neonatal blue light phototherapy and melanocytic nevi- a twin study

Neonatal Blue Light Phototherapy and MelanocyticNevi: A Twin Study

WHATS KNOWN ON THIS SUBJECT: Neonatal blue lightphototherapy is an essential therapeutic tool in the management ofneonatal jaundice to reduce the plasma concentration of bilirubin.Only a few and controversial data are available as to how blue lightphototherapy inuences melanocytic nevus development.

WHAT THIS STUDY ADDS: This is the rst survey in the literaturethat allows the precise investigation of the impact of blue lightphototherapy on cutaneous and uveal melanocytic nevusdevelopment with its possible genetic aspects in a homogenousstudy population of twins.

abstractBACKGROUND: Neonatal blue light phototherapy (NBLP) has beenwidely and successfully used for the treatment of neonatal jaundice toreduce the plasma concentration of bilirubin and, hence, to preventkernicterus. Only a few and controversial data are available in theliterature as to how NBLP inuences melanocytic nevus development.

OBJECTIVE: Our goal was to conduct a twin study with the aim of betterunderstanding the role of NBLP in melanocytic nevus development. Wealso investigated the roles of other environmental and constitutionalfactors in nevus formation.

METHODS: Fifty-nine monozygotic and dizygotic twins were included inthis cross-sectional study. One of the twinmembers received NBLP, andthe other did not. A whole-body skin examination was performed todetermine the density of melanocytic skin lesions. The prevalence ofbenign pigmented uveal lesions was evaluated during a detailed oph-thalmologic examination. A standardized questionnaire was used toassess data relating to constitutional, sun-exposure, and other vari-ables. To search for possible gene-environmental interactions involvedin the appearance of pigmented lesions, the melanocortin 1 receptorvariants and the I439V polymorphismof histidine ammonia-lyase geneswere also determined in the enrolled twins.

RESULTS: NBLP was associated with a signicantly higher prevalenceof both cutaneous and uveal melanocytic lesions. No association wasfound between the examined gene polymorphisms and the number ofpigmented alterations in the examined study group.

CONCLUSIONS: Our data suggest that NBLP couldwell be a risk factor formelanocytic nevus development. Phototherapy with blue-light lamps is astandard and essential therapeutic modality in neonatal care; therefore,additional in vivo and in vitro studies are necessary to establish its poten-tial long-term adverse effects. Pediatrics 2011;128:e856e864

AUTHORS: Zsanett Csoma, MD, PhD,a Edit Tth-Molnr,MD, PhD,b Klra Balogh, MD,a Hilda Polynka, MSc,c

Hajnalka Orvos, MD, PhD,d Henriette csai, MD,a LajosKemny, MD, DSc,a,c Mrta Szll, DSc,c and Judit Olh, MD,PhDa

aDepartment of Dermatology and Allergology, cDermatologicalResearch Group of the Hungarian Academy of Sciences, anddDepartment of Obstetrics and Gynecology, University of Szeged,Szeged, Hungary; and bNovotalex Ltd, Szeged, Hungary

KEY WORDSneonatal blue light phototherapy, cutaneous melanocytic nevi,benign pigmented ocular lesions, MC1R and HAL polymorphisms

ABBREVIATIONSCMNcommon melanocytic nevusCAMNclinically atypical melanocytic nevusBPULbenign pigmented uveal lesionNBLPneonatal blue light phototherapyMC1Rmelanocortin 1 receptorHALhistidine ammonia-lyaseUCAurocanic acidIFiris freckleINiris nevusCNchoroidal nevusSNPsingle nucleotide polymorphismRHCred hair color

Dr Csoma designed and organized the study, participated in theclinical skin examinations, was responsible for attaining the oralsputum samples from the twin pairs, evaluated the results ofdermatologic examinations, and wrote the main manuscript; DrTth-Molnr performed all ophthalmologic examinations andwrote the manuscript; Dr Balogh was responsible for thesequence analysis of the MC1R gene; Ms Polynka wasresponsible for preparation of genomic DNA samples,polymerase chain reactions, and purication of polymerasechain reaction products for sequence analysis; Dr Orvos wasresponsible for attaining data relating to the neonatal history ofthe participants and analyzed data; Dr csai participated in theclinical skin examinations; Dr Kemny designed the study,analyzed data, and revised the manuscript; Dr Szll designed thegenetic experiments, evaluated the results of geneticexperiments, and wrote the manuscript; and Dr Olh was theindependent dermato-oncologist in the course of the entiresurvey who designed the study and revised the entiremanuscript.

