- Email: [email protected]
Allelic variation in the serotonin transporter (5HTT) gene contributes to idiopathic pulmonary hypertension in children
BBRC Biochemical and Biophysical Research Communications 334 (2005) 376–379 www.elsevier.com/locate/ybbrc
Allelic variation in the serotonin transporter (5HTT) gene contributes to idiopathic pulmonary hypertension in children Akshaya Vachharajani, Scott Saunders * Department of Pediatrics, Washington University School of Medicine, St. Louis ChildrenÕs Hospital, St. Louis, MO 63110, USA Received 3 June 2005 Available online 28 June 2005
Abstract Pulmonary hypertension is a potentially lethal condition, which affects adults and children alike. Genetic factors are implicated in the causation of primary pulmonary hypertension. We investigate the role of polymorphism in the 5HTT gene in the etiology of pulmonary hypertension in children aged 1–18.8 years. We have tested the hypothesis that the 5HTT gene does contribute to the pathogenesis of this disease in children by comparing the allelic frequencies of both the long and short variants between children with idiopathic pulmonary hypertension and pulmonary hypertension secondary to underlying pulmonary disease. We found that homozygosity for the long variant of 5HTT was highly associated with idiopathic pulmonary hypertension in children, suggesting perhaps a more important role for 5HTT gene function in the pathogenesis of early onset disease. Ó 2005 Elsevier Inc. All rights reserved. Keywords: Serotonin transporter; 5HTT; Pulmonary hypertension; Smooth muscle; Lung; Pulmonary arteries; BMP
Primary pulmonary hypertension is a progressive medical condition that can occur at any age, and has a prevalence of 1–2 per 1,000,000 individuals [1]. It can be classified broadly based on whether it is primary, and therefore not arising as a result of another underlying condition, or secondary when it is the results of an underlying disease process, usually involving the lungs or the heart. In the case of secondary pulmonary hypertension, a number of specific factors have been defined to contribute to the evolution of the disease. These include as examples, environmental factors such as perinatal asphyxia and meconium aspiration syndrome in the newborn. In addition, a number of anatomic factors have also been causally linked with the development of pulmonary hypertension including those resulting from developmental abnormalities as seen in association with the pulmonary hypoplasia of congenital diaphragmatic
*
Corresponding author. Fax: +1 314 286 2893. E-mail address: [email protected] (S. Saunders).
0006-291X/$ - see front matter Ó 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.06.107
hernia, and those secondary to other disease processes such as interstitial lung disease and emphysema. Recently, significant advances have occurred in the understandings of the genetic contribution to the development of pulmonary hypertension. In particular, a role for defective signaling by members of the TGF-b super family has been clearly associated with familial pulmonary hypertension. Haploinsufficient mutations in the BMPR2 receptor have been found to be the major inherited mechanism of familial primary pulmonary hypertension [2,3], as well as a significant contributor to sporadic cases of this disease [4]. While the molecular details are currently still being investigated, it is speculated that this phenotype results from a loss in the inhibition of smooth muscle cell proliferation in response to a dominant negative mutation of the receptor [5,6]. One important issue that has arisen is the consideration of the role of modifier genes in the evolution of primary pulmonary hypertension, in as much as only 10–20% of individuals with BMP receptor mutations develop phenotypic disease. Another significant genetic
A. Vachharajani, S. Saunders / Biochemical and Biophysical Research Communications 334 (2005) 376–379
contributor to primary pulmonary hypertension is related to the functions of the serotonin 5-hydroxy tryptamine transporter (5HTT) gene. Function of 5HTT has been closely associated with the smooth muscle hyperplasia seen in pulmonary hypertension. Specifically, increased expression of the 5HTT gene has been identified in the remodeling pulmonary arteries of animals developing pulmonary hypertension in response to chronic exposure to hypoxia, and the severity of this pulmonary hypertension can be modified by either the administration of 5HTT inhibitors [7,8], or by genetically modifying 5HTT gene expression [8,9]. Further supporting the functional role for 5HTT in the evolution of pulmonary hypertension in humans, its activity has been found to be increased in cultured pulmonary artery smooth muscle cells from human patients with this disease [10,11]. A genetic polymorphism is known to exist in the 5HTT gene that effects the transcript level of this gene in vivo [12]. This polymorphism is the result of a 40 base pair deletion that diagnostically results in both a short (484 bp) and a long (528 bp) fragment when this