Associations between the risk of tooth agenesis and single-nucleotide polymorphisms of MSX1 and PAX9 genes in nonsyndromic cleft patients

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1 Originl Article Associtions between the risk of tooth genesis nd single-nucleotide polymorphisms of MSX1 nd PAX9 genes in nonsyndromic cleft ptients Yu-Jin Seo ; Ji Wn Prk b ; Young Ho Kim c ; Seung-Hk Bek d ABSTRACT Objective: To investigte the ssocition between the risk of tooth genesis nd single-nucleotide polymorphisms (SNPs) of MSX1 nd PAX9 genes in nonsyndromic cleft ptients. Mterils nd Methods: The subjects were 126 Koren nonsyndromic cleft ptients. Tooth genesis type (TAT) ws clssified s none (0); cleft re (1); cleft re + other re (2); nd other re (3) bsed on genesis of the mxillry lterl incisor (MXLI) nd nother tooth within or outside the cleft re. TAT ws further grouped into two subctegories (0 nd 1) nd four subctegories (0, 1, 2, nd 3). Three SNPs of MSX1 nd 10 SNPs of PAX9 were investigted using Fisher s exct test nd logistic regression nlysis. Results: Although the ssocition between genotype distribution of PAX9-rs nd TAT ws significnt (P,.05 in four subctegories), genotypic odds rtios (GORs) of SNPs in ech TAT were not meningful. However, for MSX1-rs12532 nd PAX9-rs , ssocitions between genotypic distribution nd TAT were significnt (P,.01 in four subctegories nd P,.05 in two subctegories; P,.01 in two subctegories, respectively). In cleft re, GORs of MXLI genesis in genotypes GA of MSX1-rs12532 nd CT of PAX9-rs were incresed by 3.14-fold nd 4.15-fold compred with genotype GG of MSX1-rs12532 nd CC of PAX9-rs , respectively (P,. 01; P,.05). In cleft re + other re, the GOR of genesis of MXLI nd nother tooth in genotype AA of MSX1-rs12532 ws incresed by fivefold compred with genotype GG (P,.05). Conclusion: Genetic disturbnces of MSX1 nd PAX9 genes re ssocited with tooth genesis within nd outside the cleft re. (Angle Orthod. 2013;83: ) KEY WORDS: SNP; MSX1; PAX9; Tooth genesis; Nonsyndromic cleft; Assocition nlysis INTRODUCTION Grdute student (PhD), Deprtment of Orthodontics, School of Dentistry, Seoul Ntionl University, Fellow Doctor, Deprtment of Orthodontics, School of Dentistry, Kyung Hee University, Seoul, South Kore. b Associte Professor, Deprtment of Medicl Genetics, College of Medicine, Hllym University, Chuncheon, Gngwon Province, South Kore. c Associte Professor nd Chir, Deprtment of Orthodontics, The Institute of Orl Helth Science, Smsung Medicl Center, Sungkyunkwn University School of Medicine, Seoul, South Kore. d Professor, Deprtment of Orthodontics, School of Dentistry, Dentl Reserch Institute, Seoul Ntionl University, Seoul, South Kore. Corresponding uthor: Dr Seung-Hk Bek, Deprtment of Orthodontics, School of Dentistry, Dentl Reserch Institute, Seoul Ntionl University, Yeonkun-dong #28, Jongro-ku, Seoul, , South Kore (e-mil: drwhite@unitel.co.kr) Accepted: April Submitted: Februry Published Online: My 29, 2013 G 2013 by The EH Angle Eduction nd Reserch Foundtion, Inc. Becuse development of the orl cleft nd formtion of the tooth germ re closely relted in terms of timing nd ntomic position, dentl nomlies including tooth genesis both within nd outside the cleft re hve been reported to be more frequent in persons with nonsyndromic cleft lip with or without cleft plte (NS-CL 6 P) thn in individuls with nonsyndromic cleft lip with or without cleft plte (NS-CL 6P) thn in the non-cleft individuls. 1 6 Tooth genesis in cleft ptients ffects esthetics, function, nd periodontl helth; cuses collpse of the dentl rch; nd cretes psychosocil problems. Therefore, n interdisciplinry pproch is required to llow ptients to receive more effective nd efficient tretment. The MSX1 nd PAX9 genes re known to contribute to tooth genesis of the posterior teeth nd the mxillry lterl incisor The MSX1 genes with homeodomin nd the PAX9 genes with pired domin encode trnscription fctors tht re essentil for crniofcil nd dentl development of the mesenchyme Generlly, muttions in MSX1 nd PAX DOI: /