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e856 CSOMA et al

The number of people with large num-bers of common (CMN) and clinicallyatypical (CAMN) melanocytic nevi hasrecently been continuously increas-ing.1 Numerous epidemiologic studieshave revealed that the number of cuta-neous nevi is affected by different con-stitutional, environmental, hormonal,and genetic factors.27 However, thereare only a few reports on endogenousand exogenous factors that inuencethe development of benign pigmenteduveal lesions (BPULs). The presence oflarge numbers of CMN and CAMN is awell-established independent pheno-typic marker of a highly increased riskof the development of both cutaneousand uveal malignant melanoma,816

and the identication of any additionalfactor that might contribute to nevusformation is therefore of great impor-tance. We previously investigated theprevalence of melanocytic nevi and as-sociated factors in a large study popu-lation of adolescents and young adults,and we found that neonatal blue lightphototherapy (NBLP) was associatedwith a signicantly higher prevalenceof CAMN.17,18

NBLP is an essential therapeutic tool inthe management of neonatal jaundiceto reduce the plasma concentration ofbilirubin and hence to prevent ker-nicterus.1921 Its potential acute, short-term adverse effects are well knownand can be adequately treated in neo-natal practice. Much less is known onits long-term adverse effects. Of these,only a few and controversial data areavailable as to how NBLP inuencesmelanocytic nevus development,2224

and there is a lack of surveys in theliterature as concerns pigmented ocu-lar alterations in such patients.

On the basis of our previous results, ourgoal was to conduct a twins study withthe aim of a better understanding of therole of NBLP in melanocytic nevus devel-opment.Wealso investigated the roles of

other environmental and constitutionalfactors in nevus formation.

To understand the complex nature ofmelanocytic nevus development, it is ofpivotal interest to investigate the gene-environment interactions that causethis melanoma-predisposing condi-tion. It is well established that variantsof the melanocortin 1 receptor (MC1R)gene are associated with the combina-tion of red hair, freckling, and sun sen-sitivity.2527 Histidase, encoded by theHAL (histidine ammonia-lyase) gene,catabolizes the amino acid L-histidineto trans-urocanic acid (trans-UCA);then, on UV-B induction, trans-UCA pho-toisomerizes to cis-UCA, which plays abasic role in UV-induced immunosup-pression.2830 It has recently been dem-onstrated31 that the interaction of sun-burn with the I439V polymorphism ofHAL is associated with nonmelanomaskin cancers such as basal cell car-cinoma and squamous cell carci-noma. To search for possible gene-environmental interactions involvedin the appearance of pigmented le-sions, we determined the MC1R vari-ants and the I439V polymorphism ofHAL in the enrolled twins.

METHODS

Patients

Fifty-eight pairs of twins and 1 set oftriplets of white origin, aged 3 to 30years, were included in our study,which was performed between Janu-ary 5, 2008, and April 12, 2008, in theDepartment of Dermatology and Aller-gology at the University of Szeged(Szeged, Hungary). The distribution ofthe participating twin pairs was as fol-lows: 15 monozygotic pairs (7 femaleand 8 male pairs), 11 dizygotic femalepairs, 11 dizygotic male pairs, 21 dizy-gotic pairs of different genders, and 1dizygotic female triplet. After approvaland permission had been obtainedfrom the institutional review board ofAlbert Szent-Gyrgyi Medidal Center at

the University of Szeged, all the partic-ipants or their parents gave their writ-ten consent before the start of the sur-vey. As we put special emphasis on theinvestigation of the effect of NBLP onnevus development, we enrolled mo-nozygotic and dizygotic twin pairs,where 1 of the twins had received pho-totherapy for neonatal jaundice andthe other had not. Data relating to theneonatal history of the subjects (pre-maturity, icterus, and NBLP) were ob-tained from the ofcial neonatal medi-cal charts.

The study was based on 4 major ele-ments: a clinical skin examination, astandardized questionnaire, an oph-thalmologic examination, and DNAsampling. Neither the dermatologistsnor the ophthalmologist knewwhetherthe investigated subjects had receivedNBLP.

Skin Examinations

All twin pairs underwent a whole-body skin examination, excluding thescalp and the anogenital area. Mela-nocytic nevi were counted as in thestandardized international protocolaccording to English et al.32 Pig-mented lesions with the morphologicfeatures of CMN, CAMN, congenitalmelanocytic nevi, blue nevi, Spitznevi, nevi spili, halo nevi, lentigines,and caf-au-lait macules werecounted separately, and the pres-ence of freckles was also recorded ineach subject.