region is amplified by PCR [12]. The long allelic variant is associated with increased transcriptional activity of the 5HTT gene, which leads to increased 5HT levels and subsequent enhanced 5HT transport into pulmonary smooth muscle cells. This has been shown to result in increased smooth muscle cell proliferation in cell culture [11]. There has been a reported increased association of this particular polymorphism in adult patients with idiopathic primary pulmonary hypertension, presumably due to an increased capacity for the proliferation of smooth muscle cells in the pulmonary arteries in these individuals in vivo [11]. Given that, by definition, adult and childhood primary pulmonary hypertension have vastly different time courses of presentation we thought it reasonable to speculate that the underlying genetic predisposition might well be distinct between these two presentation of this disease. Indeed, in support of this hypothesis is the recently reported findings that in 13 children with primary pulmonary hypertension (age at diagnosis, 6 months to 13 years), no mutations in the BMPR2 gene could be found nor any linkage in fact to the BMP receptor loci [13]. In the current study, we have tested the hypothesis that the 5HTT gene does contribute to the pathogenesis of this disease in children by comparing the allelic frequencies of both the long and short variants between children with idiopathic pulmonary hypertension and pulmonary hypertension secondary to underlying pulmonary disease. We found that homozygosity for the long variant of 5HTT was highly associated with idiopathic pulmonary hypertension in children, suggesting perhaps a more important role for 5HTT gene function in the pathogenesis of early onset disease.
377
Materials and methods Institutional human studies committee approval was obtained for this study. Both the DNA extraction and PCR amplification were performed and confirmed in duplicate. Tissue sample acquisition and DNA extraction. Pathology accession numbers of children who underwent lung transplant for primary pulmonary hypertension obtained from pulmonologists at SLCH. Pathology accession numbers of age, race, and sex matched controls that underwent lung transplants for pulmonary hypertension secondary to other causes were also obtained. Paraffin embedded lung blocks of both groups obtained from pathologists. Ten micrometer shavings of the paraffin blocks with the embedded tissues were obtained. The paraffin embedded lung tissues were deparafinized using xylene and 100% ethanol. DNA extracted from the lungs by using Qiagen Blood Mini DNA Kit. PCR performed to amplify the isolated DNA. Polymerase chain reaction amplification of the 5HTT linked polymorphism. DNA isolated from 11 controls and 10 experimental subjects. Oligonucleotide primers flanking the 5HTT polymorphic region were used to generate 484/528-bp fragments as previously described [11,12]. PCR amplification was initially carried out in 30 ll reactions using 3 ll klentaq buffer, 1.2 ll of 25 mM magnesium chloride, 9.75 ll betaine, 1 ll of 10 mM deoxyribosenucleotides, and sense and antisense primers and klentaq polymerase enzyme. The cycling conditions for this primary amplification were: 95 °C for 1 min, 95 °C for 30 s, 62 °C for 1 min, and 68 °C for 1 min. Step 2 was repeated 30 times. The products were electrophoresed on 1.2% agarose gel with ethidium bromide and the gel was cut in the region of 484 and 528 bp regions after placing the gel on UV light. The gel with the amplified DNA was centrifuged using columns. Secondary amplification was performed using the primary PCR product in a 25 ll reaction. Two microliters of the template was used with betaine, klentaq buffer, nested forward and reverse primers and klentaq polymerase enzyme. The cycling conditions were: 95 °C for 30 s, 56 °C for 30 s, and 68 °C for 1 min. Step 2 was repeated 29 times. The product was electrophoresed on 3% Nusieve gel with ethidium bromide to obtain two fragments sized 487 bp (the long allele) and 442 bp (the short allele).
Results Patient characteristics Identity of the patients was known only to the pulmonologists so as to preserve patient anonymity. There were 11 samples in the experimental group and 10 in the control group, and as can be seen in Table 1 the study patients were well matched for age and sex, with a slight discrepancy in race. The age range in the control group was from 1.4 to 18.6 years with mean of 12.3 years, while the age range in the experimental group was from 1 to 18.8 years with a mean age of 12.4 years. In the experimental group, 100% of the subjects were Caucasian, while in the control group 80% of the subjects were Caucasian, 10% African American, and 10% Asians. The experimental group consisted of 63% female subjects and the control group had 60% female subjects. Cystic fibrosis caused pulmonary hypertension in 9 and infantile interstitial pneumonitis in 1 out of the 10 subjects in the control group. Idiopathic pulmonary hypertension was the diagnosis in all of the 11 subjects in the experimental group.