2 MSX1/PAX9 SNPS AND TOOTH AGENESIS IN CLEFT 1037 cuse loss of function becuse of hploinsufficiency nd reduce the mount of functionl protein vilble to mintin tooth development; this results in bnormlities in odontogenesis, such s rrest of the tooth bud To dte, only few studies hve ddressed the genetic bsis of orl cleft with or without tooth genesis in humns Vn den Boogrd et l. 20 nd Ling et l. 21 suggested tht tooth genesis nd orl cleft were ssocited with nonsense muttions of MSX1, such s Ser104stop in exon 1 in Dutch fmily nd Q189X in exon 2 in Chinese fmily, respectively. However, Ling et l. 21 lso reported tht sequence nlysis of PAX9 did not revel muttion in ny of the ffected individuls studied. Modesto et l. 22 investigted singlenucleotide polymorphisms (SNPs) in MSX1 of CL6P with or without tooth genesis compred with non-cleft individuls nd reported tht the 101C.G vrint occurred more frequently in ptients with both NS- CL6P nd tooth genesis, wheres the *6C.T vrint ws found more often in those with NS-CL6P. However, these studies hve severl limittions, such s smll smple size, inclusion of non-cleft individuls, or no clssifiction of tooth genesis within nd outside the cleft re. Given tht not ll ptients with NS-CL6P hve tooth genesis, it is necessry to investigte the genetic bsis of NS-CL6P with tooth genesis compred with NS-CL6P without tooth genesis. The MSX1 nd PAX9 genes re known to contribute to both cleft nd tooth genesis. 16,18,20 23 However, no study hs investigted whether tooth genesis within the cleft re is ssocited with isolted genetic disturbnce or locl tissue defect s prt of clefting. Therefore, the purpose of this study ws to investigte the ssocition between the risk of tooth genesis nd the SNPs of MSX1 nd PAX9 genes in Koren nonsyndromic cleft ptients. The null hypothesis ws tht there ws no significnt ssocition between genotypic distribution of SNPs in the MSX1 nd PAX9 genes nd tooth genesis type (TAT) in ptients with cleft lip nd lveolus (CLA) nd cleft lip nd plte (CLP). MATERIALS AND METHODS The study smples consisted of 126 Koren nonsyndromic cleft ptients (82 mles nd 44 femles; 28 ptients with CLA nd 98 ptients with CLP). Subjects with longitudinl seril records nd pnormic rdiogrphs were selected from the Deprtment of Orthodontics, Seoul Ntionl University Dentl Hospitl (SNUDH). The study protocol ws pproved by the Institutionl Review Bord t SNUDH (IRB CRI- G07002). Subjects were clssified ccording to cleft type nd the sttus nd loction of missing teeth. Dignosis of nonsyndromic CLA nd CLP ws mde through clinicl inspections by highly trined orthodontists. Tooth genesis ws identified from seril pnormic rdiogrphs bsed on the ge of the subject nd considering the fct tht the men dely in tooth formtion of the cleft children ws pproximtely 4 to 6 months reltive to tht of non-cleft children. 1,24 Regrdless of size nd morphology, ny permnent tooth on either side of the lveolr cleft between the mxillry centrl incisor nd cnine ws considered existence of the mxillry lterl incisor (MXLI). 25,26 TAT ws divided into none; cleft re only (missing of the MXLI within the cleft re only), cleft re + other re (missing of the MXLI within the cleft re nd nother mxillry tooth outside the cleft re), nd other re only (missing of nother mxillry tooth outside the cleft re only) (Figure 1). The sttus of those who were missing nother mxillry tooth outside the cleft re did not include genesis of the mxillry third molr. TAT ws further grouped into two subctegories (none nd cleft re only) nd four subctegories (none, cleft re only, cleft re + other re, nd other re only). Peripherl venous blood smples of ptients were collected t SNUDH fter obtining written informed consent. Genomic DNA smples were extrcted from peripherl venous blood lymphocytes by the protein precipittion method using commercil DNA extrction kit (Quigen Inc, Vlenci, Clif) nd were genotyped using the VerCode Technology progrm (Illumin Inc, Sn Diego, Clif) t SNP Genetics Inc (Seoul, Kore). SNP mrkers locted from 2kb, 59 to 2kb, 39 of the MSX1 nd PAX9 genes were obtined from literture review nd the Ntionl Center for Biotechnology Informtion dbsnp dtbse ( nlm.nih.gov/snp/). Three SNP mrkers of the MSX1 gene (rs , rs12532, nd rs ) nd 10 SNP mrkers of the PAX9 gene (rs , rs , rs , rs , rs , rs , rs , rs , rs , nd rs ) with minor llele frequency (MAF) greter thn 1% in the Jpnese popultion were selected using the Web-bsed progrm, TAG SNP selection (TgSNP; htm#snptg) Fisher s exct test ws used to investigte the correltion between TAT nd genotypic distribution of SNPs in MSX1 nd PAX9 genes. Logistic regression nlysis ws performed to clculte the genotypic odds rtio of SNPs in MSX1 nd PAX9 genes ccording to the genotypes in ech TAT.

