A study of the genetic etiology of nonsyndromic cleft lip and             palate            Triin Jagomägi       Departmen...
INTRODUCTION
Definitions• Cleft lip with or without cleft palate  (CL/P):     ICBDMS, International Clearinghouse for Birth Defect Moni...
Definitions• Cleft palate without cleft lip (CP):       ICBDMS, International Clearinghouse for Birth Defect Monitoring Sy...
• 660 children/ per day• 1 child per 2 minutes• 235,000 children per year• an additional 3,200 cleft children per year
Prevalence•Oral clefts (OC) 1:700 live births.•CL/P 9.92 per 10,000•CL 3.28 per 10,000•CLP 6.64 per 10,000•CP 6,2 per 10,0...
Surveillance system          OC in 2011• National statistics – non-existent  • PERH - 8 patients (4male, 4 female)    • 3 ...
Specific exposures• Cigarette smoking• Medication and drugs• Alcohol• Diet and vitamins• Solvents and pesticides
Gene discovery in     nonsyndromic clefts• The Online Mendelian Inheritance in  Man catalog (OMIM) - 720 syndromes• 75% CL...
Genetic approaches• Linkage analysis• Association studies• Identification of chromosomal anomalies  or microdeletions in c...
Candidate genes for oral clefts Gene         Evidence                        References                         Confirmed ...
Gene             Evidence                           References                                 LikelyABCA4 (locus only)   ...
Gene       Evidence                              References                     Intensively studied                       ...
Aims of the study
Aims of the study• To record the occurrence rate of OC, on the  basis of records of patients treated in the  Department of...
Aims of the study• To determine the rate of occurrence between  different cleft types on the basis of gender and  location.
Aims of the study• To record the epidemiological factors which  may influence the development of OC, and to  evaluate thei...
Aims of the study• To investigate the possible contribution of  recognized candidate genes in the  development of nonsyndr...
Study population
candidate genes
GENOTYPED    GENOTYPED    GENOTYPEDGENE      CHROMOSOME         SNPs         SNPs         SNPs                            ...
GENOTYPED    GENOTYPED    GENOTYPEDGENE     CHROMOSOME           SNPs         SNPs         SNPs                           ...
Genotyping• DNA extraction• High-salt extraction method (Estonian sample)• Phenol-chloroform method (Latvian,  Lithuanian ...
Statistical analysis• Descriptive statistics - SPSS ver. 14.0• PLINK ver. 1.06• Haploviev ver. 4.1• CaTS Power Calculator ...
results
Overview of estonian OC        patients
The most common cleft       types
Epidemiological factors• Birthweight  • 3416g (boys 3447g, girls 3376g)  • 1992 -1997, mean birthweight increased    from ...
Epidemiological factors• Specific exposures • medical abortions (45%), • psychological stress (36%), • chemical factors (3...
Genetic susceptibility loci        for CL/P     Estonian study       MSX1       MTHFR       PVRL2
Genetic susceptibility loci for CL/P                Baltic Study                 FGF1                 FOXE1               ...
Genetic susceptibility loci         for CP       Estonian study         JAG2         MMP         FGF1         MSX1        ...
Genetic susceptibility loci         for CP        Baltic study          IRF6          COL2A1          Col11A2          FGF...
IRF6• In Baltic sample we could not demonstrate  convincing evidence of an association between  CL/P and variants in IRF6 ...
Conclusion• Overview of OC• High occurrence rate of CP  • CL:CLP:CP        1:2:2• Overview of possible enironmental factor...
Concluding remarks• Surveillance system and official statistics• Multifactorial etiology• Genetic factors and environmenta...
Etiology of oral clefts in estonia
Etiology of oral clefts in estonia
Etiology of oral clefts in estonia
Etiology of oral clefts in estonia
Etiology of oral clefts in estonia
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Etiology of oral clefts in estonia

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  • Dear (vice dean of research)( or dear dean of medical faculty), dear council members of medical faculty; dear opponent; dear colleagues and friends.
