Western blotting showed a smaller band for ALK-1 protein from one patient’s sample and indicated the truncated ALK-1 protein. There were no significant differences in the protein expression level of ALK-1 (-tubulin as the internal control) (Fig. 3b). Notably, all six symptomatic individuals had the same mutation. The ALK-1 mutation was not found in any of the seven healthy relatives or the 110 normal, unrelated controls (220 chromosomes). No mutation in the endoglin gene was found in any of the 13 family members or controls. The linkage analysis results revealed that the microsatellite markers near ALK-1, including D12s1586LODZMAX, were 1.82 (
= 0.0), D12s1677 LODZMAX was 1.74 ( = 0.0), D12s1635 LODZMAX was 1.65 ( = 0.0), D12s368 LODZMAX was 1.87 ( = 0.0), also supporting these results.
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). Plasma thrombomodulin of the patients (1194.81 ± 71.50 ng/mL, n = 6) was significantly higher (p < 0.001) than that of healthy individuals (465.60 ± 79.91 ng/mL, n = 7) in this family and in normal controls (594.16 ± 224.22 ng/mL, n = 50). No significant difference was found between healthy individuals in this family and normal controls (Fig. 4b).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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In this study, we identified a novel mutation in exon 10 of the ALK-1 gene in one Chinese family. The substitution mutation of a C to a T at the base position 1717 leads to CGA (Arginine R) changing into TGA (termination codon, R479X). It is a novel mutation of HHT, not previously reported. The identification of ALK-1 mutations in patients confirmed the clinical diagnosis of HHT type 2. Our results suggest that an early diagnosis of HHT by a molecular method may be useful for the early control of associated visceral involvement.
The disease manifestations are variable in severity and include epistaxis, gastrointestinal bleeding, pulmonary arteriovenous malformations, and both cerebral and liver arteriovenous malformations (Shovlin et al., 1997). The nasal mucosa is the most common site for telangiectasia (95%), followed by the tongue (60%), palate, lips, buccal area, gum, pharynx, and conjunctivae (Gorlin et al., 1990; Pau et al., 2000). There were 27 clinical reports of HHT from 1973 to 2003 in China, wherein 232 patients were described, with 82% having a family history and 20% having pulmonary arteriovenous malformations (Su, 1999). In our cases, some of the patients exhibited epistaxis, and the index case exhibited recurrent tongue bleeding. Not only are dental clinicians likely to be the first clinicians to identify a patient with HHT, but also routine dental treatment of unidentified HHT patients, without prophylactic treatment, could be fatal.
In the present study, we identified a novel missense mutation in exon 10 of the ALK-1 gene in one Chinese family, and this mutation caused a truncated ALK-1 protein; however, there were no significant differences in ALK-1 mRNA expression between six patients and the seven healthy members of the family, which indicated that the missense mutation occurred in genomic DNA, but did not affect ALK-1 mRNA transcription.
Thrombomodulin is a glycoprotein, present on intact endothelial cell surfaces, that plays a major role in activation of the protein C anticoagulant system. Thrombomodulin is a transmembrane receptor for thrombin (Esmon, 1995) and is down-regulated by inflammatory cytokines. Down-regulation can be prevented by retanoic acid and cyclic AMP (Dittman, 1991). At present, thrombomodulin is increasingly used as a marker of endothelial damage in a large number of diseases, such as ulcerative colitis, sepsis, pre-eclampsia, diabetes mellitus-associated micro- or macroangiopathy (Magriples et al., 1997), thrombotic-thrombocytopenic purpura, adult respiratory distress syndrome, and coronary and other atherosclerotic lesions (Boffa and Karmochkine, 1998). Increased thrombomodulin levels were also associated with disease activity in vasculitis, such as Takayasu’s arteritis, Behçet disease, giant cell arthritis, polyarteritis nodosa, and Wegener’s granulomatosis (Boehme et al., 1997). In the present study, a significant increase in plasma thrombomodulin in patients suggested that there was damage to vascular endothelial cells in these Chinese patients with HHT2.
The level of thrombomodulin in endothelial cells has been shown to be modulated by cytokines, including transforming growth factor-ß (TGF-ß). Both TGF-ß1 and TGF-ß2 have been shown to down-regulate thrombomodulin mRNA expression in cultured human endothelial cells (Ohji et al., 1995; Sandusky et al., 2002), and increased TGF-ß correlates with decreased thrombomodulin-containing vessels in sustained local endothelial dysfunction (Richter et al., 1997). Since ALK-1 is a type I TGF-ß receptor, and is exclusively expressed in vascular endothelial cells, it is suggested that the mutation found in this family produced a truncated ALK-1 protein, interrupting the receptor interaction with TGF-ß1 and causing down-regulated function of TGF-ß1, resulting in up-regulation of thrombomodulin, or truncated ALK-1 protein counteracted TGF-ß1 directly, destroying the vascular endothelial cells and causing more soluble thrombomodulin in the blood. The relationship between ALK-1 and thrombomodulin requires further study, but plasma thrombomodulin may provide an easy molecular marker in HHT patients.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received March 16, 2005; Last revision February 15, 2006; Accepted April 27, 2006
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REFERENCES |
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| Journal of Dental Research ® | Critical Reviews (1990-2004) |
indicates HHT2 patients, and 
indicates the normal family numbers. There are eight patients, including the proband, and 46 normal individuals in this family. It is an autosomal-dominant hereditary disease, according to genetic analysis.
