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Original Research: Pulmonary Vascular Disease |

EIF2AK4 Mutations in Patients Diagnosed With Pulmonary Arterial Hypertension FREE TO VIEW

D. Hunter Best, PhD; Kelli L. Sumner, BS; Benjamin P. Smith, MD; Kristy Damjanovich-Colmenares, BS; Ikue Nakayama, MD; Lynette M. Brown, MD, PhD; Youna Ha, BS; Eleri Paul, MLS (ASCP); Ashley Morris, MLS (ASCP); Mohamed A. Jama, MS, MB (ASCP); Mark W. Dodson, MD, PhD; Pinar Bayrak-Toydemir, MD, PhD; C. Gregory Elliott, MD, FCCP
Author and Funding Information

Dr Best and Ms Sumner contributed equally to this manuscript.

FUNDING/SUPPORT: The authors have reported to CHEST that no funding was received for this study.

aARUP Institute for Clinical and Experimental Pathology, ARUP Laboratories, Salt Lake City, UT

bDepartment of Pathology, University of Utah School of Medicine, Salt Lake City, UT

cDepartment of Pediatrics, University of Utah School of Medicine, Salt Lake City, UT

dDepartment of Medicine, Intermountain Medical Center, Murray, UT

eDepartment of Internal Medicine, University of Utah, Salt Lake City, UT

CORRESPONDENCE TO: C. Gregory Elliott, MD, FCCP, Department of Medicine, Intermountain Medical Center, 5121 S Cottonwood, #307, Murray, UT 84107


Copyright 2016, American College of Chest Physicians. All Rights Reserved.


Chest. 2017;151(4):821-828. doi:10.1016/j.chest.2016.11.014
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Background  Differentiating pulmonary venoocclusive disease (PVOD) and pulmonary capillary hemangiomatosis (PCH) from idiopathic pulmonary arterial hypertension (IPAH) or heritable pulmonary arterial hypertension (HPAH) is important clinically. Mutations in eukaryotic translation initiation factor 2 alpha kinase 4 (EIF2AK4) cause heritable PVOD and PCH, whereas mutations in other genes cause HPAH. The aim of this study was to describe the frequency of pathogenic EIF2AK4 mutations in patients diagnosed clinically with IPAH or HPAH.

Methods  Sanger sequencing and deletion/duplication analysis were performed to detect mutations in the bone morphogenetic protein receptor type II (BMPR2) gene in 81 patients diagnosed at 30 North American medical centers with IPAH (n = 72) or HPAH (n = 9). BMPR2 mutation-negative patients (n = 67) were sequenced for mutations in four other genes (ACVRL1, ENG, CAV1, and KCNK3) known to cause HPAH. Patients negative for mutations in all known PAH genes (n = 66) were then sequenced for mutations in EIF2AK4. We assessed the pathogenicity of EIF2AK4 mutations and reviewed clinical characteristics of patients with pathogenic EIF2AK4 mutations.

Results  Pathogenic BMPR2 mutations were identified in 8 of 72 (11.1%) patients with IPAH and 6 of 9 (66.7%) patients with HPAH. A novel homozygous EIF2AK4 mutation (c.257+4A>C) was identified in 1 of 9 (11.1%) patients diagnosed with HPAH. The novel EIF2AK4 mutation (c.257+4A>C) was homozygous in two sisters with severe pulmonary hypertension. None of the 72 patients with IPAH had biallelic EIF2AK4 mutations.

Conclusions  Pathogenic biallelic EIF2AK4 mutations are rarely identified in patients diagnosed with HPAH. Identification of pathogenic biallelic EIF2AK4 mutations can aid clinicians in differentiating HPAH from heritable PVOD or PCH.