  • Cleft lip with or without cleft palate palate is: a congenital malformation characterized by partial or complete clefting of the upper lip, with or without clefting of the alveolar ridge or the hard palate. “ Cleft of the lip arises by non-fusion of various processes that build up the face. Clefts may be unilateral, predominantly on the left side, or bilateral
  • Cleft palate is: a congenital malformation characterized by a closure defect of the hard and/or soft palate behind the foramen incisivum without cleft lip. Includes sub-mucous cleft palate. The cleft originates in the non-fusion of the maxillar palatal processes during the tenth week of embryonic development.
  • 660 children born every day with orofacial clefts Every two minutes, one child is born in the world with cleft 235,000 children with OCs born in a year The annual world population is growing by about 1.8 million; therefore, in the future an additional 3,200 cleft children are expected per year
  • Prevalence of Oral clefts is approximately 1 in every 700 live births; that is, with about the same frequency as Down syndrome, neural tube defects, polydactyly, and other so-called “common” congenital anomalies. There is a large geographical variation in the birth prevalence rate. Proportions for different cleft types for Caucasians are given by Fogh-Andersen.
  • CL/P prevalence in literature is 1:1000 or 9,92/10 000 in the word, so only some countries in Europe have so big CL/P prevalence. Mostly this is in Europe around 6-7 cases per 10 000. The highest reported prevalence rate for CP in the world is that of Finland 10-14 cases per 10 000. The prevalence for both OC main types, seems to depend largely on the same macro ethnicity, with maximum values among Mongols, lowest among Africans, and intermediate in Caucasians. For CL/P in Europe, higher prevalence rates are reported from northern than from southern countries
  • In American continent the case is different. Native Americans from south-America and Mexico have high incidence of oral clefts. This is more than 10 cases per 10 000 births. The highest reported prevalence rate in the world is that of Bolivia. Known data comes mainly from the city of La Paz, at 4000 meters above sea level, with a large proportion of its population being of Amerindian ethnic background. The role of both environmental factors, like chronic hypobaric hypoxia from altitude and Genetic factors like (Mongolic Amerindian ethnicity) their interactions are still unknown. Interestingly, a similarly high prevalence rate for CL/P seems to exist in the ethnic Mongolian population of Tibet at an almost equally high altitude.
  • National and official statistics about oral clefts in Estonia is nonexistent. Why there is no Estonian date available in world publications. Birth rate in Estonia was last year around 14 500 and we had 14 cleft cases. This makes 9,6 clefts per 10 000 newborns. CL/P mean value for the word is 9,92 but in Europe 6-7 cases per 10 000. Cleft ratio is different from Caucasians as we have more CP cases.
  • Nonsyndromic OC have multifactorial etiology, which involves both: environmental and genetic factors. Known specific exposures are: cigarette smoking; medications and drugs, alcohol; solvents and pesticide and in prevention vitamins and diet are important.
  • There are over 720 known syndromes featuring OC as cardinal symptoms. Most of orofacial clefts are belived to be nonsyndromic, with the rare syndromic cases. Evidence for genetic component has been obtained from studies of familial recurrence, which indicate that the relative risk for siblings is 30-40 times higher than average population risk. The lack of complete concordance in monozygotic twins illustrates the importance of environmental factors in the etiology of Cl/P.
  • To date, genetic approaches to non-syndromic CLP have included: Linkage analysis , using large, multiplex families or smaller but inbred families, or analysis of affected relative pairs; association studies, using case–parent trios or case–control samples; identification of chromosomal anomalies or microdeletions in cases; and direct sequencing of DnA samples from affected individuals. These methods can be applied to candidate genes or genome-wide strategies can be used. Each approach as its own advantages and disadvantages, some of which will depend on the underlying genetic architecture of the disease, as well as the realities of economics and technology. I briefly summarize successes using a range of those different approaches.
  • Recent successes in genome-wide linkage and association studies have identified novel loci that are significantly associated with CLP. Researchers are currently striving to identify the etiologic variants at these novel loci to understand the developmental disturbances leading to CLP. In this slide you can see the genes from Studies with the confirmed evidence, where at least two independent studies reaching conservative levels of significance.
  • Those are the genes that have likely the role in CLP. There are the genes where at least one study is with conservation/compelling data and other supportative studies is done.
  • Those are the genes that have intensively studied. There are a multiple studies but no consensus have found.
  • In our first study a total of 585 health files of patients with OCs had been preserver. There were more boys then girls. CP was almost as common as CLP.
