Margareta ; Ramsjö

Eleni Aklillu
email: eleni.aklillu@ki.se

Lilleba Bohman

Magnus Ingelman-Sundberg

Hyung-Keun Roh

Leif Bertilsson

Hyung- Keun Roh

M.Sc B Pharm

Hyung- Keun Roh

CYP2C19 activity comparison between Swedes and Koreans: Effect of genotype, sex, oral contraceptives use and smoking

Keywords: CYP2C19, omeprazole, gender, Korean, Swedes

come

Introduction

The enzyme CYP2C19 catalyses the metabolism of a wide variety of drugs such as antidepressants [1] , benzodiazepines [2] , proguanil [3] and proton pump inhibitors (PPIs) [4]. The major metabolites of omeprazole detected in vivo in human plasma are 5hydroxyomeprazole formed by CYP2C19 and omeprazole sulphone formed by CYP3A4 [5,6]. The metabolic disposition of omeprazole correlates well with the rate of S-mephenytoin hydroxylation [7] and those two drugs are the most commonly used probes of CYP2C19. About 3 % of Caucasians are PM of S-mephenytoin [8 , 9] and a higher incidence of PMs has been reported in Japanese (18-23 %) [10,11] Chinese (15-17%) [12,13] and in Korean subjects (13-16%) [14]. The PM phenotype is caused by the CYP2C19*2 [15] and CYP2C19*3 alleles although the CYP2C19*3 is very uncommon in Caucasian PMs [16]. Sim et al [17] identified a novel allele (CYP2C19*17) that was associated with increased CYP2C19 activity in vivo in two different ethnic populations. This allele is associated with higher levels of gene transcription and increased rates of omeprazole and mephenytoin metabolism. The frequency of this allele was 18% in both Swedes and Ethiopians but only 4 % in Chinese subjects [17].

In general previous studies reported lower CYP2C19 enzyme activity among Asians compared to White population. Several factors may contribute for the observed inter ethnic differences in enzyme activity such as differences in functional variant allele frequencies between populations and other environmental factors like dietary, oral contraceptive (OC) use or smoking habit. Indeed CYP2C19 defective variant alleles occur at a higher frequency in Asians as compared to whites and this may account partly for the observed lower CYP219 activity in this population. Furthermore previously it is reported that OC use inhibits CYP2C19 enzyme activity [18] and hence variation in OC use habit may also contribute to some extent. As to our knowledge no study has compared the enzyme activity between the genotype phenotype relationship differences using the same methodology between healthy Swedish and Korean subjects having the same CYP2C19 genotype, OC use and smoking habit. We find an important gender difference in the CYP2C19 activity in Koreans but not in Swedes and a generally lower CYP2C19 activity among Koreans in subjects homozygous for CYP2C19*1.

Materials and Methods

Study Subjects

We studied 185 healthy Swedish subjects at the Karolinska University Hospital, Huddinge, Sweden and 150 healthy Korean subjects at the Inha University Hospital, Incheon, Korea. The subjects were mainly students and staff at the two hospitals. In the Swedish subjects the mean age was 29 ± SD 8.3 years (range 18-60) with the mean weight 69 ± SD 12 kg (range 47-109) (no data on weight in two subjects). The Swedish subjects consisted of 112 women and 73 men and 39 (11 men and 28 women) were classified as smokers (definition of smokers > 2 cigarettes/ day) as described previously [19,20]. There were 44 women using OC. Among the 28 women smokers, 6 were OC user. In four women no data on OC use was available. In the Korean subjects the mean age was 25 ± 3.9 years (range with the mean weight 62 ± 12 kg (range 38-94). The Korean subjects consisted of 74 women and 76 men and 29 of the subjects were classified as smokers (26 men and 3 women of whom none were OC user). In one subject information on smoking habit was unavailable. Only one Korean woman was a user of OC. The participants were asked if they used OC but not which kind of contraceptives they used. Incheon, Korea approved the study and it was performed in accordance with the Helsinki Declaration. After written and oral information about the study, the participants were screened by clinical laboratory tests and interviewed for their medical histories. The subjects were not allowed to use other medications (except for OC) including herbal medicines one week before the study started. They refrained from alcohol, grapefruit juice during 2 days and caffeine products during 1, 5 days before the study started. The Karolinska cocktail included omeprazole 20 mg (enterotablet of Losec Mups 20 mg; Hässle, Stockholm, Sweden), losartan 25 mg, caffeine 100 mg, quinine 250 mg and debrisoquine 10 mg [21] .

Phenotyping

After an 8-hour overnight fast, the subjects took the drug omeprazole at 8 a.m. Three hours after the intake of omeprazole, blood samples were collected and centrifuged, plasma was separated and stored frozen at -20 o until analysis by reversed phase HPLC with UV detection.

The Korean plasma and DNA samples were packed on dry ice and sent to Sweden for analysis. The method for measuring omeprazole and its metabolite 5-hydroxyomeprazole was described by Tybring et al [22]and modified by Christensen et al [21].

Genotyping

Blood samples were collected using tubes containing EDTA. Genomic DNA was extracted from leukocytes by using QIAampDNA Mini Blood Kit (distributed by AB Applied Bio systems, California, USA and Chestrite, United Kingdom). Genotyping methods are based on the PCR (Polymerase-chain-reaction) technique and specific primers to different parts of the F o r P e e r R e v i e w 6 gene. Allelic discrimination was determined by using a Taqman assay. Genotyping of CYP2C19*17 was carried out by PCR with specific primers as described by Sim et al [17].

Statistical analysis

The individual metabolic ratio MR of omeprazole was determined by dividing the molar concentrations of omeprazole and 5-hydroxyomeprazole in plasma.

The MRs was log transformed before application of statistical analysis. For comparisons of Swedes and Koreans within each genotype group and between groups based on smoking and OC use the independent t-test was used. P-Values less than 0.05 were considered statistically significant. Statistical analyses were performed by Statistical version 7 (Stat Soft Scandinavia AB).