Figures in this Article

Idiopathic pulmonary arterial hypertension (IPAH) and heritable pulmonary arterial hypertension (HPAH) are both serious, but treatable, disorders mainly characterized by obstruction of small pulmonary arteries. The diagnosis of IPAH requires pulmonary artery catheterization demonstrating a mean pulmonary artery pressure (PA mean) ≥ 25 mm Hg accompanied by a pulmonary artery occlusion pressure (PAOP) ≤ 15 mm Hg with a pulmonary vascular resistance (PVR) > 3 Wood units and a careful diagnostic evaluation to exclude other conditions associated with these hemodynamic abnormalities. The diagnosis of HPAH also requires a family history of pulmonary arterial hypertension (PAH) or detection of mutations that cause PAH.,

Pulmonary venoocclusive disease (PVOD) and pulmonary capillary hemangiomatosis (PCH) are rare diseases of the pulmonary circulation that can mimic IPAH or HPAH.,, Differentiating PVOD and PCH from IPAH and HPAH is important because administration of pulmonary vasodilator agents to patients with PVOD or PCH can cause life-threatening or fatal pulmonary edema., Although PVOD and PCH have unique clinical and imaging characteristics, microscopic examination of lung tissue is required to diagnose these disorders. Unfortunately, lung biopsy is not safe for most patients with PAH, PVOD, or PCH. As a result, PVOD and PCH may be misclassified as IPAH or HPAH.

The discoveries that biallelic mutations in the eukaryotic translation initiation factor 2 alpha kinase 4 (EIF2AK4) gene cause autosomal recessive PCH and PVOD may allow identification of PVOD or PCH among patients diagnosed with IPAH or HPAH. The aim of the present study was to describe the frequency of pathogenic biallelic EIF2AK4 mutations in patients diagnosed with IPAH or HPAH.

Patients

Eligible patients were identified at the Pulmonary Hypertension Center of Intermountain Medical Center, as well as at national meetings of the Pulmonary Hypertension Association. Between June 1994 and October 2008, we enrolled 91 patients who provided written informed consent for genetic studies according to protocols approved by the institutional review board of Intermountain Healthcare’s Central Region (institutional review board no. 1007044). These patients were diagnosed with IPAH (n = 82) or HPAH (n = 9) at 30 medical centers (e-Appendix) where the diagnosis of PAH was made by using pulmonary artery catheterization to confirm a PA mean ≥ 25 mm Hg, PAOP ≤ 15 mm Hg, and PVR > 3 Wood units. Additional clinical information (eg, history of exposure to anorexigens) and diagnostic testing (eg, lung perfusion scanning and/or pulmonary arteriography) excluded associated PAH and alternative diagnoses (eg, chronic thromboembolic pulmonary hypertension). Each case was reviewed independently by an investigator (C. G. E.) who confirmed the clinical diagnosis of IPAH or HPAH (although lung biopsies were not performed to exclude PVOD or PCH).

A family history was obtained from each patient and/or a knowledgeable family member. We specifically sought information or records to confirm that > 1 member of a family had been diagnosed with PAH. HPAH was diagnosed when the medical history and available medical records indicated that clinicians had diagnosed PAH in ≥ 2 members of the same family. We maintained contact with patients or family members whenever possible to identify and confirm new diagnoses of PAH.

Molecular Analysis

DNA was isolated from peripheral blood specimens. Sanger sequencing was performed for the entire coding region and intron/exon boundaries of the bone morphogenetic protein receptor type II (BMPR2) gene (RefSeq: NM_001204.6; exons 1-13, primer sequences available upon request) on all study subjects. BMPR2 mutation-negative patients subsequently underwent Sanger sequencing of the entire coding region and intron/exon boundaries of the ACVRL1 gene (RefSeq: NM_000020.2; exons 2-10, primer sequences available upon request), the ENG gene (RefSeq: NM_001114753.1; exons 1-14c, primer sequences available upon request), the CAV1 gene (RefSeq: NM_001753.4; exons 1-3, primer sequences available upon request), and the KCNK3 gene (RefSeq: NM_002246.2; exons 1-2, primer sequences available upon request). We screened for large deletions/duplications in BMPR2, ACVRL1, and ENG by multiplex-dependent probe amplification by using the HHT/PPH1 probe mix kit from MRC Holland in all subjects negative for BMPR2 pathogenic mutations according to Sanger sequencing. Multiplex-dependent probe amplification results were analyzed by using GeneMarker version 1.85 software (SoftGenetics, LLC).

Finally, Sanger sequencing was performed for the entire coding region of EIF2AK4 (RefSeq: NM_001013703.3; exons 1-39, primer sequences available upon request) for all subjects with no identified pathogenic mutations in BMPR2, ACVRL1, ENG, CAV1, or KCNK3. Patients with a homozygous EIF2AK4 gene mutation (and those who were heterozygous for a single EIF2AK4 variant) were screened for large deletions/duplications via the CytoScan HD SNP Array (Affymetrix).