3 1038 SEO, PARK, KIM, BAEK Figure 1. A. Tooth genesis type (TAT)-0 None (no missing tooth) with bilterl cleft lip nd plte (CLP) B. TAT-1 Cleft re only [missing of the mxillry lterl incisor (MXLI) within the cleft re only] with unilterl CLP. C. TAT-2 Cleft re + Other re (missing of MXLI within the cleft re nd nother mxillry tooth outside the cleft re) with unilterl CLP. D. TAT-3 Other re only (missing of nother mxillry tooth outside the cleft re only) with unilterl CLP. An sterisk represents missing tooth. RESULTS Demogrphic Informtion for Cleft Type, TAT, nd Gender Among 126 Koren ptients with NS-CL6P (82 mles nd 44 femles; 28 ptients with CLA nd 98 ptients CLP), TAT distribution ws cleft re only (39.7%), cleft re + other re (15.9%), other re only (8.7%), nd none (35.7%) (Tble 1). Distribution of Tooth Agenesis The most frequently missing teeth were the MXLI (73.6%) nd the mxillry second premolr (16.5%) (Tble 2). Assocition Between TAT nd SNPs of MSX1 nd PAX9 Genes In the four subctegories of none, cleft re only, cleft re + other re, nd other re only, MSX1-rs12532 Tble 1. Tooth genesis type Demogrphic Informtion Regrding Cleft Type, Tooth Agenesis Type, nd Gender Cleft Type UCLA (Right) CLA (n 5 28) CLP (n 5 98) UCLA (Left) BCLA Subtotl UCLP (Right) UCLP (Left) BCLP Subtotl Sum (n 5 126) None (67.85%) (26.53%) 45 (35.71%) Cleft re only (21.43%) (44.90%) 50 (39.68%) Cleft re + other re (7.14%) (18.37%) 20 (15.87%) Other re only (3.57%) (10.20%) 11 (8.73%) Gender Mle (64.29%) (65.31%) 82 (65.08%) Femle (35.71%) (34.69%) 44 (34.92%) CLA indictes cleft lip nd lveolus; UCLA, unilterl cleft lip nd lveolus; BCLA, bilterl cleft lip nd lveolus; CLP, cleft lip nd plte; UCLP, unilterl cleft lip nd plte; BCLP, bilterl cleft lip nd plte; none, no missing teeth; cleft re only, missing of the mxillry lterl incisor within the cleft re only; cleft re + other re, missing of the mxillry lterl incisor within the cleft re nd missing of nother mxillry tooth outside the cleft re; other re only, presence of the mxillry lterl incisor within the cleft re nd missing of nother mxillry tooth outside the cleft re.

