Analysis of the steroidogenic acute regulatory protein (StAR) gene in Japanese patients with congenital lipoid adrenal hyperplasia
Analysis of the steroidogenic acute regulatory protein (StAR) gene in Japanese patients with congenital lipoid adrenal hyperplasiaJunNakae1, ToshihiroTajima1, TeruoSugawara2, FutoshiArakane2, KeiichiHanaki3, TomoyukiHotsubo4, NoboruIgarashi5, YutakaIgarashi6, TadashiIshii7, NaoyaKoda8, TakumaKondo9, HitoshiKohno10, YuichiNakagawa11, KatsuhikoTachibana12, YasuhiroTakeshima13, KohjiTsubouchi14, Jerome F.Strauss III2 and KenjiFujieda1,*
1Department of Pediatrics, Hokkaido University School of Medicine,Sapporo 060,Japan,2Department of Obstetrics and Gynecology, University of Pennsylvania,Philadelphia, PA 19104,USA,3Department of Pediatrics, Tottori University,Tottori,Japan,4Department of Pediatrics, Sapporo Medical College,Sapporo,Japan,5Department of Pediatrics, Kanazawa University,Kanazawa,Japan,6Igarashi Pediatric Clinic,Sendai,Japan,7Department of Pediatrics, Wakayama Medical College,Wakayama,Japan,8Department of Endocrinology and Metabolism, Saitama Children's Medical Center,Saitama,Japan,9Kondo Pediatric Clinic,Osaka,Japan,10Department of Endocrinology and Metabolism, Fukuoka Children's Hospital,Fukuoka,Japan,11Department of Pediatrics, Hamamatsu University School of Medicine,Hamamatsu,Japan,12Department of Endocrinology and Metabolism, Kanagawa Children's Medical Center,Kanagawa,Japan,13Department of Pediatrics, Kakogawa City Hospital,Kakogawa,Japan and14Department of Pediatrics, Gifu University,Gifu,Japan
Received November 21, 1996;Revised and Accepted January 28, 1997
Genomic DNA from 19 Japanese patients with congenital lipoid adrenal hyperplasia (lipoid CAH) representing 16 different families was examined to identify the genetic alterations of steroidogenic acute regulatory protein (StAR). Ten of 19 patients had a 46,XX karyotype and nine had a 46,XY karyotype. Six of the 46,XX patients have experienced spontaneous pubertal changes including breast development and irregular menstruation whereas none of the 46,XY subjects displayed pubertal changes. Eight different mutations were identified. Sixteen patients were either homozygotes or compound heterozygotes for the Q258X mutation. The seven other mutations identified were 189delG, 246insG, 564del13bp, 838delA, Q212X, A218V and M225T. The 189delG, 246insG, 546del13bp and Q212X mutants encode truncated proteins. COS-1 cells transfected with expression vectors encoding cDNAs for the mutant StAR proteins which affect the C-terminus, 838delA, A218V and Q258X, exhibited no steroidogenesis enhancing activity. However, the M225T mutant retained some steroidogenic activity. The patient with the M225T mutation had late onset of this disorder and some capacity to secrete testosterone in response to hCG. These findings suggest: (i) that the Q258X mutation can be used as a genetic marker for the screening of Japanese for lipoid CAH, (ii) that the C-terminus of StAR plays an important role in the protein's activity and (iii) that there are differences in the extent of functional impairment of the testis and ovaries in lipoid CAH.