The BigDye Terminator Cycle Sequencing Kit (Applied Biosystems, Life Technologies Corporation) and an Applied Biosystems 3730 DNA Analyzer were used for analyses.

Variants were classified on the basis of previous reports of pathogenic variants,,,,,, frequency in public databases, SIFT and PolyPhen scores, splicing predictions, and variant class.,, Previously reported mutations and truncating mutations were classified as pathogenic. Variants were classified as likely pathogenic when SIFT and PolyPhen were in agreement (deleterious, probably damaging), or in cases with splicing mutations where splice prediction programs showed a significant decrease in splicing efficiency. Variants were classified as uncertain when SIFT and PolyPhen were not in agreement. Variants with a frequency > 1% in public databases were excluded.

We sought and recorded clinical data from available medical records, and we reviewed source data (eg, lung perfusion scans, hemodynamic tracings, chest CT scans, lung histopathologic findings) when they were available.

Statistical Analysis

Demographic and clinical data of patients diagnosed with IPAH and HPAH are presented as mean ± SD. All analyses were conducted by using R Statistical Package version 3.2.4 (R Foundation for Statistical Computing).

Molecular studies were completed for 72 of 82 patients diagnosed with IPAH (10 patients were excluded because of poor DNA quality) and for nine of nine patients diagnosed with HPAH. The 72 patients with IPAH and nine patients with HPAH who underwent complete molecular studies were predominantly women with clinical and hemodynamic characteristics typical of these disorders (Table 1). The inheritance patterns of seven of the nine families were compatible with the inheritance patterns of HPAH (ie, autosomal dominant with incomplete penetrance) (Fig 1). Two family pedigrees (families 7 and 9) were compatible with either autosomal dominant with incomplete penetrance or autosomal recessive inheritance.

Table Graphic Jump Location
Table 1 Characteristics of Patients With IPAH and HPAH Who Underwent Successful Molecular Analyses

Data are expressed as mean ± SD or No. (%).

CO = cardiac output; CI = cardiac index; Dlco = diffusing capacity of the lung for carbon monoxide; HPAH = heritable pulmonary arterial hypertension; IPAH = idiopathic pulmonary arterial hypertension; PAP = pulmonary artery pressure; PCWP = pulmonary capillary wedge pressure; PVR = pulmonary vascular resistance; RAP = right atrial pressure; TLC = total lung capacity.

Figure 1
Figure Jump LinkFigure 1 Family pedigrees of the nine patients diagnosed with heritable pulmonary arterial hypertension (PAH) based on a family history of PAH. Seven pedigrees showed an autosomal dominant inheritance pattern characteristic of heritable PAH. Two pedigrees (families 7 and 9) were compatible, with either autosomal dominant with incomplete penetrance or autosomal recessive inheritance.Grahic Jump Location

Mutations are described and classified in Table 2 (BMPR2, ACVRL1, ENG, CAV1, or KCNK3) and in Table 3 (EIF2AK4). BMPR2 mutations were identified most often. Six of nine patients (66.7%) with HPAH had pathogenic BMPR2 mutations and eight of 72 (11.1%) patients with IPAH had pathogenic BMPR2 mutations. One patient diagnosed with IPAH carried a previously reported pathogenic ENG mutation. One patient with IPAH carried a likely pathogenic variation in ACVRL1, and one patient with IPAH carried a likely pathogenic KCNK3 mutation and a variant of uncertain clinical significance. None of the patients with HPAH or IPAH carried a pathogenic CAV1 variant.

Table Graphic Jump Location
Table 2 Pathogenic Variants, Likely Pathogenic Variants, and Variants of Uncertain Significance Identified in ACVRL1, BMPR2, CAV1, ENG, and KCNK3
a Human Genome Variation Society guidelines use ∗ to denote the introduction of a nonsense mutation at the designated amino acid.
b This study.

References for first characterization of the identified sequence variants.

FPAH = familial pulmonary arterial hypertension; NA = not applicable. See Table 1 legend for expansion of other abbreviations.