4 MSX1/PAX9 SNPS AND TOOTH AGENESIS IN CLEFT 1039 Tble 2. Distribution of the Tooth Agenesis of the Mxillry Dentition Centrl Incisor Lterl Incisor Cnine First Premolr Second Premolr First Molr Second Molr Sum CLA 0 9 (64.29%) 1 (7.14%) 2 (14.29%) 2 (14.29%) CLP 6 (5.60%) 80 (74.77%) 1 (0.93%) 0 18 (16.82%) 2 (1.87%) Sum 6 (4.96%) 89 (73.55%) 2 (1.65%) 2 (1.65%) 20 (16.53%) 2 (1.65%) CLA indictes cleft lip nd lveolus; CLP, cleft lip nd plte. nd PAX9-rs showed significnt ssocition with tooth genesis (P,.01 nd P,.05, respectively, Tble 3). In the two subctegories of none nd cleft re only, MSX1-rs12532 nd PAX9-rs were significntly ssocited with tooth genesis (P,.05 nd P,.01, respectively, Tble 3). These three cndidte SNPs (MSX1-rs12532, PAX9-rs , nd PAX9- rs ) were further exmined to ssess genotypic distribution nd genotypic odds rtios (GORs). Genotypic Distribution nd GORs of MSX1-rs12532 According to TAT For MSX1-rs12532, there ws significnt ssocition between genotypic distribution nd TAT in both the four subctegories nd the two subctegories (P,.01 nd P,.05, respectively, Tble 4). The GOR of MXLI genesis in genotype GA of MSX1-rs12532 ws significntly incresed 3.14-fold in cleft re only compred with genotype GG (95% confidence intervl [CI] , P,.01;Tble 5). The GOR of MXLI nd nother mxillry tooth genesis in genotype AA of MSX1-rs12532 ws lso significntly incresed fivefold in cleft re + other re compred with genotype GG (95% CI , P,.05, Tble 5). Tble 3. Assocition Between Tooth Agenesis Type nd Single- Nucleotide Polymorphisms (SNPs) of the MSX1 nd PAX9 Genes Gene SNP Tooth Agenesis Type (P-Vlue) Four Subctegories Two Subctegories MSX1 rs rs **.0304* rs PAX9 rs rs *.0845 rs ** rs rs rs rs rs rs rs Fisher s exct test ws performed. * P,.05; ** P,.01. The four subctegories re none, cleft re only, cleft re + other re, nd other re only; the two subctegories re none nd cleft re only. Genotypic Distribution nd GORs of PAX9- rs nd rs According to TAT Although the ssocition between genotype distribution of PAX9-rs nd TAT ws not significnt in the two subctegories (P..05, Tble 6), this ssocition ws significnt in the four subctegories (P,.05, Tble 6). However, PAX9-rs showed the opposite tendency: the genotypic distribution of PAX9-rs exhibited significnt ssocition with TAT in the two subctegories (P,.01, Tble 6), but not in the four subctegories (P..05, Tble 6). Although the GORs of genotypes of PAX9- rs in ech TAT were not meningful (Tble 7), the GOR of tooth genesis of MXLI in genotype CT of PAX9-rs ws significntly incresed 4.15-fold in cleft re only compred with genotype CC (95% CI , P,.01, Tble 7). DISCUSSION Becuse muttions in PAX9 or MSX1 re known to cuse genesis of the posterior teeth 7 10,17 nd MXLI, we ctegorized NS-CL6P smples into four types of tooth genesis none (0), cleft re only (1), cleft re + other re (2), nd other re only (3) nd grouped them into two subctegories (0 nd 1) nd four subctegories (0, 1, 2, nd 3). In this study, MSX1-rs12532 showed significnt ssocition with tooth genesis in both the four subctegories nd the two subctegories of TAT (P,.01 nd P,.05, respectively, Tble 4). MXLI genesis ws significntly higher in ptients with genotype GA compred with those with genotype GG (3.14-fold, P,.01, Tble 5). In ddition, genesis of MXLI nd nother mxillry tooth ws significntly higher in ptients with genotype AA compred with those with genotype GG (fivefold, P,.05, Tble 5). These findings suggest tht this SNP might be ssocited with genesis of MXLI nd nother mxillry tooth. However, the results of this study were somewht different from those of previous studies. In three ptients with spordic nonsyndromic oligodonti, Pwlowsk et l. 11 observed SNPs t MSX1-rs12532 in ptient with presence of MXLI nd SNP t MSX1- rs8670 in ptient with bsence of MXLI. Becuse