378
A. Vachharajani, S. Saunders / Biochemical and Biophysical Research Communications 334 (2005) 376–379
Table 1 Demographic distribution of study patients Controls
Experimental
N
10
11
Age (years) Range Mean
1.4–18.6 12.3
1.0–18.8 12.4
80% Caucasian 10% African American 10% Asian
100% Caucasian
Sex (%) Female Male
60 40
63 36
Diagnosis
Cystic fibrosis
Idiopathic pulmonary hypertension 11 cases
Race
9 cases Infantile interstitial pneumonitis One case
Polymorphisms As seen in Fig. 1, there was a markedly skewed distribution of the polymorphic alleles in patients with idiopathic pulmonary hypertension. Of the 11 patients in the experimental group, 9 were homozygous for the long allele, and 1 was heterozygous for the long and short form of the allele. In contrast, only 5 out of 10 patients in the control group were homozygous for the long allele, with the remaining five patients heterozygous for
Fig. 1. Bar chart showing the distribution of the long allelic (LL), short allelic (SS), and the heterozygous (LS) variant of the 5HTT gene. Ninety (90) percent of the patients in the experimental group have the long allele variant of the gene polymorphism, while fifty (50) percent of the controls have the long allele variant of the gene polymorphism.
the two alleles. Interestingly, none of the patients in either of the study populations were homozygous for the less transcriptionally active short allele.
Discussion There is good experimental data to support a significant role for 5HT in the molecular mechanisms underlying pulmonary hypertension. When pulmonary artery smooth muscle cells are exposed to hypoxia in vitro, a marked enhancement in the proliferative activity of these cells is seen in correlation with a rapid increase in 5HTT expression [14]. These in vitro observations also correlate well with in vivo studies in both mice and humans. In mice, an increase in 5HTT gene expression is seen in the remodeled pulmonary arteries from animals that have developed pulmonary hypertension in response to chronic hypoxia [7,8], while in humans, patients with pulmonary hypertension have been shown to have pulmonary artery smooth muscle cells with elevated levels of 5HTT expression, increased 5HT uptake, and enhanced proliferation in response to 5HT [10,11]. As evidence that 5HTT is actually a significant modulator of the evolution of pulmonary hypertension in vivo, animals bearing a targeted disruption of the 5HTT gene have been shown to develop less severe pulmonary hypertension in response to hypoxia when compared with genetically normal control animals, while those with forced over-expression display an enhanced severity of disease [8,9]. 5HTT expression in humans is genetically modulated by a polymorphism in the promoter region of the 5HTT gene affecting transcriptional activity. Both short and long allelic forms of this gene are distinguishable by PCR owing to a small deletion [12]. The short variant of the polymorphism reduces the transcriptional efficiency of the 5HTT gene promoter resulting in decreased 5HTT expression and function. Pulmonary artery smooth muscle cells from individuals with the long allele of the 5HTT have increased serotonin uptake capacity compared to the individuals with the short allele of the 5HTT, and therefore are susceptible to increased smooth muscle cell proliferation, which should put then at increased risk for pulmonary hypertension. This hypothesis is supported by the finding that the frequency of each of these alleles is skewed in adult patients with pulmonary hypertension [11]. Compared to controls, adult patients with pulmonary hypertension more frequently are homozygous for the long variant of 5HTT and, as expected, they show higher platelet uptake of serotonin, and higher levels of expression of 5HTT mRNA expression and immunoreactivity in lung tissue [11]. The current study looked at the frequency of 5HTT alleles in children with pulmonary hypertension. Although the sample size in the current study was small,
A. Vachharajani, S. Saunders / Biochemical and Biophysical Research Communications 334 (2005) 376–379
the finding that 90% of pediatric patients with idiopathic pulmonary hypertension were homozygous for the long allele of 5HTT suggests that 5HTT may play a significant role in the biology of pulmonary hypertension in children as well as adults. Clearly, however, other genetic or environmental factors must explain the earlier onset of disease in children who develop idiopathic pulmonary hypertension, when compared with the late onset in adults. This suggests that there are likely polygenetic risk factors for this disorder, and implies that other genes which might put patients at risk should be compared between pediatric and adult patients. Recent studies have implied that BMP receptor mutations, unlike in adults, are not a significant contributor to the pathogenesis of sporadic cases of primary pulmonary hypertension in children [13]. This suggests the need to search for other genes associated with early onset disease. Interestingly, none of the patients with pulmonary hypertension in our study were found to be homozygous for the short form of the 5HTT gene. This implies that the long form of 5HTT may be a risk factor for the onset of pulmonary hypertension in children due to secondary causes as well. Further studies with larger patient populations, normal children, and a more diverse range of etiologies of pulmonary hypertension will be required to address these issues.