Interview

After the clinical skin examinations, astandardized questionnaire was com-pleted by all the participants or theaccompanying parents. The question-naire sought information on sunbath-ing habits, sun protection methods,other sun-exposure variables, and afamily history of a large number ofmelanocytic nevi, melanoma, or non-melanoma skin cancers. Pigmentary

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PEDIATRICS Volume 128, Number 4, October 2011 e857

traits such as eye color, hair color,skin color, and skin phototype wereevaluated in each subject. Skin photo-type was assessed on the Fitzpatrickscale, which is based on a persons re-action to 30 minutes of midday sun-light for the rst time in the summer (I always burns, never tans; II al-ways burns, sometimes tans; III sometimes burns, always tans; IV never burns, always tans). Skin colorwas described on a 3-grade scale(dark, medium, or fair).

Ophthalmologic Examination

Detailed ophthalmologic examinationswere conducted, including slit-lamp bio-microscopic examination of the anteriorsegmentwithoutdilationof thepupil (us-ing the Inami L-0189 slit-lamp, Inami &Co. Ltd., Tokyo, Japan) and applanationtonometry (using the Inami L-5130 appla-nation tonometer, Inami & Co. Ltd., To-kyo, Japan). Complete indirect ophthal-moscopic examinations of the fundiwere performed after maximal dilationof the pupil with cyclopentolate 0.5%, us-ing the Heine Omega 100 (Heine Opto-technik GmbH&Co. KG., Herrsching, Ger-many) indirect ophthalmoscope.

For all participants, a standardizedform was used to record the iris color,the presence and location of conjunc-tival nevi, the numbers and distribu-tion of iris freckles (IF), iris nevi (IN),choroidal nevi (CHN), or any pigmentedlesions of other ocular structures. Le-sions identied were dened accord-ing to the Shields system of classica-tion.33 Exclusion criteria were (1)media opacity that precluded examina-tion of the choroid, (2) iris heterochro-mia, (3) disorders or medication thatcould alter the iris color (eg, iris neo-vascularization, anamnestic uveitis orocular injury, the use of prostaglandinanalogue eye drops), and (4) ocular oroculodermal melanocytosis or neuro-bromatosis as known factors predis-posing to ocular nevus formation.

Determination of MC1R GeneVariants and the I439VPolymorphism of the HAL Gene

Genomic DNA was isolated from oralsputum sample of monozygotic and dizy-gotic twin pairs and triplets using BioRo-bot EZ1 and Qiagen EZ1 DNA InvestigatorKit (Qiagen, Hilden, Germany). Determi-nation of MC1R gene variants were per-formed by using the full-length sequenc-ingof thegeneasdescribedpreviously,34

and the I439V polymorphism of HAL genewas investigated by amethod devised byWelsch et al.31

Statistical Analyses

The correlations between the preva-lence of melanocytic nevi and possibleendogenous and exogenous risk fac-tors were initially assessed univari-ately by using the nonparametricKruskal-Wallis test, and the Wilcoxonsigned rank test. The Spearman rankcorrelation test was conducted toevaluate the correlations betweenthe numbers of CMN, CAMN, andBPUL. All P values calculated were2-sided, and a signicance level of0.05 was assumed. All variables wereentered into multivariate logistic orlinear regression analyses to evalu-ate the simultaneous effect of differ-ent factors on melanocytic nevus de-velopment. For pigmented cutaneouslesions, the dependent variable wasthe number of nevi with a logarith-mic transformation; the natural log-arithm of the nevus count demon-

TABLE 1 Prevalence of Common and Clinically Atypical Melanocytic Nevi According to Age GroupsAmong Twin Pairs

Age No. ofSubjects

Median No. of CMN(Lower, UpperQuartile)

Median No. of CAMN(Lower, UpperQuartile)

Median No. of MelanocyticNevi (CMN CAMN)(Lower, Upper Quartile)

Monozygotic twin pairs(n 30)

36 y 4 1 (0.52) 0 (00) 1 (0.52)710 y 6 3.5 (36) 0 (00) 4 (36)1114 y 8 12.5 (8.526.5) 2.5 (03.5) 15 (1028.5)1518 y 2 3 (37) 0 (01) 4 (37)1922 y 2 11.5 (1112) 0.5 (01) 12 (1113)2326 y 6 12 (921) 1 (03) 14 (922)2730 y 2 7.5 (312) 8 (313) 15.5 (625)

Dizygotic twin pairs(n 89)

36 y 18 2 (14) 0 (00) 2 (14)710 y 20 9 (5.5135) 0 (01) 9 (614.5)1114 y 14 6 (39) 1 (02) 7 (410)1518 y 11 21 (1023) 3 (19) 22 (1233)1922 y 12 22.5 (6.534) 2.5 (0.55) 25.5 (6.540)2326 y 6 17 (834) 2 (02) 19 (936)2730 y 8 29.5 (13.535) 1 (03.5) 30.5 (15.536.5)