5 1040 SEO, PARK, KIM, BAEK Tble 4. Genotypic Distributions of MSX1-rs12532 According to Tooth Agenesis Type in Four Subctegories nd Two Subctegories MSX1-rs12532 P-Vlue Tooth Agenesis Type GG GA AA Four Subctegories Two Subctegories None 25 (55.56%) 17 (37.78%) 3 (6.67%).0021**.0304* Cleft re only 15 (30.0%) 32 (64.00%) 3 (6.00%) Cleft re + other re 10 (50.00%) 4 (20.00%) 6 (30.00%) NA Other re only 3 (27.27%) 6 (54.55%) 2 (18.18%) NA Fisher s exct test ws performed. * P,.05; ** P,.01; NA, not pplicble. these SNPs my be reltively common, they concluded tht these polymorphisms would not be expected to hve ny pronounced phenotypic effect. 11 In ddition, lthough Pixão-Côrtes et l. 13 found three polymorphic sites in the untrnslted region of MSX1 exon 2 (rs8670, rs1095, nd rs12532), there ws no sttisticl difference in llele nd genotype distributions between ptients nd control subjects. The polymorphism t rs8670 ws not included in the present study, but it hs previously been observed in individuls with MXLI genesis. 11,14 However, becuse this polymorphism t rs8670 is lso reltively common, dditionl genes might be involved in this phenotype. 14 The discordnce between previous studies nd this study seem to originte from differences between oligodonti ptients nd cleft ptients. For PAX9-rs , significnt ssocition ws found in the four subctegories of TAT (P,.05, Tble 6) but not in the two subctegories (Tble 6). However, meningful GOR ws not demonstrted in either ctegory (Tble 7), suggesting tht this SNP might not be relted to MXLI genesis. However, the low percentge of genotype CA in the other re only compred with tht of the cleft re only (9.1% vs 60.0%, respectively, Tble 6) suggests possible ssocition between this SNP nd TAT in cleft ptients (GOR , P in cleft re only; GOR , P in other re only, compred with none, Tble 7). These results indicte tht sttisticl power might be incresed s the smple size increses. Therefore, further studies re needed to investigte the reltionship between PAX9-rs nd MXLI genesis using lrger smple size. In contrst, PAX9-rs showed significnt ssocition in the two subctegories of TAT (P,.01, Tble 6), but not in the four subctegories (Tble 6). In prticulr, the frequency of genotype CT ws significntly incresed in cleft re only (4.15-fold, P,.01, Tble 7). These results indicte tht PAX9-rs might be potentil cndidte mrker for MXLI genesis susceptibility in Koren cleft ptients. However, PAX9-rs nd other polymorphisms locted djcent to this locus in the promoter region re lso known to be ssocited with third molr genesis. Sito et l. 30 nd Binchi et l. 31 reported tht the CC genotype of the PAX9-rs showed positive ssocition with third molr genesis. Peres et l. 32 lso suggested tht the GT hplotype of PAX9-rs nd rs polymorphisms ws ssocited with hypodonti, in most cses including third molr genesis. Therefore, further studies re needed to investigte the reltionship between PAX9-rs nd MXLI genesis. In previous studies on the ssocition between NS- CL6P nd SNPs of the MSX1 nd PAX9 genes, the A llele t MSX1-rs ws ssocited with significntly incresed risk of NS-CL6P (GOR , 95% CI , P,.05, dditive model) 28 nd the G/A heterozygote t PAX9-rs showed significnt ssocition with NS-CL6P (GOR , 95% CI into 5.84, P,.01). 29 However, in the present study, these two SNPs of MSX1 nd PXA9 did not show ny significnt ssocition with tooth genesis (Tbles 3 through 7), wheres MSX1-rs12532 nd PAX9-rs nd rs did. These findings suggest tht occurrence of orl cleft or tooth genesis might depend on Tble 5. Genotypic Odds Rtios (GORs) of Genotypes GA nd AA Compred with Genotype GG of MSX1-rs12532 in Ech Tooth Agenesis Type MSX1-rs12532 Tooth Agenesis Type Genotypic Odds Rtio GG (Intercept) GA AA None Cleft re only GOR (95% CI) 3.14 (1.32, 7.48) 1.67 (0.3, 9.34) P-vlue.0099**.5613 Cleft re + other re GOR (95% CI) 0.59 (0.16, 2.19) 5 (1.04, 23.98) P-vlue * Other re only GOR (95% CI) 2.94 (0.65, 13.4) 5.56 (0.65, 47.83) P-vlue Logistic regression nlysis ws performed. CI indictes confidence intervl. * P,.05; ** P,.01.