[4]
[5]
[6]
[7]
[8]
[9]
Acknowledgments [10]
These studies were supported by NIH Grants DK56063 and HD39952 (S.S.) and March of Dimes Birth Defects Foundation Grant 6-FY99-441 (S.S.). We thank Dr. Stuart Sweet, MD for providing the pathology accession numbers of children who underwent lung transplant for primary pulmonary hypertension and Dr. Frances White, MD for providing paraffin embedded lung blocks.
[11]
[12]
References [13] [1] L.J. Rubin, Primary pulmonary hypertension, N. Engl. J. Med. 336 (1997) 111–117. [2] K.B. Lane, R.D. Machado, M.W. Pauciulo, J.R. Thomson, J.A. Phillips 3rd, J.E. Loyd, W.C. Nichols, R.C. Trembath, Heterozygous germline mutations in BMPR2, encoding a TGF-beta receptor, cause familial primary pulmonary hypertension. The International PPH Consortium, Nat. Genet. 26 (2000) 81–84. [3] R.D. Machado, M.W. Pauciulo, J.R. Thomson, K.B. Lane, N.V. Morgan, L. Wheeler, J.A. Phillips 3rd, J. Newman, D. Williams, N. Galie, A. Manes, K. McNeil, M. Yacoub, G. Mikhail, P.
[14]
379
Rogers, P. Corris, M. Humbert, D. Donnai, G. Martensson, L. Tranebjaerg, J.E. Loyd, R.C. Trembath, W.C. Nichols, BMPR2 haploinsufficiency as the inherited molecular mechanism for primary pulmonary hypertension, Am. J. Hum. Genet. 68 (2001) 92–102. J.R. Thomson, R.D. Machado, M.W. Pauciulo, N.V. Morgan, M. Humbert, G.C. Elliott, K. Ward, M. Yacoub, G. Mikhail, P. Rogers, J. Newman, L. Wheeler, T. Higenbottam, J.S. Gibbs, J. Egan, A. Crozier, A. Peacock, R. Allcock, P. Corris, J.E. Loyd, R.C. Trembath, W.C. Nichols, Sporadic primary pulmonary hypertension is associated with germline mutations of the gene encoding BMPR-II, a receptor member of the TGF-beta family, J. Med. Genet. 37 (2000) 741–745. N. Rudarakanchana, J.A. Flanagan, H. Chen, P.D. Upton, R. Machado, D. Patel, R.C. Trembath, N.W. Morrell, Functional analysis of bone morphogenetic protein type II receptor mutations underlying primary pulmonary hypertension, Hum. Mol. Genet. 11 (2002) 1517–1525. A. Nishihara, T. Watabe, T. Imamura, K. Miyazono, Functional heterogeneity of bone morphogenetic protein receptor-II mutants found in patients with primary pulmonary hypertension, Mol. Biol. Cell 13 (2002) 3055–3063. E. Marcos, S. Adnot, M.H. Pham, A. Nosjean, B. Raffestin, M. Hamon, S. Eddahibi, Serotonin transporter inhibitors protect against hypoxic pulmonary hypertension, Am. J. Respir. Crit. Care Med. 168 (2003) 487–493. S. Eddahibi, B. Raffestin, M. Hamon, S. Adnot, Is the serotonin transporter involved in the pathogenesis of pulmonary hypertension?, J. Lab. Clin. Med. 139 (2002) 194–201. M.R. MacLean, G.A. Deuchar, M.N. Hicks, I. Morecroft, S. Shen, J. Sheward, J. Colston, L. Loughlin, M. Nilsen, Y. Dempsie, A. Harmar, Overexpression of the 5-hydroxytryptamine transporter gene: effect on pulmonary hemodynamics and hypoxia-induced pulmonary hypertension, Circulation 109 (2004) 2150– 2155. S. Eddahibi, M. Humbert, E. Fadel, B. Raffestin, M. Darmon, F. Capron, G. Simonneau, P. Dartevelle, M. Hamon, S. Adnot, Serotonin transporter overexpression is responsible for pulmonary artery smooth muscle hyperplasia in primary pulmonary hypertension, J. Clin. Invest. 