Table Graphic Jump Location
Table 3 Likely Pathogenic Variants and Variants of Uncertain Significance Identified in EIF2AK4
a Frequency data from the Exome Aggregation Consortium database (available at http://exac.broadinstitute.org/).
b Observed in a single individual.
c This silent mutation results from a change in the last nucleotide of the exon and is predicted to impact splicing by computational splicing prediction programs.

EIF2AK4 = eukaryotic translation initiation factor 2 alpha kinase 4.

One of three patients with HPAH without a pathogenic BMPR2 mutation carried a novel homozygous EIF2AK4 variant (c.257+4A>C) (Table 3). The c.257+4A>C intronic mutation is predicted to alter gene splicing and is therefore likely to result in a truncated or absent protein, and thus is likely pathogenic for PVOD or PCH. This patient had severe PAH (mean PA pressure, 43 mm Hg; PAOP, 6 mm Hg; PVR, 7.6 Wood units) without vasoreactivity or pulmonary edema during acute vasoreactivity testing and subtle centrilobular nodules on chest radiographs when she presented at 62 years of age. She had a 30 pack-year history of exposure to tobacco smoke. Spirometry results were normal; however, her diffusing capacity of the lung for carbon monoxide was 21% of predicted. Lung ventilation and perfusion scans were normal. High-resolution chest CT scanning and lung biopsy were not performed. An echocardiogram showed normal left ventricular function and no aortic or mitral valve abnormalities, although there were signs of severe pulmonary hypertension. PAH was diagnosed clinically. She refused treatment with epoprostenol and died at 64 years of age; no autopsy was performed. This patient had a sister who had been diagnosed 3 years earlier with IPAH (mean PA pressure, 52 mm Hg; PAOP, 9 mm Hg; PVR, 7.6 Wood units) and died at 62 years of age; no autopsy was performed. Sequencing of this sister’s DNA identified the same homozygous EIF2AK4 variant (c.257+4A>C); seven unaffected siblings were either heterozygotes for the c.257+4A>C EIF2AK4 variant or lacked the variant altogether (Fig 2).

Figure 2
Figure Jump LinkFigure 2 Family pedigree of two sisters diagnosed with heritable pulmonary arterial hypertension demonstrates segregation of severe pulmonary hypertension with the novel biallelic eukaryotic translation initiation factor 2 alpha kinase 4 mutation c.257+4A>C. The pedigree, with two of nine siblings affected by severe pulmonary hypertension, typifies inheritance of autosomal recessive pathogenic mutations.Grahic Jump Location

An additional two EIF2AK4 gene variants, one likely pathogenic and one of uncertain clinical significance, were identified in two unrelated patients with IPAH (Table 3). Both were heterozygous mutations. None occurred with a second pathogenic variant, and single nucleotide polymorphism array testing eliminated the possibility of a large deletion (undetected by Sanger sequencing) on the opposite allele in these two EIF2AK4 mutation-positive patients. Sequencing of EIF2AK4 in the patient cohort also identified several low-frequency variants that are likely benign (Table 4).

Table Graphic Jump Location
Table 4 Rare, Likely Benign Variants Identified in EIF2AK4
a Frequency data from the Exome Aggregation Consortium database (available at http://exac.broadinstitute.org/).

Variants classified as likely benign based on computational prediction and presence of variant homozygous individuals in larger population databases. See Table 3 for expansion of abbreviation.

To the best of our knowledge, we provide the first report of biallelic pathogenic EIF2AK4 mutations in patients diagnosed clinically with HPAH. We identified a novel pathogenic homozygous EIF2AK4 mutation (c.257+4A>C) in one of nine patients diagnosed with HPAH. The homozygous EIF2AK4 mutation was a previously unreported splice donor site mutation. This newly identified pathogenic mutation adds to the compilation of EIF2AK4 mutations predicted to cause PCH or PVOD. The index patient was a member of one of three families in our cohort diagnosed with HPAH for whom a pathogenic BMPR2 mutation was not identified by using Sanger sequencing and multiplex-dependent probe amplification. The inheritance pattern of pulmonary hypertension in the family suggested either autosomal recessive inheritance (which would be consistent with the inheritance pattern of PVOD or PCH due to EIF2AK4 mutations) or autosomal dominant inheritance with incomplete penetrance (which would be consistent with the inheritance pattern of PAH due to mutations in BMPR2, ACVRL1, ENG, CAV1, or KCNK3). In this family, two sisters with a homozygous EIF2AK4 gene mutation presented with PAH and severe right ventricular failure, and they died without undergoing a lung biopsy or having an autopsy performed. The presentation in this family highlights the potential utility of genetic testing for pathogenic biallelic EIF2AK4 mutations in establishing a precise diagnosis for patients who appear to have HPAH without gene mutations known to cause HPAH (eg, BMPR2). Identification of pathogenic homozygous or compound heterozygous EIF2AK4 mutations is important to establish the autosomal recessive mode of inheritance and to clarify which family members might benefit from genetic counseling and testing.