TABLE 2 Prevalence of Other Pigmented SkinLesions Among Monozygotic andDizygotic Twin Pairs (N 119)

Lesion No. of SubjectsWith Lesions

No. ofLesions

Congenital nevus 19 28Caf au lait macules 20 24Nevus spilus 2 2Becker nevus 1 1

TABLE 3 Summary of Statistical Analysis (Wilcoxon Signed Rank Test, P Values) for the Differencein Prevalence of Melanocytic Nevi Between Blue LightExposed and Nonexposed TwinMembers (N 119)

Subjects CMN CAMN All Melanocytic Nevi

Monozygotic twin pairs (n 15) .025 .017 .014Dizygotic twin pairs (n 44) .042 .12 .038All twin pairs (n 59) .010 .016 .005

e858 CSOMA et al

strated a normal distribution with theKolmogorov-Smirnov 1-sample test,and multivariate linear regressionanalysis was performed. The numberof BPUL did not show normal distribu-tion by the Kolmogorov-Smirnov test,and because of the high numbers of 0values in the survey, multivariate logis-tic regression analysis was conducted.Statistical analyses were performedwith SPSS 15.0 (SPSS Inc, Chicago, IL).

RESULTS

Skin Examinations

The prevalence of CMN and CAMN ispresented by median nevus countswith interquartile ranges in differentage groups in Table 1. The number ofCAMNwas strongly associatedwith theprevalence of CMN (the Spearmanrank correlation test, r 0.589). Theprevalence of other pigmented cutane-ous lesions is presented in Table 2.

On univariate analysis, NBLP was asso-ciated with a signicantly higher prev-alence of both CMN and CAMN in theexamined twin pairs. When the analy-sis was focused separately on the mo-nozygotic and the dizygotic twin pairs,a statistically signicant difference inthe number of nevi was still observedbetween the exposed and nonexposedsubjects in the monozygotic twins. Inthe case of dizygotic twin pairs, thenumber of CMN and the whole numberof melanocytic nevi differed in a statisti-cally signicant manner between thetreated and the untreated twin mem-bers (Wilcoxonsignedrank test; Table 3).

The associations between gender, con-stitutional and sun-exposure vari-ables, and the prevalence of melano-cytic nevi with the nonparametricKruskall-Wallis test are presented inTable 4. In multivariate linear regres-sion analysis, the number of melano-cytic nevi was signicantly and inde-pendently associated with age, withthe number of summer holidays be-side the sea in the Mediterranean or in

TABLE 4 Associations Between Gender, Constitutional, and Sun-Exposure Variables and thePrevalence of Melanocytic Nevi Among Monozygotic and Dizygotic Twin Pairs (N 119)(Nonparametric Kruskall-Wallis Test)

Factors n Median No. ofMelanocytic Nevi

P

GenderMale 59 7 (416)Female 60 9 (421) .46

Eye colorBrown 57 6 (315.5)Hazel, greenish-brown 12 8 (530.75)Green, gray, blue 50 12.5 (426.25) .049

Hair colorBlack, dark-brown 25 12 (5.530.5)Medium-brown, light-brown 81 7 (417.5)Blond 13 12 (219.5) .33

Skin colorDark, medium 62 8 (320.25)Fair 57 9 (421) .46

Skin phototypeIII 32 8 (413.25)IIIIV 87 9 (423) .36

Frequency of use of sunscreensNever 11 8 (313)Occasionally 43 9 (424)Always at the beginning of the summer, then occasionally 31 10 (617)Regularly 34 5 (314) .12

Duration of use of sunscreensNever 18 8 (3.7513.75)15 y 47 9 (216)610 y 30 7.5 (418.5)1020 y 24 17.5 (632.75) .048

SPF0 11 8 (313)110 20 18 (8.2532.25)1020 43 13 (424)20 45 6 (2.510)

No. of severe painful sunburns during childhood0 62 6 (315.25)12 46 9 (424.25)35 11 16 (1127) 0.014

No. of severe painful sunburns during adolescence0 39 10 (530)12 21 24 (13.533)35 4 17.5 (9.551.75) 0.043

No. of severe painful sunburns during adulthood0 25 17 (735.5)12 13 14 (11.530.5)35 2 15 (327) 0.69

Frequency of sunbathing episodes between April andSeptember

0 17 9 (421)110 32 8 (517.75)1020 19 17 (835)20 47 6 (316) 0.020

Duration of 1 sunbathing episode30 min 23 7 (313)30 min to 1 h 31 17 (733)13 h 41 6 (2.517.5)3 h 22 9 (5.7514.5) 0.005

No. of days per week when4 h was spent outdoors duringchildhood

01 17 5 (2.512.5)23 44 11.5 (4.526.75)45 31 13 (421)67 27 6 (310) 0.08

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a subtropical or a tropical climate, andwith a history of NBLP (see Table 5).