108 (2001) 1141–1150. E. Marcos, E. Fadel, O. Sanchez, M. Humbert, P. Dartevelle, G. Simonneau, M. Hamon, S. Adnot, S. Eddahibi, Serotonininduced smooth muscle hyperplasia in various forms of human pulmonary hypertension, Circ. Res. 94 (2004) 1263–1270. K.P. Lesch, D. Bengel, A. Heils, S.Z. Sabol, B.D. Greenberg, S. Petri, J. Benjamin, C.R. Muller, D.H. Hamer, D.L. Murphy, Association of anxiety-related traits with a polymorphism in the serotonin transporter gene regulatory region, Science 274 (1996) 1527–1531. E. Grunig, R. Koehler, G. Miltenberger-Miltenyi, R. Zimmermann, M. Gorenflo, D. Mereles, K. Arnold, B. Naust, H. Wilkens, A. Benz, A. von Hippel, H.E. Ulmer, W. Kubler, H.A. Katus, C.R. Bartram, D. Schranz, B. Janssen, Primary pulmonary hypertension in children may have a different genetic background than in adults, Pediatr. Res. 56 (2004) 571–578. S. Eddahibi, V. Fabre, C. Boni, M.P. Martres, B. Raffestin, M. Hamon, S. Adnot, Induction of serotonin transporter by hypoxia in pulmonary vascular smooth muscle cells. Relationship with the mitogenic action of serotonin, Circ. Res. 84 (1999) 329–336.
Allelic variation in the serotonin transporter (5HTT) gene contributes to idiopathic pulmonary hypertension in children Akshaya Vachharajani, Scott Saunders * Department of Pediatrics, Washington University School of Medicine, St. Louis ChildrenÕs Hospital, St. Louis, MO 63110, USA Received 3 June 2005 Available online 28 June 2005
Abstract Pulmonary hypertension is a potentially lethal condition, which affects adults and children alike. Genetic factors are implicated in the causation of primary pulmonary hypertension. We investigate the role of polymorphism in the 5HTT gene in the etiology of pulmonary hypertension in children aged 1–18.8 years. We have tested the hypothesis that the 5HTT gene does contribute to the pathogenesis of this disease in children by comparing the allelic frequencies of both the long and short variants between children with idiopathic pulmonary hypertension and pulmonary hypertension secondary to underlying pulmonary disease. We found that homozygosity for the long variant of 5HTT was highly associated with idiopathic pulmonary hypertension in children, suggesting perhaps a more important role for 5HTT gene function in the pathogenesis of early onset disease. Ó 2005 Elsevier Inc. All rights reserved. Keywords: Serotonin transporter; 5HTT; Pulmonary hypertension; Smooth muscle; Lung; Pulmonary arteries; BMP
Primary pulmonary hypertension is a progressive medical condition that can occur at any age, and has a prevalence of 1–2 per 1,000,000 individuals [1]. It can be classified broadly based on whether it is primary, and therefore not arising as a result of another underlying condition, or secondary when it is the results of an underlying disease process, usually involving the lungs or the heart. In the case of secondary pulmonary hypertension, a number of specific factors have been defined to contribute to the evolution of the disease. These include as examples, environmental factors such as perinatal asphyxia and meconium aspiration syndrome in the newborn. In addition, a number of anatomic factors have also been causally linked with the development of pulmonary hypertension including those resulting from developmental abnormalities as seen in association with the pulmonary hypoplasia of congenital diaphragmatic
*
Corresponding author. Fax: +1 314 286 2893. E-mail address: [email protected] (S. Saunders).