  • Estonian study group for candidate genes included 153 patients : 100 patients with CL/P and 53 patients with CP. Estonian controls were selected from the Biobank of the Estonian Genome Center.
  • The Latvian subjects included 32 patients with CP and 108 patients with CL/P. The latvian control group consisted of 182 randomly selected individuals collected at the Latvian Biomedical research and study center   The Lithuanian subjects included 19 patients with CP and 92 patients with CL/P. The Lithuanian control group consisted of 219 individuals selected from six ethnolinguistic group of Lithuania with an equal male-to female ratio.
  • 18 candidate genes for study II and 40 candidate genes for study III and IV were selected on the basis of previously published findings : from association and linkage studies, from gene expression patterns during craniofacial development, from cleft phenotype in knockout or transgenic mouse models, from genes that underlie mendelian syndromic forms of clefting, and from studies of chromosomal rearrangements associated with orofacial cleft phenotypes in humans.
  • The selected genes are encoding for a variety of molecules implicated in craniofacial morphogenesis: transcription factors (IRF6, MSX1, TBX22), growth factors (FGF1, 2), polarizing signals (BMP2 and 4 or WNT genes), cell adhesion moleculs (PVRL1 and 2) and extracellular matrix moleculs (COL genes; MMP-s and TIMP2)
  • genomic DNA was extracted from peripheral blood lymphocytes. Genotyping in a Baltic sample was performed according to the principles of an arrayed primer extension-based genotyping method.
  • Descriptive statistics was performed using Statistical package for social sciences software. Statistical analyses were conducted using PLINK software which is a free, open-source whole genome association analysis toolset. LD measures were calculated and the haplotype blocks were defined using the confidence interval method, and haplotype frequencies were estimated with Haploview software. Power analysis was performed with Power Calculator for Two Stage Association Studies
  • Fogh-Andersen was the first to emphasize the proportions of occurrence of different cleft types in the Caucasians. The prevalence of CP varies significantly in Europe, not only between registers but also within countries. Our study find a high occurrence rate for CP, which is similar to the studies conducted in Finland and Sweden, but not so high as in Finland. The reason for this finding need further research. Different ethnic groups have different occurrence proportions for different clefts. From last year clefts, the same proportion was seen as I showed previously.
  • The most common cleft type was incomplete CP and the least frequent was BCL. The most common cleft type for boys was left side CLP and the most common for girls was CP. Left side of the face was affected 2,2 times more frequently then right side. No definite explanation for the difference in left and right side occurrences is given in literature. Johnston and Brown have suggested that blood vessels supplying the right side of the fetal head leave the aortic arch closer to the heart and may be better perfuse by blood than those on the left side. Men have CLP twice as often as female and CP is more common among women. There is no definite scientific explanation for the differences in clefts, between genders. One reason given is that the development of clefts occur at different stages of development in male and female fetuses in the critical stage, but there is no justification for this claim.
  • Many risk factors and mechanisms have been described in the literature for OC. Several studies have shown that the birth weight of children with clefts is similar to the birth weight of children without clefts, which was also confirmed by the present study. During the last 20 years, maternal and paternal ages have increased. Some malformations are clearly associated with older maternal age, but the effect of paternal old age is less certain. Increased maternal age is a risk factor for both chromosomal and non-chromosomal abnormalities.
  • There is little information regarding the temporal sequence between exposures and the outcome of the environmental risk factors and dose response relationship cannot be demonstrated. Not enough information is available to draw any conclusions about the role of these exposures and the risk of oral cleft formation. There is the list of different exposures, reported in patients case histories. As most of those patients were born during soviet time the contact of the mothers during or before pregnancy with toxic substantsis was marked We could not analyze that information because those groups were small compared with the all study. We only documented these findings.
  • Results from genome scans suggest that severe regions may contain genes predisposing to the development of NS clefts. We analyzed the role of 18 genes in the Estonian sample for a possible association with CL/P and 26 NPs in 9 genes showed nominal P-value less than 0.05. The most significant associations with CL/P were found for SNPs in MSX1, in methylentetrahydrofolate reductase and in poliovirus receptor-related 2 genes.
  • Concerning the Baltic study, the most significant associations were found for SNPs in the FGF1, FOXE1 and WNT9B genes. The strongest evidence of association was found for IIIII this SNP..... In the FGF1 gene. Additional evidence from haplotype analysis demonstrating the involvement of following gene variants in CL/P predisposition.