Results

CYP2C19 genotype:

The study encompassed 185 Swedes and 150 Koreans. We evaluated results after administering the Karolinska cocktail with the use omeprazole as a probe drug for phenotyping of CYP2C19. The genotype analyses revealed allele frequencies of CYP2C19*2, and *17 in the Swedes of 16 and 20 %, respectively and 28, 11 and 0.3 % of *2, *3 and *17 in the Koreans, respectively (Table 1). In both Koreans and Swedes, there was no significant difference between the observed and expected genotype frequency according to Hardy-Weinberg law (χ² test, p>0.05).

CYP2C19 phenotype:

The subjects were phenotyped for omerazole and Figure 1 shows histograms of the MR (omeprazole/5-hydroxyomeprazole). A bimodal distribution especially in Koreans with an antimode of about 10 was observed. The median MR in Koreans was substantially higher (2.57) as compared to the Swedes (0.71) (p-value <0.00001). The mean

CYP2C19 phenotype comparison between Swedes and Koreans:

For statistical analysis and comparison of CYP2C19 enzyme activity, subjects were first stratified based on CYP2C19 genotype. The subjects homozygous for CYP2C19*1 were further stratified for gender and intake of OC (Figure 2). Interestingly the Koreans homozygous for CYP2C19*1 had significantly higher MR for omeprazole; the MR in Korean men was 1.91 vs. 0.56 in Swedish men (p<0.000001) and an MR of 0.91 was seen in Korean females as compared to 0.58 in Swedish women who were none OC users (p=0.022). In Koreans a gender difference was seen where men (n=30) had higher MR (1.91) as compared to females (n=24) (0.91) (p<0.0001). By contrast, no gender differences were seen among the Swedes. Among the Swedish women, OC had a pronounced inhibitory effect (p<0.00001), whereas no Korean female on OC was included in the study since OC usage was only reported by one Korean woman.

The results of the phenotyping of the two populations are also shown stratified according to the number of active genes (with CYP2C19*17 excluded) with the two genders together (Figure 3). Korean subjects homozygous for CYP2C19*1 had much higher MR (1.48) than the Swedish individuals (0.58) (p<0.000001). A similar difference was also noted when comparing those with one functional allele (2.88 vs 1.10, p<0.0012) and also a small difference (28.8 vs 20.9, p<0.028) was noted among those homozygous for null alleles although the number of subjects in the Swedish *0/*0 group is very low. [17].

There was no effect of smoking on MR neither in Swedes (p=0.75) nor in Koreans (p=0.50) in the genotype CYP2C19 *1/ *1 by using the independent t-test. Considering smokers and non-smokers separately, effect of CYP2C19 genotype on enzyme activity remained significant in both Koreans (p<0.0001) and Swedes (P<0.0003). We further investigated for interaction between genotype, oral contraceptive use and smoking using factorial ANOVA in Swedes. Since there was only one OC user in Koreans we did not perform this analysis in Koreans. The result indicated only oral contraceptive use (p<0.001) and CYP2C19 genotype (p<0.001) but not smoking affects CYP2C19 activity. Regardless of smoking habit, oral contraceptive use inhibited and CYP2C19 genotype influenced CYP2C19 enzyme activity.

Discussion

In the present study we compared CYP2C19 enzyme activity between Koreans and Swedes using the same methodology and taking into consideration effect of genotype, sex, OC use.

Our major finding include quite unexpected and very interesting differences in the CYP2C19 phenotype between Koreans and Swedes belonging to the same genotype group, namely for those homozygous for CYP2C19*1. Phenotyping data based on the probe drug omeprazole revealed that the Korean men had a much higher MR for omeprazole than the Swedish men.

Interestingly Korean women were significantly more rapid for omeprazole metabolism than the Korean men. Still the Korean women were slower than Swedish women without intake of oral contraceptives, but the difference was much less than observed between Swedish and Korean men. Oral contraceptive intake did decrease the rate of omeprazole metabolism in Swedish women to approach a similar MR for omeprazole as Korean men. Korean subjects carrying one null allele were also slower as compared to Swedes and a slight difference was seen between men and women homozygous for null alleles (Figure 3), but this difference was very small and the number of Swedish subjects in this group was also very low. Stratifying the phenotype data for smokers or nonsmokers revealed no influence of smoking habit on CYP2C19 enzyme activity.

Previously a small hardly significant gender difference in CYP2C19 activity was observed in Chinese using S-mephenytoin as a probe drug [23]. Here the S/R-ratio for mephenytoin was lower in homozygous extensive metabolizers (EM) females than in homozygous EM males (0.22 +/-0.14 vs. 0.33 +/-0.09; p = 0.046). By using omeprazole as a probe drug in the current study in Koreans, much higher significance values were obtained for a gender difference indicating that such a gender difference is true in some Asian populations.

The mechanism behind the gender difference in Koreans in CYP2C19 activity is unknown.

One could imagine environmental, epigenetic or genetic reasons. Regarding environmental influence differences in diets or drug habits between Korean men and women are unlikely, but could not be excluded. Also one could hypothesize that hormonal or environmental influence should result in gender specific epigenetic control of the CYP2C19 gene expression which would influence the activity. This cannot be excluded and a true evaluation would require liver samples from both sexes where the pattern of gene methylation as well as histone acetylation and methylation could be investigated [24]. Perhaps the most likely explanation is of true genetic origin. The differences in sequence between the Caucasian and Korean CYP2C locus as revealed from whole genome sequencing in the CYP2C locus is extensive and encompasses several hundreds of mutations [25]. One could therefore suggest that the Korean variant of the CYP2C19*1 allele carries mutations that makes it less expressed as compared to the Swedish variant, and at the same time there are mutations in this locus that makes the Korean CYP2C19*1 gene more sensitive for the estrogen receptor or other nuclear factors controlled by the action of estrogens, thereby causing an increased rate of CYP2C19 activity in the Korean females as compared to males. Further studies have to be designed that investigate these alternatives before the basis for the gender difference is understood.