Ophthalmologic Examination

The following pigmented ocular le-sions were documented during theophthalmologic examination: IF in 18subjects; IN in 2 subjects; and CHN in 3subjects (Table 6). A statistically signif-icant correlation was found betweenthe prevalence of BPUL and the num-ber of CAMN (the Spearman rank cor-relation test, r 0362).

When all of the melanocytic ocularndings were examined together,

NBLP was associated with a substan-tially higher prevalence of these le-sions. IF were observed in 16 subjectswith a history of NBLP, and in 2 personswho did not receive NBLP. The differ-ence between the 2 groups in the rateof occurrence of IF also proved to bestatistically signicant (Wilcoxonsigned rank test; Table 7).

In univariate analysis, the frequency ofsunbathing and a history of severe pain-ful sunburns during childhoodwere alsosignicantly related to the density ofBPUL (nonparametric Kruskall-Wallistest; Table 8). Multivariate logistic re-

gression analysis conrmed the signif-icant correlation between NBLP andthe prevalence of BPUL (Table 9).

MC1R and HAL Polymorphisms

Genetic analysis was performed on 75subjects (36 twin pairs and 1 set oftriplets). For this analysis, we selectedtwin pairs in whom the difference innevus count was signicant. Withinthis selected subgroup, there was anunequivocal association of NBLP bothwith the CMN count (P .010; Wilcoxonsigned ranks test) and with the CAMNcount (0.055, Wilcoxon signed rankstest). When the number of cutaneouspigmented lesions (CMN CAMN) wasanalyzed as a function of NBLP, a highlysignicant effect was detected (P

TABLE 4 Continued

Factors n Median No. ofMelanocytic Nevi

P

No. of days per week when4 h was spent outdoors duringadolescence

01 11 9 (432)23 22 21.5 (13.533.5)45 23 13 (730)67 6 17.5 (7.533.75) 0.33

No. of days per week when4 h was spent outdoors duringadulthood

01 6 20.5 (566.25)23 13 30 (938.5)45 10 16 (1126.25)67 8 10.5 (6.533.25) 0.67

No. of summer holidays beside the sea in the Mediterraneanor in a subtropical or tropical climate

0 63 8 (314)12 25 6 (416)34 14 13 (424.25)57 14 25 (943.75)8 3 33 (635) 0.008

Use of sunbedsNever 106 8 (417.25)Occasionally 10 20 (11.530.75)Regularly 3 17 (9109) 0.013

Family history of large numbers of melanocytic neviNo 52 9.5 (421)Yes 56 7.5 (3.2522.5) 0.49

TABLE 5 Factors Associated With the Prevalence of Melanocytic Nevi: Results of Multivariate Linear Regression Analysis

Variable UnstandardizedCoefcients, B

StandardizedCoefcients,

P 95% Condence Limits for B

Lower Bound Upper Bound

Age 0.076 0.503 .000 0.051 0.101No. of summer holidays beside the sea in the Mediterranean, or in asubtropical or tropical climate

0.172 0.178 .035 0.012 0.332

NBLP 0.177 0.158 .047 0.003 0.352

The multivariate linear regression (stepwise method) model included gender, eye color, hair color, skin color, skin phototype, the frequency of use of sunscreens, a history of severe painfulsunburns, the frequency and duration of sunbathing, the number of days per week when4 hours was spent outdoors, the number of summer holidays beside the sea in theMediterranean,or in a subtropical or tropical climate, the use of sunbeds, a family history of large numbers of melanocytic nevi, a history of NBLP, and age. The number of melanocytic nevi did not shownormal distribution by the Kolmogorov-Smirnov test. Multivariate linear regression was therefore performed on the natural logarithm of the nevus count.