  • The strongest associations with nonsyndromic CP in Estonian study group was found for following genes....... Considering the small sample size of our patient group, which was 53 cases, we must emphasize that our study carries the risk of false-positive findings as a result of the large number of comparisons performed. Controversy we cannot exclude the possibility that a modest effect of polymorphisms or haplotypes in disease predisposition may become apparent in a large sample.
  • In Baltic sample, we genotyped 591 tag SNPs in 40 genes in 104 patients with CP. 35 SNPs in 17 genes showed nominal P value less than 0,05. IIIIIII The strongest evidence of association was found in IRF6 and in collagen type II alfa 1 genes. Additional evidence from haplotype analysis demostrated the possible involvement of those.... gene variants in predisposition to CP. We can assume that the described polymorphisms are not functionally significant variants that actually contribute to disease susceptibility, and our findings need confirmation by replication in other independent cohorts or by resequencing the selected candidate genes in patients to identify the causal variants.
  • IRF6 is one of the CL/P candidate genes with the most accordant results across studies. In Baltic sample we could not demonstrate convincing evidence of an association between CL/P and variants in IRF6 but., In the study from Baltic CP sample, the IRF6..showed evidence of association, which is a novel implication of IRF6 in nonsyndromic CP susceptibility IRF6 is essential for oral epithelial differentiation and plays a key role in the control of palatal adhesion and fusion.
  • With the study we documented the situation of Estonian OC, We found high occurrence of CP. We got the overview of possible environmental factors which may influence oral clefts in Estonia Our data provide additional confirmation for the role of MSX1 and MTHFR in the etiology of NS CL/P in Estonian sample. The results of this study provide further evidence that variations in FOXE1; TIMP2 and FGF and WNT signaling pathway genes are likely involved in the development of NS CL/P in North-Eastern European populations. Moreover, we have demonstrated that variations in cartilage collagen type II and 11 genes, IRF6 and FGF and Wnt signaling pathway genes are likely involved in the etiology of CP in Northeastern European populations.
  • Registration and classification of oral clefts is of paramount importance in providing a solid basis for epidemiologic, clinical and/or fundamental research. Official statistics of orla clefts in estonia does not exist. There is no doubt modern genetics have greatly influenced our professional and personal lives during the last decade. Uncovering genetic causes of many medical and dental pathologies is helping to narrow the diagnosis and select a treatment plan that would provide the best outcome. Importantly, having an understanding of multifactorial etiology helps direct our attention toward prevention. We now understand much better our own health problems. In some cases, we can modify our lifestyle and diet in order to prevent "environmental factors" from triggering the mutated genes inherited from our parents. One needs to understand genetic and environmental causes of nonsyndromic orofacial clefts in order to prevent them. With all this in mind,. It has been leading to a better understanding of etiology of nonsyndromic orofacial clefts. It has been learned that genetic factors and environmental factors are ethnicity-specific and, in many places throughout the world, location-specific. Thus, a specific protocol for cleft prevention has to be worked out based on genetic and environmental or lifestyle studies of each specific population group in order to be effective. This is our ultimate goal. And my work is just a small drop in big ocean and a first attempt to bring Estonian oral clefts into word map.