The Swedish females taking oral contraceptives had lower CYP2C19 activity as compared to women without. Tamminga et al. have also describes an effect of OCs using S-mephynotoin as a probe drug [26]. The oral contraceptives may contain progestin with or without ethinyl oestradiol and it is not known whether it is the oestrogen or the progestin component that inhibits drug metabolism [10]. The inhibition of CYP2C19 activity by OC seems to be due to the ethinyloestradiol component of OCs [27,28]. The mechanism behind this effect is unknown but has been suggested to be interaction at the active site between the hormones and the CYP2C19 substrates. A mechanism where the oral contraceptives directly inhibit the rate of CYP2C19 expression can however not be excluded.

Our result indicated no significant effect of cigarette smoking on CYP2C19 enzyme activity in both populations. Even controlling for the effect of genotype and OC use there was no significant difference between smokers and non-smokers on enzyme activity. Among cytochrome P450 drug metabolizing enzymes that expressed in the liver, CYP1A2 is the only one whose activity is affected by smoking habit [20]. Previous studies reported interaction between smoking and CYP2C19 genotype in influencing H.Pylori eradication success and suggested interaction between smoking and CYP2C19 should be examined in the future [START_REF] Suzuki | Influence of smoking and CYP2C19 genotypes on H. pylori eradication success[END_REF].

Apparently effect of smoking on H.Pylori eradication might be not by directly altering CYP2C19 enzyme activity and hence omeprazole metabolism. Our study also can be a base for further investigation to identify the underlying genetic, epigenetic or environmental factors responsible for the observed wide differences in CYP2C19 enzyme activity between Koreans and Swedes.

Limitation of present study includes that subjects were genotyped for the most common variant alleles in the respective population and other variants of the CYP2C19 alleles were not analyzed because of their low frequencies. However contribution of these rare variant alleles to the observed difference in enzyme activity between the two populations cannot be ruled out. In summary, the present study describes an interesting gender difference in the CYP2C19 enzyme activity in Koreans which cannot be seen in Swedes. Further studies are needed to evaluate the mechanisms behind this difference, which however, would drastically influence the pharmacokinetics of CYP2C19 drugs as to motivate gender specific dosing regimens in Koreans. -Interaction between smoking habit, oral contraceptive use and CYP2C19 genotype were performed using factorial ANOVA. Regardless of smoking habit, oral contraceptive use and genotype affected CYP2C19 enzyme activity.

F

Effect of oral contraceptive on enzyme activity was tested only in Swedes since there was only on oral contraceptive user in Koreans. The result is now described under "Result" Section, last paragraph on page 7

2. Were quality controls included during the transport of the samples from Korea to Sweden to ensure that the samples were not affected by transport conditions?

Authors reply:

-We did not use any specific controls on the Korean samples as they were sent by courier packed with dry ice to Sweden. Upon arrival to Stockholm, the dry ice was still intact. Furthermore the samples were analyzed by the same methods and during the same conditions as the Swedish samples in the same laboratory with the same laboratory personnel.

3. Which oral contraceptives were used by Swedish subjects? This information should be added to the text.

Authors reply:

-The participants were asked if they used contraceptives, not which kind of contraceptives they used.

-This information is now included under "Materials and Methods" section on the 1 st paragraph, last sentence on page 4. This information should be added.

F

Authors reply:

-We have now inserted the respective 95% CI for allele frequency in Table 1.

-Statement about compliance to the Hardy Weinberg equation is now described under "Result" Section, 1 st paragraph on page 6 as follows Limitation of present study includes that subjects were genotyped for the most common variant alleles in the respective population and other variants of the CYP2C19 alleles were not analyzed because of their low frequencies. However contribution of these rare variant alleles to the observed difference in enzyme activity between the two populations cannot be ruled out.

6. Definition of smoking was defined as ">2 cigarettes/day". A reference or logic for this assumption could be provided.

Authors reply:

-This definition is a subjective definition of smoking and this factor as smoking -We have now cited these papers as a reference under "Materials and Methods" section on the 1 st paragraph, last sentence on page 4.

7. OC should be stated as "oral contraceptive" at the first place in the text.

Authors reply:

-

Authors reply:

the following sentence is now inserted "Result" Section, on page 7, 1 st paragraph -"an MR of 0.91 was seen in Korean females as compared to 0.58 in Swedish women who were none OC users (p=0.022)

Reviewer: 2

General comments:

The article entitled "Interethnic and gender dependent differences in CYP2C19

Authors reply:

We agree and now have changed the title to "CYP2C19 activity comparison between Swedes and Koreans: Effect of genotype, sex, oral contraceptives use and smoking".

2. Present layout of the article is somewhat confusing and need improving:

Abstract:

Objectives, conclusions and the article title are not in accordance with one another.

Authors reply:

Introduction:

In this part, the authors should state the grounds for conducting the study and refer to what new information this article would provide. There needs a strong scientific rationale to conduct the study.

Authors reply:

-The rational to conduct the study and aims of the study is now clearly sated in the introduction part "Introduction" section on the last paragraph on page 4 as follows.

"In general previous studies reported lower CYP2C19 activity among Asians compared to White population. Several factors may contribute for the observed inter ethnic differences in enzyme activity such as differences in functional defective variant alleles occur at a higher frequency in Asians as compared to whites and this may account partly for the observed lower CYP219 activity in this population. Furthermore previously it is reported that OC use inhibits CYP2C19

enzyme activity [18] and hence variation in OC use habit may also contribute to some extent. As to our knowledge no study has compared the enzyme activity between the two populations controlling for the effect of confounding factors such as oral contraceptive use and CYP2C19 genotype. The aim of the present study was to investigate the CYP2C19 genotype phenotype relationship differences using the same methodology between healthy Swedish and Korean subjects having the same CYP2C19 genotype, OC use and smoking habit by. and to assess effect of sex, cigarette smoking and OC use on enzyme activity We evaluated results after administering the Karolinska cocktail with the use omeprazole as a probe drug for phenotyping of CYP2C19. We find an important gender difference in the CYP2C19 activity in Koreans but not in Swedes and a generally lower CYP2C19 activity among Koreans in subjects homozygous for CYP2C19*1.