TABLE 6 Prevalence of Benign PigmentedOcular Lesions Among Monozygoticand Dizygotic Twin Pairs (N 113)

Lesion No. of SubjectsWith Lesions

No. ofLesions

IN 2 5CN 3 4IF 18 123

TABLE 7 Prevalence of Benign PigmentedOcular Lesions in Blue LightExposedand Nonexposed Twin Members(Wilcoxon Signed Rank Test)

Lesion No. of Lesionsin SubjectsWith no NBLP

No. of Lesionsin SubjectsWith NBLP

P

IN 1 4 NAa

CHN 0 4 NAa

IF 18 105 .009IN CHN IF 19 114 .006a In view of the low rate of occurrence of uveal nevi in thestudy population, statistical analyses were not possible.

e860 CSOMA et al

.006; Wilcoxon signed ranks test). Oursequencing survey detected 9 MC1Rpolymorphisms: the synonymousT413T SNP (single nucleotide polymor-phism) in heterozygous form in 3 sub-jects, the rare I120T polymorphism in atwin pair as a heterozygous variant, 2red hair color (RHC) variants (R151Cand R160W), 4 frequent non-RHC vari-ants (V60L, V92M, I155T, and W163Q),and a new variant, W169R, in 1 subject.

Statistical analyses were performedwith various groupings of the MC1Rpolymorphisms: (1) the presence of ei-ther of the RHC alleles (R151C andR160W) was considered; (2) the pres-ence of any of the most frequent 6MC1R polymorphisms (V60L, V92M,R151C, R160W, W163Q, and I155T SNP6) was considered; (3) the pres-ence of any of the SNP6 group and therare I120T polymorphism ( SNP7)was considered; or (4) the presence ofeither of SNP7 and the newly identiedW169R polymorphism ( SNP8) wasconsidered. It was unambiguouslydemonstrated that the MC1R polymor-phisms have a signicant effect on theskin type of the examined twins (R151Cand R160W, P .001; SNP6, P .013;SNP7, P .013; SNP8, P .023), but theI439V HAL polymorphism did not ex-hibit any association with the skin typein our study population.

The effect of the polymorphisms on thenumbers of pigmented skin lesionsand the presence of pigmented uveallesions was assessed by using univar-iate and multivariate statistical analy-ses. Neither the univariate (Mann-Whitney test; Table 10) nor themultivariate (analysis of variance; Ta-ble 11) analysis revealed any effects ofthe studied polymorphisms on the skinand uveal pigmented lesions.

DISCUSSION

Physiologic jaundice develops in a no-tably high proportion of otherwisehealthy newborn infants as a result of

TABLE 8 Associations Between Gender, Constitutional, and Sun-Exposure Variables and thePrevalence of Benign Pigmented Ocular Lesions Among Monozygotic and Dizygotic TwinPairs (N 113) (Nonparametric Kruskall-Wallis Test)

Factors No. of Subjects P

GenderMale 66/56Female 66/57 .66

Eye colorBrown 63/51Hazel, greenish-brown 34/11Green, gray, blue 35/51 .19

Hair colorBlack, dark-brown 18/21Medium-brown, light-brown 89/80Blond 25/12 .71

Skin colorDark, medium 49/58Fair 83/55 .47

Skin phototypeIII 59/31IIIIV 73/82 .33

No. of severe painful sunburns during childhood0 49/5912 80/4435 3/10 .042

No. of severe painful sunburns during adolescence0 60/3412 24/2135 0/3 .61

No. of severe painful sunburns during adulthood0 24/2012 1/1135 7/3 .09

Frequency of sunbathing episodes between April and September0 0/17110 43/311020 40/1620 44/45 .040

Duration of 1 sunbathing episode30 min 24/2430 min1 h 30/3113 h 62/373 h 14/19 .16

No. of days per week when4 h was spent outdoors during childhood01 12/1723 70/4345 18/2767 32/26 .45

No. of days per week when4 h was spent outdoors during adolescence01 12/723 24/2345 26/2167 14/6 .45

No. of days per week when4 h was spent outdoors during adulthood01 2/623 16/945 0/967 14/8 .21

No. of summer holidays beside the sea in the Mediterranean or in asubtropical or tropical climate

0 58/5912 41/2434 21/1557 12/138 0/2 .88

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excessive bilirubin formation. Withoutadequate treatment, the lipid-soluble,unconjugated bilirubin crosses theblood-brain barrier. The deposition ofbilirubin in the basal ganglia andbrainstem nuclei can result in very se-vere, permanent central nervous sys-tem damage (ie, acute and chronic bil-irubin encephalopathy). Phototherapy,

applying the clinically most effectiveblue emission spectrum (425475nm), has been widely and successfullyused for the treatment of neonataljaundice to reduce the plasma concen-tration of bilirubin and hence to pre-vent kernicterus.1921