Congenital lipoid adrenal hyperplasia (lipoid CAH) is an autosomal recessive disorder which is characterized by neonatal onset of severe adrenal insufficiency and male pseudohermaphroditism due to defects in the production of adrenal and testicular steroidal hormones(1 ). Lipoid CAH was previously thought to be caused by a defect in the cholesterol side-chain system consisting of the cholesterol side-chain cleavage enzyme (P450scc) and two electron transfer proteins. However, the P450scc gene sequences were reported to be normal and the protein normally expressed in two patients with lipoid CAH (2 ,3 ). In addition, adrenodoxin reductase and adrenodoxin are generic electron transport proteins for all mitochondrial P450s, and not just for those involved in steroidogenesis (4 ). Furthermore, northern blotting revealed the normal expression of adrenodoxin reductase and adrenodoxin mRNAs in patients with lipoid CAH (3 ). These data eliminated the hypothesis that mutations in the genes for P450scc, adrenodoxin reductase or adrenodoxin are responsible for lipoid CAH. Recently, mutations in the steroidogenic acute regulatory protein (StAR) gene were found to be responsible for lipoid CAH (5 ). The human StAR gene was mapped to chromosome 8p11.2, spanning 8 kb and consisting of seven exons interrupted by six introns (6 ). The human StAR cDNA cloned from a human adrenal cDNA library encodes a 285 amino acid protein with a mitochondrial import sequence, which is cleaved from the N-terminus after transport into mitochondria. The human StAR mRNA is expressed abundantly in the adrenal glands and gonads (7 ). The reduced synthesis of steroids in patients with lipoid CAH results from an inability to transfer cholesterol to the inner mitochondrial membrane where the cholesterol side-chain cleavage complex is located (5 ). Thus, affected individuals with lipoid CAH have a marked impairment in the synthesis of all steroids in these organs. However, the precise mechanism of action of the StAR protein is not yet known. The molecular analysis of the StAR gene in patients with lipoid CAH might provide insight into the structure and function of the StAR protein.
Summary of genetic analysis of StAR gene in Japanese patients with lipoid CAH
In the present study, we performed a molecular analysis of the StAR gene in 19 Japanese patients from 16 different families with lipoid CAH. We identified eight different mutations including nonsense, missense and frameshift mutations and performed a functional analysis on some of the mutant StARs using a transient transfection approach in COS-1 cells. The significance of the molecular analysis of the StAR gene in Japanese patients with lipoid CAH and the relationships between genetic alterations in the StAR gene and clinical features of lipoid CAH are discussed.
Eight different mutations including two nonsense, four frameshift and two missense mutations were identified in each of the exons except exon 3. Of these mutations, the Q258X mutation was the most frequent. Sixteen patients (81%) were homozygous or heterozygous for the Q258X mutation (Table1 ). Homozygous Q258X mutations were found in six patients, while 10 were heterozygous for the Q258X mutation and another mutation. These were 189delG, 246insG, 564del13bp, 838delA, Q212X, A218V and M225T (Table1 ). The 189delG mutation introduces a premature stop codon at codon 23. The 246insG mutation introduces a premature stop codon at codon 46. The 564del13bp mutation introduces a premature stop codon at codon 181. The 838delA mutation causes a frameshift which extends the open reading frame and introduces a stop codon at codon 320. Homozygous mutations of 246insG and compound heterozygous mutations of 246insG and A218V were also identified. A mutation in one allele in patient 14 with a 246insG mutation was not identified using our genetic analysis strategies which included sequence analysis of the exon-intron boundaries. When DNA was available from parents, we found that the parents were heterozygous for the mutations in the StAR gene carried by their respective children. This confirmed the Mendelian inheritance of the mutant alleles.
Co-transfection of the wild type StAR protein with the cholesterol side chain cleavage system in COS-1 cells resulted in an ~7-fold increase in pregnenolone production. The Q258X mutant protein was reported previously to be biologically inactive (5 ). In the present study, we re-tested the functional activity of the Q258X mutant protein. Co-transfection of expression vectors for mutant StAR proteins (A218V and Q258X) resulted in no enhancement of the production of pregnenolone (Table2 ). These results indicate that these mutant StAR proteins are inactive. The 838delA mutant is also inactive (8 ). However, the M225T StAR mutant displayed some steroidogenic activity (43% of wild type StAR protein) (Table2 ).
Functional studies of mutant StAR protein in COS-1 cells
Vector
Pregnenolone (ng/dish)
pSPORT
14 ± 6
pStAR
98 ± 7
pQ258X
12 ± 3
pA218V
14 ± 0.1
pM225T
43 ± 10
COS-1 cells were transfected with a plasmid expressing the human cholesterol side-chain cleavage enzyme as a fusion protein and the indicated plasmids. Pregnenolone secreted into the culture medium was assayed by radioimmunoassay. Values presented are means ± SE from three separate experiments.