In contrast to our observations among patients diagnosed with HPAH, we found no pathogenic biallelic EIF2AK4 mutations in 72 patients diagnosed with IPAH. Pathogenic BMPR2 mutations were identified in eight of 72 patients diagnosed with IPAH, consistent with previous reports that approximately 10% to 20% of patients with IPAH harbor germ line mutations that are pathogenic for PAH. Previous investigations identified pathogenic EIF2AK4 mutations in 20% of patients diagnosed with sporadic PCH and 25% of patients diagnosed with sporadic PVOD., Our failure to detect pathogenic biallelic EIF2AK4 mutations in 72 patients with IPAH seems consistent with the low rate of pathogenic EIF2AK4 mutations in patients diagnosed with sporadic PVOD or PCH and with estimates that PVOD histopathology occurs in only 5% to 10% of patients initially suspected to have IPAH. Our data, combined with previous observations, suggest that tests for EIF2AK4 mutations are of limited value in detecting heritable PVOD or PCH among patients diagnosed with IPAH unless other features suggest PVOD or PCH.

Two patients diagnosed with IPAH were identified who were heterozygous carriers of EIF2AK4 gene variants. It is possible that heterozygous variants in EIF2AK4 are genetic modifiers that contribute to disease development in a subset of patients with IPAH. Additional studies are necessary to investigate this possibility.

Clinicians may have difficulty distinguishing PVOD and PCH from IPAH and HPAH because the history, physical examination, hemodynamics, lung perfusion scans, and echocardiographic findings are often similar at initial presentation. Differentiation of these disorders is important because of the poor response of PVOD and PCH to PAH-specific therapies and because of the risk of life-threatening pulmonary edema when pulmonary vasodilators are given to patients with PVOD or PCH., Diffusing capacity of the lung for carbon monoxide < 55% of predicted and centrilobular ground glass opacities, septal lines, pleural effusions, and mediastinal lymph node enlargement on high-resolution CT scans of the chest are characteristics of PVOD or PCH that aid in differential diagnosis., A definitive diagnosis formerly required microscopic examination of lung tissue. However, lung biopsies are associated with a high risk for morbidity and mortality in patients with severe pulmonary hypertension. Current guidelines state that identification of pathogenic homozygous or compound heterozygous EIF2AK4 mutations is sufficient to confirm a diagnosis of PVOD or PCH. The absence of EIF2AK4 mutations does not exclude PVOD or PCH.

We acknowledge limitations in the present study. First, it is possible that a correct diagnosis of PVOD or PCH would have been made if one of the affected family members had presented at a younger age with less advanced disease so that PVOD or PCH was suspected and a lung biopsy was performed. Unfortunately, both sisters presented late in life with advanced pulmonary hypertension and right ventricular failure, precluding lung biopsy. Furthermore, high-resolution CT scanning of the chest was scheduled but not performed on either of the sisters who had pathogenic homozygous EIF2AK4 mutations. It is possible that identification of septal lines, nodular ground glass opacities, or mediastinal lymphadenopathy on high-resolution CT scanning of the chest would have led physicians to suspect PVOD or PCH rather than PAH.

Pathogenic biallelic EIF2AK4 mutations may be identified in patients diagnosed with HPAH when pathogenic mutations associated with HPAH (eg, BMPR2 mutations) are not identified. Identification of pathogenic biallelic EIF2AK4 mutations can aid clinicians in differentiating HPAH from heritable PVOD or PCH and can facilitate genetic counseling and genetic testing of families affected by severe pulmonary hypertension.