• L52-60 on P3 should sit in the part of results

Authors reply: Agreed. We have moved this sentence to the Result section, 1 st paragraph on page 6 as follows

The study encompassed 185 Swedes and 150 Koreans. We evaluated results after administering the Karolinska cocktail with the use omeprazole as a probe drug for phenotyping of CYP2C19.

• Results:

In order to avoid unnecessary confusion, recommend the author present the results in two parts, first the results of genotyping and then that of phenotyping. • The first paragraph states nothing new other than repeating the results. I recommend the author condense this paragraph.

Authors reply:

Authors reply:

We have now modified the first paragraph of the discussion as suggested (page 8)

• As to the reasons of gender difference, is it possible that the difference is related to the different probe drugs used?

Authors reply: We used the same probe drug omeprazole

• No effect of smoking was mentioned in this part.

Authors reply: Effect of smoking is now presented in the "Result" on page 8 and discussed in the "Discussion# part on page 10-11 as follows

Result:

There was no effect of smoking on MR neither in Swedes (p=0.75) nor in Koreans (p=0.50) in the genotype CYP2C19 *1/ *1 by using the independent t-test. Considering smokers and non-smokers separately, effect of CYP2C19 genotype on enzyme activity remained significant in both Koreans (p<0.0001) and Swedes (P<0.0003). We further investigated for interaction between genotype, oral contraceptive use and smoking using factorial ANOVA in Swedes. Since there was only one OC user in Koreans we did not perform this analysis in Koreans. The result indicated only oral contraceptive use (p<0.001) and CYP2C19 genotype (p<0.001) but not smoking affects CYP2C19 activity.

Regardless of smoking habit, oral contraceptive use inhibited and CYP2C19 genotype influenced CYP2C19 enzyme activity.

Discussion:

Our result indicated no significant effect of cigarette smoking on CYP2C19 enzyme activity in both populations. Even controlling for the effect of genotype and OC use there was no significant difference between smokers and non-smokers on enzyme activity. Among cytochrome P450 drug metabolizing enzymes that expressed in the liver, CYP1A2 is the only one whose activity is affected by smoking habit [20]. Previous studies reported interaction between smoking and CYP2C19 genotype in influencing H.Pylori eradication success and suggested interaction between smoking and CYP2C19 should be examined in the future [START_REF] Suzuki | Influence of smoking and CYP2C19 genotypes on H. pylori eradication success[END_REF]. Apparently effect of smoking on H.Pylori eradication might be not by directly altering CYP2C19 enzyme activity and hence omeprazole metabolism.

• Clinical implications of the present study should be discussed. The author discussed very little about this issue.

Authors reply:

The clinical implications of our study is now discussed in the "Discussion# part on page 11 as follows

The finding of significantly lower CYP2C19 enzyme activity in Koreans compared to Swedes that is not accountable or explained by the common CYP2C19 genotype or OC use may have clinical relevance. In clinical practice the common CYP2C19 SNPs that are recommended or practiced to genotype for include CYP2C19*2 and *3 in Asians. Our

Study indicates genotyping for these SNPs only may not provide full information about individual's metabolic capacity or phenotype. Alternatively, phenotyping with a safe probe drug to reflect an individual´s metabolic capacity or therapeutic drug monitoring may need to be considered. Our study can be a base for further investigation to identify the underlying genetic, epigenetic or environmental factors responsible for the observed wide differences in CYP2C19 enzyme activity between Koreans and Swedes.

• Limitation of present study should be discussed briefly

Authors reply: Limitation of present study now described under "Discussion" section Last paragraph on page 11 as follows.

Limitation of present study includes that subjects were genotyped for the most common variant alleles in the respective population and other variants of the CYP2C19 alleles were not analyzed because of their low frequencies. However contribution of these rare Authors reply: In the revised manuscript we have done our best to correct spelling errors and improve the linguistics.

Minor:

• ? "indeed was the case, i.e. that the reporting was true."(Line 51, page 7)

Authors reply: This sentence is now removed from the Discussion part 1 st paragraph on page 9

• "EM female", EM here should be written in full for the first appearance in the article (Line 10, page 8)

Authors reply: This abbreviation EM is now fully stated where it first appears in the text Discussion part 1 st paragraph on page 9

• "….could be investigated cf. [21].", cf should be deleted. (Line 39 and 46, page 8)

"indeed" is superfluous (Line 15, page 9)

"effect)" should be "effect" (Line 22, page 9) ?"would not drastically", here would or would not? (Line39, page 9)

Authors reply: We agree and now made the correction in the revised manuscript as indicated in the Discussion part on page 9 and 10.

F

Introduction

The enzyme CYP2C19 catalyses the metabolism of a wide variety of drugs such as antidepressants [1] , benzodiazepines [2] , proguanil [3] and proton pump inhibitors (PPIs) [4]. The major metabolites of omeprazole detected in vivo in human plasma are 5hydroxyomeprazole formed by CYP2C19 and omeprazole sulphone formed by CYP3A4 [5,6]. The metabolic disposition of omeprazole correlates well with the rate of S-mephenytoin hydroxylation [7] and those two drugs are the most commonly used probes of CYP2C19.

About 3 % of Caucasians are PM of S-mephenytoin [8 , 9] and a higher incidence of PMs has been reported in Japanese (18-23 %) [10,11] Chinese (15-17%) [12,13] and in Korean subjects (13-16%) [14]. The PM phenotype is caused by the CYP2C19*2 [15] and CYP2C19*3 alleles although the CYP2C19*3 is very uncommon in Caucasian PMs [16]. Sim et al [17] identified a novel allele (CYP2C19*17) that was associated with increased CYP2C19 activity in vivo in two different ethnic populations. This allele is associated with higher levels of gene transcription and increased rates of omeprazole and mephenytoin metabolism. The frequency of this allele was 18% in both Swedes and Ethiopians but only 4

% in Chinese subjects [17].