Western blot analyses were carried out on the two C-terminal amino acid replacement mutants expressed in transfected COS-1 cells. The A218V and the M225T mutant protein had the same molecular weight as the wild-type StAR mature protein (Fig.1 ). The preprotein was detected in cells transfected with wild-type StAR and the M225T and A218V mutants, but the abundance of the M225T preprotein was consistently greater than that for wild-type StAR and the A218V mutant. These data suggest that the A218V and M225T mutant proteins can enter the mitochondria and be processed. Western analysis on the Q258X and the 838delA mutants have been reported previously (5 ,8 ). The Q258X mutant yields a major truncated protein that migrates between the preprotein and the mature protein. The 838delA mutant yields species migrating with a molecular weight larger than the preprotein and mature protein, reflecting the presence of the C-terminal extension. These patterns were confirmed in the present study (data not shown).
The present study revealed that at least eight different mutations of the StAR gene including seven novel mutations are responsible for lipoid CAH in Japanese. Of these mutations, the Q258X mutation was the most prevalent, representing 56% of the mutant alleles. Sixteen patients were either homozygous or heterozygous for the Q258X mutation; homozygous Q258X mutations were found in six patients and heterozygous Q258X mutations were found in 10 patients. The percentage of families carrying at least one Q258X allele in this study is 81% (13 of 16 families). The Q258X mutation has been reported in one Japanese and one Korean patient with lipoid CAH who live in the United States (5 ) and, recently, three other Japanese subjects (9 ). The Q258X mutation results in a truncated StAR protein which has no stimulatory effect on the conversion of cholesterol into pregnenolone in COS-1 cells transiently co-expressing the cholesterol side chain cleavage system (5 ).
We also identified another nonsense mutation (Q212X) in exon 5 and several frameshift mutations. The 189delG mutation, which is a deletion of one nucleotide (G) at codon 20 in exon 1, introduces a premature stop codon at codon 23. The 246insG mutation, which is an insertion of one nucleotide (G) at codon 41 in exon 2, produces a premature stop codon at position 46. The 564del13bp mutation, which is a deletion of 13 nucleotides from codon 146 to 150 in exon 4, introduces a premature stop codon at position 181. These frameshift mutations and the Q212X nonsense mutation all introduce a premature stop codon resulting in the production of a truncated StAR protein.
The 838delA mutation, which is a deletion of one nucleotide (A) at codon 238 in exon 6, causes a frameshift that adds a novel extension to the StAR C-terminus. Transfection studies using COS-1 cells demonstrated that this mutant protein has no steroidogenesis enhancing activity. This indicates that amino acids beyond codon 238 encode important residues for StAR functional activity or possibly that the novel extension peptide inhibits StAR's steroidogenic activity.
The two missense mutations we identified, A218V and M225T, are both in exon 6. These mutations provide insight into the structure-function relationship of the StAR protein. The COS-1 cells transiently expressing these mutant StAR cDNAs and cholesterol side-chain cleavage system produced less pregnenolone from cholesterol than those expressing the wild-type StAR cDNA. Thus, these two missense mutations are responsible for lipoid CAH. The alanine at codon 218 and methionine at codon 225 are conserved in mouse, bovine and ovine StAR (10 ). Thus, these amino acids may play an important role in the biological action of the StAR protein. Notably, they are both located near the C-terminus. Western blot analysis indicated that these two mutant StAR proteins are processed normally. This confirms the expression and processing of inactive StAR proteins with C-terminal amino acid replacements (9 ). These data imply that the A218V and the M225T mutant proteins can enter into the mitochondria and be cleaved proteolytically. Therefore, these observations suggest that StAR's steroidogenic activity is not obligatorily linked to the mitochondrial importation of the protein. They direct focus to the protein's C-terminus rather than its N-terminal mitochondrial import sequence for insight into the key structural features of StAR. Indeed, our recently published work demonstrates that StAR retains steroidogenic activity in the absence of a functional mitochondria import sequence (9 ,11 ).
Clinical characteristics in Japanese patients with lipoid CAH
*CS, cortisol; 17-OHCS, urinary 17-hydroxycorticosteroid; 17-KS, urinary 17-ketosteroid.*1Units are mg/dl.NT, not tested; ND, not detected.Hyponatremia was defined as serum Na+ <130 mEq/l. Hyperkalemia was defined as serum K+ >6.0 mEq/l.