  • Etiology of oral clefts in estonia

    1. 1. A study of the genetic etiology of nonsyndromic cleft lip and palate Triin Jagomägi Department of Stomatology University of Tartu
    2. 2. INTRODUCTION
    3. 3. Definitions• Cleft lip with or without cleft palate (CL/P): ICBDMS, International Clearinghouse for Birth Defect Monitoring Systems
    4. 4. Definitions• Cleft palate without cleft lip (CP): ICBDMS, International Clearinghouse for Birth Defect Monitoring Systems
    5. 5. • 660 children/ per day• 1 child per 2 minutes• 235,000 children per year• an additional 3,200 cleft children per year
    6. 6. Prevalence•Oral clefts (OC) 1:700 live births.•CL/P 9.92 per 10,000•CL 3.28 per 10,000•CLP 6.64 per 10,000•CP 6,2 per 10,000•CL:CLP:CP for Caucasians 1:2:1International Perinatal Database of Typical Orofacial Clefts 2011
    7. 7. Surveillance system OC in 2011• National statistics – non-existent • PERH - 8 patients (4male, 4 female) • 3 CL; 4 CLP; 1CP • SA TÜK – 6 patients (2 male, 4 female) • 1CLP; 5 CP • 3CL:5CLP:6CP 1:2:2
    8. 8. Specific exposures• Cigarette smoking• Medication and drugs• Alcohol• Diet and vitamins• Solvents and pesticides
    9. 9. Gene discovery in nonsyndromic clefts• The Online Mendelian Inheritance in Man catalog (OMIM) - 720 syndromes• 75% CL/P and 50% CP are nonsyndromic• Multifactorial etiology • environmental • genetic factors
    10. 10. Genetic approaches• Linkage analysis• Association studies• Identification of chromosomal anomalies or microdeletions in cases• Direct sequencing of DNA samples
    11. 11. Candidate genes for oral clefts Gene Evidence References Confirmed Zucchero et al., 2004; Blanton et al., 2005; Ghassibe IRF6 GWA, LD, L, M et al., 2005; Scapoli et al., 2005; Srichomthong et al., 2005; Rahimov et al., 2008;8q24 locus GWA, LD Marazita et al., 2009; Birnbaum et al., 2009. VAX1 GWA, LD Beaty et al., 2010; Mangold et al., 2010.
    12. 12. Gene Evidence References LikelyABCA4 (locus only) GWA Beaty et al., 2010. Van der Boogaard et al., 2000; Maestri et al., 1997; Mitchell et MSX1 LD, M al., 2001; Romitti et al., 1999; Jezewski, 2003; Lidral et al., 1998; Vieira et al., 2003; Suzuki et al., 2004. FOXE1 L, LD, M Vieira et al., 2005b; Venza et al., 2006; Moreno et al., 2009a. FGFR2 M Riley et al., 2007; Riley and Murray, 2007; Osoegawa, 2008. BMP4 M Lin et al., 2008; Suzuki et al., 2009; Jianyan et al., 2010. 17q22 locus LD Beaty et al., 2010; Mangold et al., 2010. MAFB GWA Beaty et al., 2010. Birnbaum et al., 2009; Martinelli et al., 2007; Chiquet et al., MYH9 GWA 2009; Jia et al., 2010.
    13. 13. Gene Evidence References Intensively studied Martinelli et al., 2001; Blanton et al., 2000; Botto and Yang, 2000;MTHFR LD Jugessur et al., 2003b; Jagomägi et al., 2010. Marazita and Mooney, 2004; Mitchell, 1997; Hwang et al.,1995; Maestri TGF-α LD et al.,1997; Miettinen et al., 1989; Suzuki et al., 2004; Carter et al., 2010. Alkuraya et al., 2006; Shi et al., 2009; Mostowska et al., 2010; Carter et SUMO1 M al., 2010. PDGFC LD, M Ding et al., 2004; Choi et al., 2009; Jugessur et al., 2009. FGF8 M Riley and Murray, 2007; Riley et al., 2007. PVRL1 M, LD Avila et al., 2006; Sözen et al., 2001; Sözen et al., 2009. Miettinen et al.,1999; Maestri et al.,1997; Hwang, 1992; Lidral et TGF-β3 LD, M al.,1998; Suzuki et al., 2004; Beaty et al., 2002; Suazo et al., 2010.CRISPLD2 LD Chiquet et al., 2007; Letra et al., 2010 GSTT1 LD Shi et al., 2007
    14. 14. Aims of the study
    15. 15. Aims of the study• To record the occurrence rate of OC, on the basis of records of patients treated in the Department of Oral and Maxillofacial Surgery of the Tartu University Hospital, during the period of 1910–2000.
    16. 16. Aims of the study• To determine the rate of occurrence between different cleft types on the basis of gender and location.
    17. 17. Aims of the study• To record the epidemiological factors which may influence the development of OC, and to evaluate their occurrence regularities.
    18. 18. Aims of the study• To investigate the possible contribution of recognized candidate genes in the development of nonsyndromic OC in an Estonian population and in the Baltic region (Estonia, Latvia, Lithuania).