Christensen et al [18] developed the Karolinska cocktail based on five probe drugs to phenotype the enzymes CYP1A2, CYP2C9, CYP2D6, CYP3A4 and CYP2C19. In general previous studies reported lower CYP2C19 enzyme activity among Asians compared to White population. Several factors may contribute for the observed inter ethnic differences in enzyme activity such as differences in functional variant allele frequencies between populations and other environmental factors like dietary, oral contraceptive (OC) use or smoking habit. Indeed CYP2C19 defective variant alleles occur at a higher frequency in Asians as compared to whites and this may account partly for the observed lower CYP219 activity in this population.

Furthermore previously it is reported that OC use inhibits CYP2C19 enzyme activity [18] and

Materials and Methods

Study Subjects

We studied 185 healthy Swedish subjects at the Karolinska University Hospital, Huddinge, Sweden and 150 healthy Korean subjects at the Inha University Hospital, Incheon, Korea. The subjects were mainly students and staff at the two hospitals. In the Swedish subjects the mean age was 29 ± SD 8.3 years (range 18-60) with the mean weight 69 ± SD 12 kg (range 47-109) (no data on weight in two subjects). The Swedish subjects consisted of 112 women and 73 men and 39 (11 men and 28 women) were classified as smokers (definition of smokers > 2 cigarettes/ day) as described previously [19,20]. There were 44 women using OC. Among the 28 women smokers, 6 were OC user. In four women no data on OC use was available. In the Korean subjects the mean age was 25 ± 3.9 years (range 20-46) with the mean weight 62 ± 12 kg (range 38-94). The Korean subjects consisted of 74 women and 76 men and 29 of the subjects were classified as smokers (26 men and 3 women of whom none were OC user). In one subject information on smoking habit was unavailable. Only one Korean woman was a

Phenotyping

After an 8-hour overnight fast, the subjects took the drug omeprazole at 8 a.m. Three hours after the intake of omeprazole, blood samples were collected and centrifuged, plasma was separated and stored frozen at -20 o until analysis by reversed phase HPLC with UV detection.

The Korean plasma and DNA samples were packed on dry ice and sent to Sweden for analysis. The method for measuring omeprazole and its metabolite 5-hydroxyomeprazole was described by Tybring et al [22]and modified by Christensen et al [21].

Genotyping

Blood samples were collected using tubes containing EDTA. Genomic DNA was extracted from leukocytes by using QIAampDNA Mini Blood Kit (distributed by AB Applied Bio Allelic discrimination was determined by using a Taqman assay. Genotyping of CYP2C19*17 was carried out by PCR with specific primers as described by Sim et al [17].

Statistical analysis

The individual metabolic ratio MR of omeprazole was determined by dividing the molar concentrations of omeprazole and 5-hydroxyomeprazole in plasma. 1). In both Koreans and Swedes, there was no significant difference between the observed and expected genotype frequency according to Hardy-Weinberg law (χ² test, p>0.05).

CYP2C19 phenotype:

The subjects were phenotyped for omerazole and Figure 1 shows 2). Interestingly the Koreans homozygous for CYP2C19*1 had significantly higher MR for omeprazole; the MR in Korean men was 1.91 vs. 0.56 in Swedish men (p<0.000001) and an MR of 0.91 was seen in Korean females as compared to 0.58 in Swedish women who were none OC users (p=0.022). In Koreans a gender difference was seen where men (n=30) had higher MR (1.91) as compared to females (n=24) (0.91) (p<0.0001). By contrast, no gender differences were seen among the Swedes. Among the Swedish women, OC had a pronounced inhibitory effect (p<0.00001), whereas no Korean female on OC was included in the study since OC usage was only reported by one Korean woman.

histograms
The results of the phenotyping of the two populations are also shown stratified according to the number of active genes (with CYP2C19*17 excluded) with the two genders together (Figure 3). Korean subjects homozygous for CYP2C19*1 had much higher MR (1.48) than the Swedish individuals (0.58) (p<0.000001). A similar difference was also noted when comparing those with one functional allele (2.88 vs 1.10, p<0.0012) and also a small difference (28.8 vs 20.9, p<0.028) was noted among those homozygous for null alleles although the number of subjects in the Swedish *0/*0 group is very low.

F

Discussion

In the current investigationpresent study we compared CYP2C19 enzyme activity between Koreans and Swedes using the same methodology and taking into consideration effect of genotype, sex, OC use. Our major finding include we found quite unexpected and very interesting differences in the CYP2C19 phenotype between Koreans and Swedes belonging to the same genotype group, namely for those homozygous for CYP2C19*1. Phenotyping data based on the probe drug omeprazole revealed that the Korean men had a much higher MR for omeprazole than the Swedish men. (p<0.000001, Figure 2). Interestingly Korean women were significantly more rapid for omeprazole metabolism than the Korean men (p<0.0001).

Still the Korean women were slower than Swedish women without intake of oral contraceptives, but the difference was much less than observed between Swedish and Korean men. Oral contraceptive intake did decrease the rate of omeprazole metabolism in Swedish women to approach a similar MR for omeprazole as Korean men. Only one Korean women was reported to take oral contraceptives and the relatively low MRs seen in among female Koreans indicate that this indeed was the case, i.e. that the reporting was true. Korean subjects carrying one null allele were also slower as compared to Swedes and a slight difference was seen between men and women homozygous for null alleles (Figure 3), but this difference was very small and the number of Swedish subjects in this group was also very low. Stratifying the phenotype data for smokers or nonsmokers revealed no influence by of smoking habit on CYP2C19 enzyme activity.