6 MSX1/PAX9 SNPS AND TOOTH AGENESIS IN CLEFT 1041 Tble 6. Genotypic Distributions of PAX9-rs nd rs According to Tooth Agenesis Type in Four Subctegories nd Two Subctegories Tooth Agenesis Type PAX9-rs CC CA AA Four Subctegories P-Vlue Two Subctegories PAX9-rs CC CT TT Four Subctegories P-Vlue Two Subctegories None 20 (44.44%) 17 (37.78%) 8 (17.78%).0365* (33.33%) 14 (31.11%) 16 (35.56%) ** Cleft re only 16 (32.00%) 30 (60.00%) 4 (8.00%) 8 (16.0%) 31 (62.00%) 11 (22.00%) Cleft re + other re 10 (50.00%) 8 (40.00%) 2 (10.00%) NA 6 (30.00%) 9 (45.00%) 5 (25.00%) NA Other re only 8 (72.73%) 1 (9.09%) 2 (18.18%) NA 4 (36.36%) 4 (36.36%) 3 (27.27%) NA Fisher s exct test ws performed. * P,.05; ** P,.01; NA indictes not pplicble. the locus of the SNP within the MSX1 gene or PAX9 gene. The results of this study might be helpful for improving our understnding of the effects of genetic vrition of MSX1 nd PAX9 genes on tooth genesis within nd outside the cleft re. However, there re severl fctors to consider when interpreting the results obtined in this study. First, it would be better to increse the smple size to increse the sttisticl power nd to void unnecessry sttisticl errors. Second, it is necessry to exmine the genotypic distribution in non-cleft individuls with nd without tooth genesis to verify the exct role of SNPs in tooth genesis. Third, functionl consequences of these SNPs should be verified. Fourth, further studies re needed to investigte the influence of interctions of these genes with other genes, environmentl fctors, or ethnic differences. CONCLUSIONS N Becuse n ssocition between the risk of tooth genesis nd SNPs of MSX1 nd PAX9 genes ws found in nonsyndromic cleft ptients, the null hypothesis ws rejected. N Genetic disturbnces of MSX1 nd PAX9 genes re ssocited with tooth genesis within nd outside the cleft re in ddition to the locl tissue defect of clefting. ACKNOWLEDGMENTS This reserch ws supported by the Bsic Science Reserch Progrm, the Ntionl Reserch Foundtion of Kore (NRF ) funded by the Ministry of Eduction, Science nd Technology, Republic of Kore. The uthors thnk ll prticipnts who donted smples nd cknowledge Jung Sun Cho (Hllym University College of Medicine) for ssistnce during this work, s well s Duk Hwn Kim, Yong Ick Ji, Eunhyun Jung, nd Se Young Cho for their contributions to DNA preprtion for genotyping (Center for Genome Reserch, Smsung Biomedicl Reserch Institute, Seoul, Kore). REFERENCES 1. Rnt R. A review of tooth formtion in children with cleft lip/ plte. Am J Orthod Dentofcil Orthop. 1986;90: Shpir Y, Lubit E, Kuftinec MM. Hypodonti in children with vrious types of clefts. Angle Orthod. 2000;70: Lrmour CJ, Mossey PA, Thind BS, Forgie AH, Stirrups DR. Hypodonti retrospective review of prevlence nd etiology. Prt I. Quintessence Int. 2005;36: Tble 7. Genotypic Odds Rtios (GORs) of Genotypes CA nd AA Compred with Genotype CC of PAX9-rs nd of Genotypes CT nd TT Compred with Genotype CC of PAX9-rs in Ech Tooth Agenesis Type PAX9-rs PAX9-rs Tooth Agenesis Type CC (Intercept) CA AA CC (Intercept) CT TT None Cleft re only GOR (95% CI) 2.21 (0.91, 5.35) 0.63 (0.16, 2.46) 4.15 (1.43, 12.05) 1.29 ( ) P-vlue **.6656 Cleft re + other re GOR (95% CI) 0.94 (0.3, 2.92) 0.5 (0.09, 2.81) NA NA P-vlue Other re only GOR (95% CI) 0.15 (0.02, 1.3) 0.63 (0.11, 3.61) NA NA P-vlue Logistic regression nlysis ws performed. CI indictes confidence intervl; NA, not pplicble. ** P,.01.

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