0006-291X/$ - see front matter Ó 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.06.107
hernia, and those secondary to other disease processes such as interstitial lung disease and emphysema. Recently, significant advances have occurred in the understandings of the genetic contribution to the development of pulmonary hypertension. In particular, a role for defective signaling by members of the TGF-b super family has been clearly associated with familial pulmonary hypertension. Haploinsufficient mutations in the BMPR2 receptor have been found to be the major inherited mechanism of familial primary pulmonary hypertension [2,3], as well as a significant contributor to sporadic cases of this disease [4]. While the molecular details are currently still being investigated, it is speculated that this phenotype results from a loss in the inhibition of smooth muscle cell proliferation in response to a dominant negative mutation of the receptor [5,6]. One important issue that has arisen is the consideration of the role of modifier genes in the evolution of primary pulmonary hypertension, in as much as only 10–20% of individuals with BMP receptor mutations develop phenotypic disease. Another significant genetic
A. Vachharajani, S. Saunders / Biochemical and Biophysical Research Communications 334 (2005) 376–379
contributor to primary pulmonary hypertension is related to the functions of the serotonin 5-hydroxy tryptamine transporter (5HTT) gene. Function of 5HTT has been closely associated with the smooth muscle hyperplasia seen in pulmonary hypertension. Specifically, increased expression of the 5HTT gene has been identified in the remodeling pulmonary arteries of animals developing pulmonary hypertension in response to chronic exposure to hypoxia, and the severity of this pulmonary hypertension can be modified by either the administration of 5HTT inhibitors [7,8], or by genetically modifying 5HTT gene expression [8,9]. Further supporting the functional role for 5HTT in the evolution of pulmonary hypertension in humans, its activity has been found to be increased in cultured pulmonary artery smooth muscle cells from human patients with this disease [10,11]. A genetic polymorphism is known to exist in the 5HTT gene that effects the transcript level of this gene in vivo [12]. This polymorphism is the result of a 40 base pair deletion that diagnostically results in both a short (484 bp) and a long (528 bp) fragment when this region is amplified by PCR [12]. The long allelic variant is associated with increased transcriptional activity of the 5HTT gene, which leads to increased 5HT levels and subsequent enhanced 5HT transport into pulmonary smooth muscle cells. This has been shown to result in increased smooth muscle cell proliferation in cell culture [11]. There has been a reported increased association of this particular polymorphism in adult patients with idiopathic primary pulmonary hypertension, presumably due to an increased capacity for the proliferation of smooth muscle cells in the pulmonary arteries in these individuals in vivo [11]. Given that, by definition, adult and childhood primary pulmonary hypertension have vastly different time courses of presentation we thought it reasonable to speculate that the underlying genetic predisposition might well be distinct between these two presentation of this disease. Indeed, in support of this hypothesis is the recently reported findings that in 13 children with primary pulmonary hypertension (age at diagnosis, 6 months to 13 years), no mutations in the BMPR2 gene could be found nor any linkage in fact to the BMP receptor loci [13]. In the current study, we have tested the hypothesis that the 5HTT gene does contribute to the pathogenesis of this disease in children by comparing the allelic frequencies of both the long and short variants between children with idiopathic pulmonary hypertension and pulmonary hypertension secondary to underlying pulmonary disease. We found that homozygosity for the long variant of 5HTT was highly associated with idiopathic pulmonary hypertension in children, suggesting perhaps a more important role for 5HTT gene function in the pathogenesis of early onset disease.