Previously a small hardly significant gender difference in CYP2C19 activity was observed in Chinese using S-mephenytoin as a probe drug [23]. Here the S/R-ratio for mephenytoin was lower in homozygous EM extensive metabolizers (EM) females than in homozygous EM males (0.22 +/-0.14 vs. 0.33 +/-0.09; p = 0.046). By using omeprazole as a probe drug in the current study in Koreans, interestingly much higher significance values were obtained for a gender difference indeed indicating that such a gender difference indeed is true in some Asian populations.

The mechanism behind the gender difference in Koreans in CYP2C19 activity is unknown.

One could imagine environmental, epigenetic or genetic reasons. Regarding environmental influence differences in diets or drug habits between Korean men and women are unlikely, but could not be excluded. Also one could hypothesize that hormonal or environmental influence should result in gender specific epigenetic control of the CYP2C19 gene expression which would influence the activity. This cannot be excluded and a true evaluation would require acetylation and methylation could be investigated cf. [24]. Perhaps the most likely explanation is of true genetic origin. The differences in sequence between the Caucasian and Korean CYP2C locus as revealed from whole genome sequencing in the CYP2C locus is extensive and encompasses several hundreds of mutations cf.- [25]. One could therefore suggest that the Korean variant of the CYP2C19*1 allele carries mutations that makes it less expressed as compared to the Swedish variant, and at the same time there are mutations in this locus that makes the Korean CYP2C19*1 gene more sensitive for the estrogen receptor or other nuclear factors controlled by the action of estrogens, thereby causing an increased rate of CYP2C19 activity in the Korean females as compared to males. Further studies have to be designed that investigate these alternatives before the basis for the gender difference is understood.

The Swedish females taking oral contraceptives had lower CYP2C19 activity as compared to women without. Tamminga et al. have also describes an effect of OCs using S-mephynotoin as a probe drug [26]. The oral contraceptives may contain progestin with or without ethinyl oestradiol and it is not known whether it is the oestrogen or the progestin component that inhibits drug metabolism [10]. The inhibition of CYP2C19 activity by OC seems to be due to the ethinyloestradiol component of OCs [27,28]. The mechanism behind this effect) is unknown but has been suggested to be interaction at the active site between the hormones and the CYP2C19 substrates. A mechanism where the oral contraceptives directly inhibit the rate of CYP2C19 expression can however not be excluded.

Our result indicated no significant effect of cigarette smoking on CYP2C19 enzyme activity in both populations. Even controlling for the effect of genotype and OC use there was no 11 significant difference between smokers and non-smokers on enzyme activity. Among cytochrome P450 drug metabolizing enzymes that expressed in the liver, CYP1A2 is the only one whose activity is affected by smoking habit [20]. Previous studies reported interaction between smoking and CYP2C19 genotype in influencing H.Pylori eradication success and suggested interaction between smoking and CYP2C19 should be examined in the future [START_REF] Suzuki | Influence of smoking and CYP2C19 genotypes on H. pylori eradication success[END_REF].

Apparently effect of smoking on H.Pylori eradication might be not by directly altering CYP2C19 enzyme activity and hence omeprazole metabolism.

The finding of significantly lower CYP2C19 enzyme activity in Koreans compared to Swedes that is not accountable or explained by the common CYP2C19 genotype or OC use may have some clinical relevance. In clinical practice the common CYP2C19 SNPs that are recommended or practiced to genotype for include CYP2C19*2 and *3 in Asians. Our Study indicates genotyping for these SNPs only may not provide full information about individual's metabolic capacity or phenotype. Alternatively, phenotyping with a safe probe drug to reflect an individual´s metabolic capacity or therapeutic drug monitoring may need to be considered.

Our study also can be a base for further investigation to identify the underlying genetic, epigenetic or environmental factors responsible for the observed wide differences in CYP2C19 enzyme activity between Koreans and Swedes.

Limitation of present study includes that subjects were genotyped for the most common variant alleles in the respective population and other variants of the CYP2C19 alleles were not analyzed because of their low frequencies. However contribution of these rare variant alleles to the observed difference in enzyme activity between the two populations cannot be ruled out. In summary, the present study describes an interesting gender difference in the CYP2C19

  for the effect of confounding factors such as oral contraceptive use and CYP2C19 genotype. The aim of the present study was to investigate the CYP2C19

  committees at Karolinska Institutet, Stockholm, Sweden and Inha University,

  was 3.04 while in Swedes 0.86.

  significantly lower CYP2C19 enzyme activity in Koreans compared to Swedes that is not accountable or explained by the common CYP2C19 genotype or OC use may have some clinical relevance. In clinical practice the common CYP2C19 SNPs that are recommended or practiced to genotype for include CYP2C19*2 and *3 in Asians. Our Study indicates genotyping for these SNPs only may not provide full information about individual's metabolic capacity or phenotype. Alternatively, phenotyping with a safe probe drug to reflect an individual´s metabolic capacity or therapeutic drug monitoring may need to be considered.

Fig. 1

 1 Fig.1

Fig. 2 3 or * 17 .

 2317 Fig.2

Fig. 3 -

 3 Fig.3

  has been studied before (Eur J Clin Pharmacol 2007: 63;537-546, Eur J Clin Pharmacol 2008;64:381-385) with the definition as a smoker; "smoking is to smoke between two or forty cigarettes per day".

-section on the last paragraph on page 3 . 8 .

 38 The abbreviation for oral contraceptives (OC) is stated in the Abstract under Objectives It is now sated also at the first place in the text under "Introduction" Page 6 (Results, lines[18][19]: The mean values should also be provided and a p value should be given.

-

  The p value and mean value for Swedes and Koreans is now described under "Result" Section, 1 st paragraph on page 6 as follows (p-value <0.00001). The mean MR among Koreans was 3.04 while in Swedes 0.86. 9. Page 6 (Results, lines: 30-32): It should be stated that subjects are non oral contraceptive.

1 .