We could not identify a mutation in one allele of patient 14. Because we sequenced only exons and exon-intron boundaries, we would fail to detect mutations in regulatory regions including the promoter and internal intron sequences in these patients. We can conclude, however, that the majority of Japanese patients with lipoid CAH have mutations in exons of the StAR gene.
The manifestations of adrenal insufficiency in most patients with lipoid CAH appear during the neonatal or early infantile period. Interestingly, the age at onset in patient 7 was much later, at 10 months of age. Patient 7 is a compound heterozygote for the Q258X and M225T mutations. The Q258X mutation produces a completely inactive protein. However, transfection studies reveal that the M225T mutant retains some steroidogenic activity. It is noteworthy that this patient had clitomegaly at the time of diagnosis and a moderate elevation of serum testosterone in response to hCG. Thus, these clinical findings suggest that the mutant StAR protein (M225T), which retains some biological function, only partially impairs steroidogenesis in the adrenal and gonads. However, since this patient subsequently developed adrenocortical insufficiency, the partial activity of the M225T mutant is evidently not able to prevent the loss of glandular function that results from the massive accumulation of cholesterol in the steroidogenic cells as a consequence of inefficient translocation of cholesterol to the inner mitochondrial membranes (9 ). This patient suggests the possibility that late onset lipoid CAH results from mutations that do not completely inactivate StAR.
Furthermore, the pubertal aged patients with a 46,XX karyotype that we studied spontaneously developed secondary sexual characteristics and menstruation with detectable serum estradiol. These findings suggest that there exists StAR-independent pathways for ovarian steroidogenesis. Therefore, there is heterogeneity in the degree to which gonadal steroidogenesis is functionally impaired in lipoid CAH.
In conclusion, various mutations in the StAR gene are responsible for lipoid CAH in Japanese patients and genetic alterations in the StAR gene are the major cause of lipoid CAH. The Q258X mutation could be used as a genetic marker for screening of Japanese for lipoid CAH because of the high frequency of this allele. Our molecular analysis of the StAR gene in Japanese patients with lipoid CAH confirms the importance of the C-terminus of the StAR protein for steroidogenesis enhancing activity. Knowledge of these additional mutations provides a basis to study the domains involved in the transport of cholesterol into the inner mitochondrial membrane. Finally, our analysis reveals the heterogeneous pattern of gonadal steroidogenesis impairment in lipoid CAH.
Clinical characteristics of the patients in this study are detailed in Tables3 and4 . Nineteen Japanese patients with lipoid CAH (13 unrelated patients and three pairs of siblings) were studied. Ten patients had a 46,XX karyotype and nine had a 46,XY karyotype. No consanguineous marriages were noted. The age of onset of adrenal insufficiency was during the neonatal and early infantile period in all subjects except patient 7 whose diagnosis of adrenal insufficiency was at 10 months of age. All patients had generalized pigmentation at the time of diagnosis, elevated levels of ACTH and plasma renin activity (PRA), decreased levels of cortisol, urinary 17-hydroxycorticosteroids and urinary 17-ketosteroids. None of the patients with a 46,XY karyotype manifested any pubertal changes despite their chronological age. In contrast, six pubertal aged patients (patient 1-1, 1-2, 6-2, 9, 10 and 16) of the 10 patients with a 46,XX karyotype manifested secondary sexual characteristics with the development of breast tissue, pubic hair and irregular menstruation. Their serum estradiol levels ranged from 10 to 30 pg/ml. The clinical findings of patients 1-1 and 9 are described in detail elsewhere (8 ).
*T, testosterone; E2, estradiol; NT, not tested; ND, not detected.Normal ranges of basal serum LH and FSH in male are 0.02-0.15 and 0.38-1.11 mIU/ml in the prepubertal stage (<10 years of age), 0.04-0.25 and 0.13-0.91 mIU/ml in the prepubertal stage (>10 years of age), 0.44-1.63 and 1.73-4.27 mIU/ml in the pubertal stage (Tanner II-IIIo), and 1.64-3.53 and 4.21-8.22 mIU/ml in the pubertal stage (Tanner IVo). Normal ranges of serum LH and FSH in female are 0.01-0.09 and 0.54-2.47 mIU/ml in the prepubertal stage (<10 years of age), 0.02-0.11 and 1.16-3.65 mIU/ml in the prepubertal stage (>10 years of age), and 0.05-2.44 and 1.49-5.95 mIU/ml in the pubertal stage (Tanner II-IIIo). Serum level of testosterone at basal and stimulated states in response to hCG in male (Tanner Io) are 18 ±4 and 173 ± 27, respectively. Normal range of serum estradiol in the pubertal female is 20-60 pg/ml.