377
Materials and methods Institutional human studies committee approval was obtained for this study. Both the DNA extraction and PCR amplification were performed and confirmed in duplicate. Tissue sample acquisition and DNA extraction. Pathology accession numbers of children who underwent lung transplant for primary pulmonary hypertension obtained from pulmonologists at SLCH. Pathology accession numbers of age, race, and sex matched controls that underwent lung transplants for pulmonary hypertension secondary to other causes were also obtained. Paraffin embedded lung blocks of both groups obtained from pathologists. Ten micrometer shavings of the paraffin blocks with the embedded tissues were obtained. The paraffin embedded lung tissues were deparafinized using xylene and 100% ethanol. DNA extracted from the lungs by using Qiagen Blood Mini DNA Kit. PCR performed to amplify the isolated DNA. Polymerase chain reaction amplification of the 5HTT linked polymorphism. DNA isolated from 11 controls and 10 experimental subjects. Oligonucleotide primers flanking the 5HTT polymorphic region were used to generate 484/528-bp fragments as previously described [11,12]. PCR amplification was initially carried out in 30 ll reactions using 3 ll klentaq buffer, 1.2 ll of 25 mM magnesium chloride, 9.75 ll betaine, 1 ll of 10 mM deoxyribosenucleotides, and sense and antisense primers and klentaq polymerase enzyme. The cycling conditions for this primary amplification were: 95 °C for 1 min, 95 °C for 30 s, 62 °C for 1 min, and 68 °C for 1 min. Step 2 was repeated 30 times. The products were electrophoresed on 1.2% agarose gel with ethidium bromide and the gel was cut in the region of 484 and 528 bp regions after placing the gel on UV light. The gel with the amplified DNA was centrifuged using columns. Secondary amplification was performed using the primary PCR product in a 25 ll reaction. Two microliters of the template was used with betaine, klentaq buffer, nested forward and reverse primers and klentaq polymerase enzyme. The cycling conditions were: 95 °C for 30 s, 56 °C for 30 s, and 68 °C for 1 min. Step 2 was repeated 29 times. The product was electrophoresed on 3% Nusieve gel with ethidium bromide to obtain two fragments sized 487 bp (the long allele) and 442 bp (the short allele).
Results Patient characteristics Identity of the patients was known only to the pulmonologists so as to preserve patient anonymity. There were 11 samples in the experimental group and 10 in the control group, and as can be seen in Table 1 the study patients were well matched for age and sex, with a slight discrepancy in race. The age range in the control group was from 1.4 to 18.6 years with mean of 12.3 years, while the age range in the experimental group was from 1 to 18.8 years with a mean age of 12.4 years. In the experimental group, 100% of the subjects were Caucasian, while in the control group 80% of the subjects were Caucasian, 10% African American, and 10% Asians. The experimental group consisted of 63% female subjects and the control group had 60% female subjects. Cystic fibrosis caused pulmonary hypertension in 9 and infantile interstitial pneumonitis in 1 out of the 10 subjects in the control group. Idiopathic pulmonary hypertension was the diagnosis in all of the 11 subjects in the experimental group.
378
A. Vachharajani, S. Saunders / Biochemical and Biophysical Research Communications 334 (2005) 376–379
Table 1 Demographic distribution of study patients Controls
Experimental
N
10
11
Age (years) Range Mean
1.4–18.6 12.3
1.0–18.8 12.4
80% Caucasian 10% African American 10% Asian
100% Caucasian
Sex (%) Female Male
60 40
63 36
Diagnosis
Cystic fibrosis
Idiopathic pulmonary hypertension 11 cases
Race
9 cases Infantile interstitial pneumonitis One case
Polymorphisms As seen in Fig. 1, there was a markedly skewed distribution of the polymorphic alleles in patients with idiopathic pulmonary hypertension. Of the 11 patients in the experimental group, 9 were homozygous for the long allele, and 1 was heterozygous for the long and short form of the allele. In contrast, only 5 out of 10 patients in the control group were homozygous for the long allele, with the remaining five patients heterozygous for
Fig. 1. Bar chart showing the distribution of the long allelic (LL), short allelic (SS), and the heterozygous (LS) variant of the 5HTT gene. Ninety (90) percent of the patients in the experimental group have the long allele variant of the gene polymorphism, while fifty (50) percent of the controls have the long allele variant of the gene polymorphism.
the two alleles. Interestingly, none of the patients in either of the study populations were homozygous for the less transcriptionally active short allele.