 1 activity. A comparison between Swedes and Koreans" by Margareta Ramsjö et al.studied the interethnic difference in CYP2C19 activity and investigated the effect of gender, smoking and oral contraceptives (OC). The authors found that in the genotype CYP2C19 *1/ *1 group, gender difference of CYP 2C19 activity was seen in Koreans where men had higher MR as compared to females (1.91 vs 0.91, p<0.0001). No gender difference was seen among the Swedes. OC had a pronounced inhibitory effect among the Swedish women (p<0.00001). No effect of smoking was found in Swedes and Koreans.In my opinion, this study is well designed and the results are well presented with tables and figures. However the writing needs considerable improvement. Specific comments: The title "Interethnic and gender dependent differences in CYP2C19 activity. A comparison between Swedes and Koreans" dose not reflect the objectives of the study. I recommend the author revise the title and try to make it in accord with the objectives, main results and final conclusion. Providing there is no major change of the content, I would suggest the title be revised as "CYP2C19 activity comparison between Swedes and Koreans. Effect of gender, oral contraceptives (OC) and smoking".

  between populations and other environmental factors such as dietary, oral contraceptive (OC) use or smoking habit. Indeed CYP2C19

  Agreed: Now we have structured the result section under subheadings

  the observed difference in enzyme activity between the two populations cannot be ruled out.

3 .

 3 There are some spelling errors and the linguistics need improving. The authors should seek the services of those fluent in English before submitting the next version.

  OC use habit may also contribute to some extent. As to our knowledge no study has compared the enzyme activity between the two populations controlling for the effect of confounding factors such as oral contraceptive use and CYP2C19 genotype. The aim of the present study was to investigate the CYP2C19 genotype phenotype relationship differences using the same methodology between healthy Swedish and Korean subjects having the same CYP2C19 genotype, OC use and smoking habit. and to assess effect of sex, cigarette smoking and OC use on enzyme activity. We evaluated results after administering the Karolinska cocktail with the use omeprazole as a probe drug for phenotyping of CYP2C19 We find an important gender difference in the CYP2C19 activity in Koreans but not in Swedes and a generally lower CYP2C19 activity among Koreans in subjects homozygous for CYP2C19*1.

  . The participants were asked if they used OC but not which kind of contraceptives they used.The local Ethics committees at Karolinska Institutet, Stockholm, Sweden and Inha University, Incheon, Korea approved the study and it was performed in accordance with the Helsinki Declaration. After written and oral information about the study, the participants were screened by clinical laboratory tests and interviewed for their medical histories. The subjects were not allowed to use other medications (except for OC) including herbal medicines one week before the study started. They refrained from alcohol, grapefruit juice during 2 days and caffeine products during 1, 5 days before the study started. The Karolinska cocktail included omeprazole 20 mg (enterotablet of Losec Mups 20 mg; Hässle, Stockholm, Sweden), losartan 25 mg, caffeine 100 mg, quinine 250 mg and debrisoquine 10 mg[21] .

  , USA and Chestrite, United Kingdom). Genotyping methods are based on the PCR (Polymerase-chain-reaction) technique and specific primers to different parts of the gene.

  both sexes where the pattern of gene methylation as well as histone

Fig. 1

 1 Fig.1

Fig. 2 3 or * 17 .

 2317 Fig.2

Fig. 3

 3 Fig.3

  The CYP2C19*17 allele was only present in one Korean subject. Analysis of the MR for omeprazole among Swedes revealed that the MR in *17/ *17 was significantly lower (0.18,

	n=4) than in *1/ *1 (0.58, n=56, p=0.016) and *1/ *17 (0.51, n=35; p=0.048), in line with our
	previous results
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Table 1

 1 Distribution of CYP2C19 allele frequencies with the respective 95% CI in the Swedish Korean populations. CYP2C19*1 denotes alleles not identified as *2, *3 or *17. The *3 allele was not analysed in the Swedish cohort due to its very rare occurrence.

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Suzuki T, Matsuo K, Sawaki A, Wakai K, Hirose K, Ito H, Saito T, Nakamura T, Yamao K, Hamajima N, Tajima K (2007) Influence of smoking and CYP2C19 genotypes on H. pylori eradication success. Epidemiol Infect 135 (1): 171-176

Table 1 :

 1 95% confidence intervals should be added for the allele frequencies. Are the allele frequencies distributed in accordance with the Hardy Weinberg equation?

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Table 1

 1 Distribution of CYP2C19 allele frequencies with the respective 95% CI in the Swedish and Korean populations analysed. CYP2C19*1 denotes alleles not identified as *2, *3 or *17. The *3 allele was not analysed in the Swedish cohort due to its very rare

	occurrence
	CYP2C19 allele	Koreans (n=150)	Swedes (n=185)
	CYP2C19*1	0.61 (0.55 -0.66)	0.64 (0.59 -0.69)
	CYP2C19*2	0.28 (0.23 -0.33)	0.16 (0.12 -0.20)
	CYP2C19*3	0.11 (0.08 -0.15)	n.d
	CYP2C19*17	0.003 (-0.003 -0.01) 0.20 (0.16 -0.24)
	5. There are 26 different variants of CYP2C19 listed in the official website of allele
	nomenculature committee (www.cypalleles.ki.se). The *2, *3 and *17 alleles were
	analyzed in the present study. The contribution of other variants that were not
	analyzed cannot be ruled out. A statement about this matter should be added to the
	discussion.
	Authors reply:

-The following sentence is now inserted Under "

Discussion" section Last paragraph on page 11.

  The MRs was log transformed before application of statistical analysis. For comparisons of Swedes and Koreans within each genotype group and between groups based on smoking and OC use the independent t-test was used. P-Values less than 0.05 were considered statistically significant. Statistical analyses were performed by Statistical version 7 (Stat Soft Scandinavia AB).