So far, only a few and contradictorydata are available as to how NBLP in-uences melanocytic nevus develop-ment. 17,18,2224,35 Our results reveal asignicantly higher prevalence of cuta-neous melanocytic nevi among twinmembers with a history of NBLP. Astandardized questionnaire was usedto assess the data relating to constitu-tional, sun-exposure, and other vari-ables. These factors proved to be veryconsistent in the examined monozy-gotic twin pairs. The phenotypic char-acteristics of the dizygotic twins werepartly different, but the environmentalimpacts were very similar until adult-hood. The emission spectrum of theblue light lamps used in Hungary is be-tween 370 and 600 nm (maximum: 450nm). Approximately 0.3% of the emit-ted light comprises UVA radiation. The

wavelengths of blue light and UV lightare adjacent, and they might thereforeexert partly similar biological effects.In addition to inducingmelanocyte pro-liferation, UV irradiation has profoundimmunosuppressive and immuno-modulatory effects, and it is well estab-lished that immunosuppression in-creases the risk of both nevusformation and melanoma develop-ment. In view of the special character-istics of newborn skin and the immunesystem, intensive NBLP may mean anacute shock-like attack on the imma-ture melanocytes of the epidermis.

We also observed a signicantly ele-vated number of IF among participantswith anamnestic NBLP. The number ofmelanocytic lesions of the iris in ourstudy proved to be age independent,which can be explained by the time

TABLE 8 Continued

Factors No. of Subjects P

Use of sunbedsNever 125/100Occasionally 5/11Regularly 2/2 .64

Family history of large numbers of melanocytic neviNo 62/53Yes 70/50 .86

TABLE 9 Factors Associated With thePrevalence of Benign PigmentedOcular Lesions: Results ofMultivariate Logistic RegressionAnalysis

P OddsRatio

95% CondenceLimits for Odds

Ratio

Lower Upper

Blue light 0.001 3.778 1.694 8.423

CI indicates condence interval. The multivariate logisticregression (stepwisemethod)model included gender, eyecolor, hair color, skin color, skin phototype, the frequencyof use of sunscreens, a history of severe painful sunburns,the frequency and duration of sunbathing, the number ofdays per week when 4 hours was spent outdoors, thenumber of summer holidays beside the sea in the Mediter-ranean or in a subtropical or tropical climate, the use ofsunbeds, a family history of large numbers of melanocyticnevi, a history of NBLP, and age. The number of ocularlesions did not show normal distribution by theKolmogorov-Smirnov test, and due to the high numbers of0 values in the survey, multivariate logistic regressionwasperformed.

TABLE 10 Summary of Statistical Analyses (Mann-Whitney Test; P Values) for the GenePolymorphism Skin/Ocular Pigmented Lesions Associations

Variable CommonCMN

CAMN All Melanocytic Neviof the Skin

(CMN CAMN)

IF IN CHN IF IN CHN

HAL .81 .15 .63 .9 .96 .86R151C_R160W .99 .62 .92 .53 .76 .62SNP6 .95 .71 .91 .7 .74 .76SNP7 .95 .71 .91 .7 .74 .76SNP8 .65 .97 .78 .79 .68 .86

HAL indicates I439V polymorphism of the histidase gene; R151C_R160W, the presence of either of the RHC alleles (R151C andR160W) of MC1R gene; SNP6, the presence of any of the most frequent 6 MC1R polymorphisms (V60L, V92M, R151C, R160W,W163Q, and I155T); SNP7, the presence of any of the SNP6 group and the rare I120T polymorphism; SNP8, the presence ofeither of SNP7 and the newly identied W169R polymorphism.

TABLE 11 Summary of Multivariate StatisticalAnalyses

Variable PigmentedLesions of the

Skin(CMN CAMN)a

PigmentedOcular Lesions(IF IN CHN)b

HAL .62 .88Blue light .011 .010Age .000 .4

R151C_R160W .35 .56Blue light .05 .010Age .000 .45

SNP6 .82 .65Blue light .004 .010Age .000 .39



HAL indicates I439V polymorphism of the histidase gene;R151C_R160W, the presence of either of the RHC alleles(R151C and R160W) of the MC1R gene; SNP6, the presenceof any of the most frequent 6 MC1R polymorphisms (V60L,V92M, R151C, R160W, W163Q, and I155T); SNP7, the pres-ence of any of the SNP6 group and the rare I120T polymor-phism; SNP8, presence of either of SNP7 and the newlyidentied W169R polymorphism.a According to the results of the Kolgomorov-Smirnov test,the number of CMN CAMN did not show normal distri-bution. Analysis of variance was therefore performed onthe ln(CMN CAMN).b According to the results of the Kolgomorov-Smirnov test,the number of IF IN CHN did not show normal distri-bution, and because of the high numbers of 0 values in thesurvey, logistic regression was performed.