Genomic DNA was isolated from peripheral blood leukocytes using standard procedures (12 ). Each exon of the StAR gene was amplified using exon-specific primer pairs. The following primers were used: Ex1S and Ex1AS for exon 1 (9 ); PE2S 5'-AAC AAG GGT TAT TCC CTT CT-3' (from -24 to -5) and PE2AS 5'-GAG CCC AGA AGC CTC AGC ACT-3' (from +24 to +4) for exon 2; PE3S 5'-TCT CCT CGG CTG TGT ATC CA-3' (from -21 to -2) and PE3AS 5'-AGG CTT CTC CCC GAC ACT TA-3' (from +21 to +2) for exon 3; PE4S 5'-TCT GGG GGC TCC TTT CTC TGA-3' (from -24 to -4) and PE4AS 5'-GCA CCT GGA CTT TGC TCA CC-3' (from +19 to 0) for exon 4; S3 and AS3 for exon 5 (5 ); PE6S 5'-GAC TTG ACT TGC TCC ATT TGC CAG-3' (from -24 to -1) and PE6AS 5'-AGG TCC CCC TCC CAT GCC CTT CAC-3' (from +24 to +1) for exon 6; and S4 (5 ) and PE7AS 5'-ATG AGC GTG TGT ACC AGT GCA G-3' (nt 1037-1016). All nucleotide numbers except PE7AS indicate the base pairs from the beginning or the end of each exon. Minus numbers indicate the 5' site and plus numbers indicate the 3' site of each exon. The numbers for PE7AS are based on the numbering of the StAR cDNA (GenBank accession number U17280). PCR-amplification involved an initial period of denaturation for 10 min at 94oC, followed by 30 cycles consisting of denaturation at 94oC for 45 s, annealing at 64oC for 45 s and extension at 72oC for 120 s for exon 1; denaturation at 94oC for 45 s, annealing at 60oC for 45 s and extension at 72oC for 120 s for exon 2; denaturation at 94oC for 60 s, annealing at 62oC for 90 s and extension at 72oC for 90 s for exons 3, 4, 5 and 7; denaturation at 94oC for 45 s, annealing at 64oC for 30 s and extension at 72oC for 90 s for exon 6 and a final period of extension at 72oC for 10 min. After electrophoresis on 2% Nusieve agarose gel, each PCR-product was purified and sequenced directly using exon-specific primers described above by an autosequencer (Applied Biosystems Model 373A). To confirm the mutations identified, we performed direct-sequencing of PCR-products from both strands on two separate occasions for each mutated position and also on the parent's genomic DNA in all cases except for patients 4, 5-1, 5-2, 7, 8 and 12.
Transient expression of wild type or mutant StAR cDNAs in COS-1 monkey kidney cells was performed as described elsewhere (5 ,7 ). In brief, COS-1 cells were transfected with expression vectors containing mutant StAR cDNAs (A218V, M225T or Q258X) with Lipofectamine (GIBCO/BRL). The vectors included pSV-SPORT-1 without cDNA insert or pSV-SPORT-1 with the wild type 1.6 kb StAR cDNA (pStAR) or various mutant StAR cDNAs and a pECE vector encoding a fusion protein, termed F2, that consists of the human cholesterol side chain cleavage system (H2N-P450scc-adrenodoxin reductase-adrenodoxin-COOH) (13 ). F2 was kindly provided by Dr Walter L. Miller (University of California, San Francisco). Forty-eight hours after transfection, medium was collected for radioimmunoassay of pregnenolone as described (5 ). Values presented are means ± SE from three separate experiments.
Western blot analyses on mitochondrial fractions isolated from transfected COS-1 cells were carried out as previously described using an anti-peptide antibody kindly provided by Dr Douglas M. Stocco (Texas Technical University) (5 ) and a polyclonal antibody raised against recombinant human StAR which will be described elsewhere.