Author contributions: C. G. E., D. H. B., and K. L. S. had full access to all data in the study and take responsibility for the data and the accuracy of the data analysis; D. H. B., K. L. S., M. W. D., and C. G. E. contributed to conception and design, acquisition of data, analysis and interpretation of data, drafting the submitted article, and revising it critically for important intellectual content; B. P. S., I. N., and P. B.-T. contributed to the acquisition of data, drafting the submitted article, and revising it critically for important intellectual content; K. D.-C., Y. H., E. P., A. M., and M. A. J. contributed to the acquisition of data; and L. M. B contributed to reviewing and revising the manuscript critically for important intellectual content.

Financial/nonfinancial disclosures: The authors have reported to CHEST the following: K. L. S. and E. P. are employed by ARUP Laboratories. L. M. B. and C. G. E. are employed by Intermountain Healthcare. Intermountain Healthcare may receive revenue related to licensing of genetic markers and has received grants from Actelion, Bayer, Bellerophon, and Lung Biotechnology. C. G. E. has served as a consultant for Actelion, Bayer, Ikaria and Bellerophon. None declared (D. H. B., B. P. S., K. D.-C., I. N., Y. H., A. M., M. A. J., M. W. D., P. B.-T.).

Other contributions: The authors thank the patients and their family members, as well as Jana Johnson, Ken Ward, MD, and Lesa Nelson. This work would not have succeeded without their assistance.

Additional information: The e-Appendix can be found in the Supplemental Materials section of the online article.

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Figures

Figure Jump LinkFigure 1 Family pedigrees of the nine patients diagnosed with heritable pulmonary arterial hypertension (PAH) based on a family history of PAH. Seven pedigrees showed an autosomal dominant inheritance pattern characteristic of heritable PAH. Two pedigrees (families 7 and 9) were compatible, with either autosomal dominant with incomplete penetrance or autosomal recessive inheritance.Grahic Jump Location
Figure Jump LinkFigure 2 Family pedigree of two sisters diagnosed with heritable pulmonary arterial hypertension demonstrates segregation of severe pulmonary hypertension with the novel biallelic eukaryotic translation initiation factor 2 alpha kinase 4 mutation c.257+4A>C. The pedigree, with two of nine siblings affected by severe pulmonary hypertension, typifies inheritance of autosomal recessive pathogenic mutations.Grahic Jump Location

Tables

Table Graphic Jump Location
Table 1 Characteristics of Patients With IPAH and HPAH Who Underwent Successful Molecular Analyses

Data are expressed as mean ± SD or No. (%).

CO = cardiac output; CI = cardiac index; Dlco = diffusing capacity of the lung for carbon monoxide; HPAH = heritable pulmonary arterial hypertension; IPAH = idiopathic pulmonary arterial hypertension; PAP = pulmonary artery pressure; PCWP = pulmonary capillary wedge pressure; PVR = pulmonary vascular resistance; RAP = right atrial pressure; TLC = total lung capacity.

Table Graphic Jump Location
Table 2 Pathogenic Variants, Likely Pathogenic Variants, and Variants of Uncertain Significance Identified in ACVRL1, BMPR2, CAV1, ENG, and KCNK3
a Human Genome Variation Society guidelines use ∗ to denote the introduction of a nonsense mutation at the designated amino acid.
b This study.

References for first characterization of the identified sequence variants.

FPAH = familial pulmonary arterial hypertension; NA = not applicable. See Table 1 legend for expansion of other abbreviations.

Table Graphic Jump Location
Table 3 Likely Pathogenic Variants and Variants of Uncertain Significance Identified in EIF2AK4
a Frequency data from the Exome Aggregation Consortium database (available at http://exac.broadinstitute.org/).
b Observed in a single individual.
c This silent mutation results from a change in the last nucleotide of the exon and is predicted to impact splicing by computational splicing prediction programs.

EIF2AK4 = eukaryotic translation initiation factor 2 alpha kinase 4.

Table Graphic Jump Location
Table 4 Rare, Likely Benign Variants Identified in EIF2AK4
a Frequency data from the Exome Aggregation Consortium database (available at http://exac.broadinstitute.org/).

Variants classified as likely benign based on computational prediction and presence of variant homozygous individuals in larger population databases. See Table 3 for expansion of abbreviation.

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