    19. 19. Study population
    20. 20. candidate genes
    21. 21. GENOTYPED GENOTYPED GENOTYPEDGENE CHROMOSOME SNPs SNPs SNPs Ref. II Ref. III Ref. IVMTHFR 1p36.3 11 9 8 LHX8 1p31.1 9 9 9COL11A1 1p21 42 42 SKI 1q22-q24 20 19 19 IRF6 1q32.3-q41 11 10 10 TGFA 2p13 36 35 FN1 2q34 27 27 MSX1 4p16.3-p16.1 15 15 15 FGF2 4q26-q27 18 18 FGF1 5q31 31 31 MSX2 5q34-q35 6 6 6 EDN1 6p24.1 15 15 15COL11A2 6p21.3 19 19 FGFR1 8p11.2-p11.1 11 11FOXE1 9q22 4 4 4 TBX10 11q13.2 10 10 10 MMP3 11q22.3 5 5MMP13 11q22.3 19 19PVRL1 11q23.3 18 17 17COL2A1 12q13.11 32 32
    22. 22. GENOTYPED GENOTYPED GENOTYPEDGENE CHROMOSOME SNPs SNPs SNPs Ref. II Ref. III Ref. IVSPRY2 13q31.1 3BMP4 14q22-q23 4 4TGF-β3 14q24 8 8 8 JAG2 14q32 11 11 11MMP25 16p13.3 6 6MMP2 16q13-q21 20 20CDH1 16q22.1 13 13RARA 17q21 5 4 4WNT3 17q21 16 16WNT9B 17q21 11 11TIMP2 17q25 25 25OFC11’ 18q21a 25 25 BCL3 19q13.1-q13.2 4 3 2PVRL2 19q13.2 13 12 12CLPTM1 19q13.2-q13.3b 8 7 7BMP2 20p12 25 25MMP9 20q11.2-q13.1 6 5TIMP3 22q12.3 36 36TBX22 Xq21.1 5 5 5
    23. 23. Genotyping• DNA extraction• High-salt extraction method (Estonian sample)• Phenol-chloroform method (Latvian, Lithuanian sample)• SNP genotyping: APEX-2
    24. 24. Statistical analysis• Descriptive statistics - SPSS ver. 14.0• PLINK ver. 1.06• Haploviev ver. 4.1• CaTS Power Calculator www.sph.umich.edu/csg/abecasis/CaTS/
    25. 25. results
    26. 26. Overview of estonian OC patients
    27. 27. The most common cleft types
    28. 28. Epidemiological factors• Birthweight • 3416g (boys 3447g, girls 3376g) • 1992 -1997, mean birthweight increased from 3465g to 3497g (Koupilova et al.2000)• Parent’s age • >30y. mothers 25%, fathers 33% • >40y. mothers 2.6%
    29. 29. Epidemiological factors• Specific exposures • medical abortions (45%), • psychological stress (36%), • chemical factors (34%), • teratogenic substances (22%) • hard physical work (13%), • physical trauma (6%),
    30. 30. Genetic susceptibility loci for CL/P Estonian study MSX1 MTHFR PVRL2
    31. 31. Genetic susceptibility loci for CL/P Baltic Study FGF1 FOXE1 WNT9B TIMP2 PVRL2 LHX8 MMP9
    32. 32. Genetic susceptibility loci for CP Estonian study JAG2 MMP FGF1 MSX1 LOXHD1
    33. 33. Genetic susceptibility loci for CP Baltic study IRF6 COL2A1 Col11A2 FGFR1 CLPTM1 WNT3
    34. 34. IRF6• In Baltic sample we could not demonstrate convincing evidence of an association between CL/P and variants in IRF6 • CL/P candidate gene • most accordant results • VWS - Van der Woude syndrome • Baltic CP sample IRF6 SNP rs17389541/novel implication
    35. 35. Conclusion• Overview of OC• High occurrence rate of CP • CL:CLP:CP 1:2:2• Overview of possible enironmental factors• Candidate genes • MSX1, MTHFR, OFC3, IRF6, COL2A1, COL11A2, WNT3, FGF1, TIMP2, WNT9B, FOXE1
    36. 36. Concluding remarks• Surveillance system and official statistics• Multifactorial etiology• Genetic factors and environmental factors are ethnicity-specific, location-specific• A specific protocol for cleft prevention has to be worked out based on: • genetic • environmental • lifestyle studies of each specific population group

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