The authors thank Dr Walter L. Miller (University of California, San Francisco) for providing the human cholesterol side-chain cleavage enzyme construct, Dr Douglas Stocco (Texas Tech University) for providing anti-StAR antibody, and Dr Charles Strott of the National Institutes of Health for providing the antiserum to pregnenolone used in these studies. These studies were supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan and a grant from the Investigation Committee on Abnormalities in Steroidogenesis from the Ministry of Health and Welfare of Japan and USPHS grant HD 06274 and a grant from the Lalor Foundation.
1 Prader, A. and Gurtner, H.P. (1955) Das Syndrom des Pseudoherphroditismus masculinus bei kongenitaler Nebennierenrindenhyperplasie ohne Androgenuberproduktion (adrenaler Pseudohermaphroditismus masculinus). Helv. Paed. Acta., 10, 397-412.
2 Matteson, K.J., Chung, B., Urdea, M.S. and Miller, W.L. (1986) Study of cholesterol side chain cleavage (20, 22 desmolase) deficiency causing congenital lipoid adrenal hyperplasia using bovine-sequence P450scc oligodeoxyribonucleotide probes. Endocrinology., 118, 1296-1305.
3 Lin, D., Gitelman, S.E., Saenger, P. and Miller, W.L. (1991) Normal genes for the cholesterol side chain cleavage enzyme, P450scc, in congenital lipoid adrenal hyperplasia. J. Clin. Invest., 88, 1955-1962.MEDLINE Abstract
5 Lin, D., Sugawara, T., Strauss III, J.F., Clark, B.J., Stocco, D.M., Saenger, P., Rogol, A. and Miller, W.L. (1995) Role of steroidogenic acute regulatory protein in adrenal and gonadal steroidogenesis. Science., 267, 1828-1831.MEDLINE Abstract
6 Sugawara, T., Lin, D.L., Holt, J.A., Martin, K.O., Javitt, N.B., Miller, W.L. and Strauss III, J.F. (1995) Structure of the human steroidogenic acute regulatory protein (StAR) gene: StAR stimulates mitochondrial cholesterol 27-hydroxylase activity. Biochemistry, 34, 12506-12512. MEDLINE Abstract
7 Sugawara, T., Holt, J.A., Driscoll, D., Strauss III, J.F., Lin, D., Miller, W.L., Patterson, D., Clancy, K.P., Hart, I.M., Clark, B.J. and Stocco, D.M. (1995) Human steroidogenic acute regulatory protein: functional activity in COS-1 cell, tissue-specific expression, and mapping of the structual gene to 8p11.2 and a pseudogene to chromosome 13. Proc. Natl. Acad. Sci. USA, 92, 4778-4782. MEDLINE Abstract
8 Fujieda, K., Tajima, T., Nakae, J., Sageshima, S., Tachibana, K., Suwa, S., Sugawara, T. and Strauss III, J. F. (1997) Spontaneous puberty in two 46,XX patients with congenital lipoid adrenal hyperplasia: ovarian steroidogenesis is spared to some extent despite inactivating mutations in the steroidogenic acute regulatory protein (StAR) gene. J. Clin. Invest.,in press.
9 Bose, H.S., Sugawara, T., Strauss III, J. F. and Miller, W. L. (1996) The pathophysiology and genetics of congenital lipoid adrenal hyperplasia. N. Engl. J. Med., 335, 1870-1878.MEDLINE Abstract
10 Stocco, D.M. and Clark, B.J. (1996) Regulation of the acute production of steroids in steroidogenic cells. Endocrinol. Rev., 17, 221-244.
11 Arakane, F., Sugawara, T., Nishino, H., Liu Z., Holt, J. A., Pain, D., Stocco, D. M., Miller, W. L. and Strauss III, J. F. (1996) Steroidogenic acute regulatory protein (StAR) retains activity in the absence of its mitochondrial import sequence: Implications for the mechanism of StAR action. Proc. Natl. Acad. Sci. USA, 93, 13731-13736.MEDLINE Abstract
12 Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
13 Harilrishna, J.A., Black, S.M., Szklarz, G.D. and Miller, W.L. (1993) Construction and function of fusion enzymes of the human cytochrome P450scc system. DNA Cell Biol., 12, 371-379.
*To whom correspondence should be addressed
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