e862 CSOMA et al

course of iris pigmentation: the con-centration of melanin peaks duringearly childhood, thereafter usually re-maining constant throughout life,unless affected by certain oculardisorders, which can lead tohypopigmentation or hyperpigmenta-tion.36,37 The eyes of phototreated in-fants are routinely patched to excludeeye burning.38,39 Although eye patchingshields and phototherapy hoods areeffective in reducing the intensity of in-cident light, accidental exposure mayoccur.40,41 Patches are prone to slip:there may be difculties in securingeye shields effectively. Conversely, theprecise patching of an infants eyemaybe of secondary importance to moreimmediate and potentially life-preserving interventions. Although thepotential hazard of blue light is allevi-ated by the fact that neonates tend tokeep their eyes shut in bright light, it isknown that light in the visible spec-trum penetrates the skin. The level ofblue light transmission through theclosed eyelids of infants cannot be as-sessed with accuracy. With regard tothe light transmissibility prole of theneonatal cornea and crystalline lens,which allows the penetration of an ap-preciable amount of potentially harm-ful light into the eye, additional studies

are needed to clarify the possible long-term effects of neonatal blue light ex-posure on the melanocytic prolifera-tion of the uveal tract.42,43 Our resultsindicate the importance of appropri-ate eye care and eye protection of in-fants receiving phototherapy. In theevent of unavoidable phototherapytreatment, alternative methods of eyeprotection should be used to minimizeaccidental blue light exposure of theextremely vulnerable neonatal eye.

It is well established that both environ-mental and genetic factors contributeto melanoma-predisposing melano-cytic nevus development. To investi-gate whether polymorphisms knownto be associated with human pigmen-tation, melanoma predisposition2527

and skin immune functions2831 can en-hance the effects of NBLP, we investi-gated several SNPs of the MC1R andHAL genes. The statistical analysis re-vealed that the examined polymor-phisms of these genes do not contrib-ute either to an elevated number ofpigmented skin lesions or to the ap-pearance of pigmented uveal lesions.In this respect, we failed to demon-strate any gene-environmental inter-actions in our cohort, although thestudy group was sufciently large forthe effects of MC1R polymorphisms on

the skin type to be detected. We cannotexclude the possibility that, throughenlargement of our cohort, the contri-bution of some polymorphisms mightbecome apparent, but the presentndings clearly suggest that NBLP hasa much more robust effect on the de-velopment of pigmented lesions com-paredwith the possible contribution ofgenetic factors.

CONCLUSIONS

Our new epidemiologic data suggestthat NBLP couldwell be a risk factor formelanocytic nevus development. Pho-totherapy with blue light lamps is cur-rently a standard and essential thera-peutic modality in neonatal care;additional studies are therefore neces-sary to establish its potential long-termadverse effects. We suggest that a morerestricted treatment protocol should beintroduced to rule out the unnecessaryapplication of NBLP and thereby preventits possible adverse effects.

ACKNOWLEDGMENTSThis studywas supported by the ETT (Na-tional Fund of the Hungarian Ministry ofHealth) 429-07, the TMOP-4.2.2-08/1-2008-0001, the TMOP-4.2.1/B-09/1/KONV-2010-0005, and the OTKA (Hungarian Sci-entic Research Fund) 5K302 grants.

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(Continued from rst page)

www.pediatrics.org/cgi/doi/10.1542/peds.2011-0292

doi:10.1542/peds.2011-0292

Accepted for publication Jun 14, 2011

Address correspondence to Zsanett Csoma, MD, PhD, Department of Dermatology and Allergology, University of Szeged, PO Box 427, H-6701 Szeged, Hungary.E-mail: [email protected]

PEDIATRICS (ISSN Numbers: Print, 0031-4005; Online, 1098-4275).

Copyright 2011 by the American Academy of Pediatrics

FINANCIAL DISCLOSURE: The authors have indicated they have no nancial relationships relevant to this article to disclose.

e864 CSOMA et al

Neonatal Blue Light Phototherapy and Melanocytic Nevi: A Twin StudyMETHODSPatientsSkin ExaminationsInterviewOphthalmologic ExaminationDetermination of MC1R Gene Variants and the I439V Polymorphism of the HAL GeneStatistical Analyses

RESULTSSkin ExaminationsOphthalmologic ExaminationMC1R and HAL Polymorphisms

DISCUSSIONCONCLUSIONSACKNOWLEDGMENTSREFERENCES

neonatal blue light phototherapy and melanocytic nevi- a twin study

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