Discussion There is good experimental data to support a significant role for 5HT in the molecular mechanisms underlying pulmonary hypertension. When pulmonary artery smooth muscle cells are exposed to hypoxia in vitro, a marked enhancement in the proliferative activity of these cells is seen in correlation with a rapid increase in 5HTT expression [14]. These in vitro observations also correlate well with in vivo studies in both mice and humans. In mice, an increase in 5HTT gene expression is seen in the remodeled pulmonary arteries from animals that have developed pulmonary hypertension in response to chronic hypoxia [7,8], while in humans, patients with pulmonary hypertension have been shown to have pulmonary artery smooth muscle cells with elevated levels of 5HTT expression, increased 5HT uptake, and enhanced proliferation in response to 5HT [10,11]. As evidence that 5HTT is actually a significant modulator of the evolution of pulmonary hypertension in vivo, animals bearing a targeted disruption of the 5HTT gene have been shown to develop less severe pulmonary hypertension in response to hypoxia when compared with genetically normal control animals, while those with forced over-expression display an enhanced severity of disease [8,9]. 5HTT expression in humans is genetically modulated by a polymorphism in the promoter region of the 5HTT gene affecting transcriptional activity. Both short and long allelic forms of this gene are distinguishable by PCR owing to a small deletion [12]. The short variant of the polymorphism reduces the transcriptional efficiency of the 5HTT gene promoter resulting in decreased 5HTT expression and function. Pulmonary artery smooth muscle cells from individuals with the long allele of the 5HTT have increased serotonin uptake capacity compared to the individuals with the short allele of the 5HTT, and therefore are susceptible to increased smooth muscle cell proliferation, which should put then at increased risk for pulmonary hypertension. This hypothesis is supported by the finding that the frequency of each of these alleles is skewed in adult patients with pulmonary hypertension [11]. Compared to controls, adult patients with pulmonary hypertension more frequently are homozygous for the long variant of 5HTT and, as expected, they show higher platelet uptake of serotonin, and higher levels of expression of 5HTT mRNA expression and immunoreactivity in lung tissue [11]. The current study looked at the frequency of 5HTT alleles in children with pulmonary hypertension. Although the sample size in the current study was small,
A. Vachharajani, S. Saunders / Biochemical and Biophysical Research Communications 334 (2005) 376–379
the finding that 90% of pediatric patients with idiopathic pulmonary hypertension were homozygous for the long allele of 5HTT suggests that 5HTT may play a significant role in the biology of pulmonary hypertension in children as well as adults. Clearly, however, other genetic or environmental factors must explain the earlier onset of disease in children who develop idiopathic pulmonary hypertension, when compared with the late onset in adults. This suggests that there are likely polygenetic risk factors for this disorder, and implies that other genes which might put patients at risk should be compared between pediatric and adult patients. Recent studies have implied that BMP receptor mutations, unlike in adults, are not a significant contributor to the pathogenesis of sporadic cases of primary pulmonary hypertension in children [13]. This suggests the need to search for other genes associated with early onset disease. Interestingly, none of the patients with pulmonary hypertension in our study were found to be homozygous for the short form of the 5HTT gene. This implies that the long form of 5HTT may be a risk factor for the onset of pulmonary hypertension in children due to secondary causes as well. Further studies with larger patient populations, normal children, and a more diverse range of etiologies of pulmonary hypertension will be required to address these issues.
[4]
[5]
[6]
[7]
[8]
[9]
Acknowledgments [10]
These studies were supported by NIH Grants DK56063 and HD39952 (S.S.) and March of Dimes Birth Defects Foundation Grant 6-FY99-441 (S.S.). We thank Dr. Stuart Sweet, MD for providing the pathology accession numbers of children who underwent lung transplant for primary pulmonary hypertension and Dr. Frances White, MD for providing paraffin embedded lung blocks.
[11]
[12]
References [13] [1] L.J. Rubin, Primary pulmonary hypertension, N. Engl. J. Med. 336 (1997) 111–117. [2] K.B. Lane, R.D. Machado, M.W. Pauciulo, J.R. Thomson, J.A. Phillips 3rd, J.E. Loyd, W.C. Nichols, R.C. Trembath, Heterozygous germline mutations in BMPR2, encoding a TGF-beta receptor, cause familial primary pulmonary hypertension. The International PPH Consortium, Nat. Genet. 26 (2000) 81–84. [3] R.D. Machado, M.W. Pauciulo, J.R. Thomson, K.B. Lane, N.V. Morgan, L. Wheeler, J.A. Phillips 3rd, J. Newman, D. Williams, N. Galie, A. Manes, K. McNeil, M. Yacoub, G. Mikhail, P.
[14]
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