	Results
	CYP2C19 genotype: The study encompassed 185 Swedes and 150 Koreans. We evaluated
	results after administering the Karolinska cocktail with the use omeprazole as a probe drug
	for phenotyping of CYP2C19. The genotype analyses revealed allele frequencies of
	CYP2C19*2, and *17 in the Swedes of 16 and 20 %, respectively and 28, 11 and 0.3 % of *2,
	*3 and *17 in the Koreans, respectively (Table

  of the MR (omeprazole/5-hydroxyomeprazole). A bimodal distribution especially

	in Koreans with an antimode of about 10 was observed. The median MR in Koreans was
	substantially higher (2.57) as compared to the Swedes (0.71) (p-value <0.00001). The mean
	MR among Koreans was 3.04 while in Swedes 0.86.
	CYP2C19 phenotype comparison between Swedes and Koreans: For statistical analysis
	and comparison of CYP2C19 enzyme activity, subjects were first stratified based on
	CYP2C19 genotype. The subjects homozygous for CYP2C19*1 were further stratified for
	r o F gender and intake of OC (Figure
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Table 1

 1 enzyme activity in Koreans which cannot be seen in Swedes. Further studies are needed to 12 evaluate the mechanisms behind this difference, which however, would not drastically influence the pharmacokinetics of CYP2C19 drugs as to motivate gender specific dosing regimens in Koreans. Distribution of CYP2C19 allele frequencies with the respective 95% CI in the Swedish and Korean populations analysed. CYP2C19*1 denotes alleles not identified as *2, *3 or *17. The *3 allele was not analysed in the Swedish cohort due to its very rare

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European Journal of Clinical Pharmacology

Acknowledgment

The study was financially supported by the Swedish Research Council, Medicine, 3902 and the Swedish Capio Forskningsstiftelse.

Acknowledgment

The study was financially supported by the Swedish Research Council, Medicine, 3902 and the Swedish Capio Forskningsstiftelse.

(EJCP-2010-0050) RE: entitled "Interethnic and gender dependent differences in CYP2C19 activity. A comparison between Swedes and Koreans"

We thank the referees for review of our manuscript and helpful feedback. We considered the peer-review feedback in great detail and made every attempt to incorporate the suggested amendments.

We present below our itemized responses to specific comments by the referees. The revised sections and points of major emphasis are highlighted in color throughout the manuscript as they relate to the referee comments. A separate clean copy of the manuscript is also provided as part of the revised manuscript dossier.

Changes in the revised manuscript are indicated in blue color.

Reviewer: 1

COMMENTS TO THE AUTHOR: Use the space below for comments to be transmitted to the author.

The authors investigated phenotype-genotype association for CYP2C19 in two different ethnic groups from Sweden and Korea. Interethnic and gender differences in omeprazole metabolic ratio and effect of oral contraceptives are original findings in the present study.

Manuscript is well written. The following points need to be considered/answered.


Holger Gevensleben

Birgit Holl

Björn Albrecht

Dieter Schlamp

Oliver Kratz

Petra Studer

Aribert Rothenberger

Gunther H Moll

Hartmut Heinrich



ECAP-D-09-00159. R2

Keywords: ADHD, neurofeedback, randomised, controlled trial (RCT), follow-up, children

Objective: Neurofeedback (NF) could help to improve attentional and self-management capabilities in children with attention-deficit / hyperactivity disorder (ADHD). In a randomised controlled trial, NF training was found to be superior to a computerised attention skills training (Gevensleben et al., 2009). In the present paper, treatment effects at 6-month follow-up were studied. Methods: 94 children with ADHD, aged 8 to 12 years, either completed 36 sessions of NF training (n=59) or a computerised attention skills training (n=35). Pre-training, post-training and follow-up assessment encompassed several behaviour rating scales (e.g., the German ADHD rating scale, FBB-HKS) completed by parents.

Results: Follow-up information was analysed in 61 children (ca. 65%) on a per-protocol basis. 17 children (of 33 dropouts) had started a medication after the end of the training or early in the follow-up period. Improvements in the NF group (n=38) at follow-up were superior to those of the control group (n=23) and comparable to the effects at the end of the training. For the FBB-HKS total score (primary outcome measure), a medium effect size of 0.71 was obtained at follow-up. A reduction of at least 25% in the primary outcome measure (responder criterion) was observed in 50% of the children in the NF group.

Conclusions: Behavioural improvements induced by NF training in children with ADHD were maintained at a 6-month follow-up. Though treatment effects appear to be limited, the results confirm the notion that NF is a clinically efficacious module in the treatment of children with ADHD.

Trial registry: ISRCTN87071503 -Comparison of neurofeedback and computerised attention skills training in children with attention-deficit/hyperactivity disorder (ADHD).

(http://www.controlled-trials.com/ISRCTN87071503)

Introduction

For attention-deficit hyperactivity disorder (ADHD), European guidelines recommend a multimodal treatment tailored to the requirements of the child [START_REF] Taylor | European clinical guidelines for hyperkinetic disorderfirst upgrade[END_REF]. Medication (first-line treatment: methylphenidate), cognitive-behaviour therapy and parental training have proven to be effective [START_REF] The | A 14-month randomized clinical trial of treatment strategies for attention-deficit/hyperactivity disorder[END_REF][START_REF] Van Der Oord | Efficacy of methylphenidate, psychosocial treatments and their combination in school-aged children with ADHD: a metaanalysis[END_REF]. But there is still a need for further effective treatment strategies in improving attentional and self-management capabilities in children with ADHD, especially concerning long term effects [START_REF] Molina | The MTA at 8 years: prospective follow-up of children treated for combined-type ADHD in a multisite study[END_REF][START_REF] Rothenberger | ADHD beyond core symptomsnot only a European perspective[END_REF]. Recent neurofeedback (NF) studies obtained encouraging results and raise the hope of closing the gap in providing children strategies for better self-regulation and -management [START_REF] Drechsler | Controlled evaluation of a neurofeedback training of slow cortical potentials in children with attentiondeficit/hyperactivity disorder (ADHD)[END_REF]12,[START_REF] Strehl | Self-regulation of slow cortical potentials: a new treatment for children with attention-deficit/hyperactivity disorder[END_REF].

NF aims at acquiring self-control over certain brain activity patterns, deriving self-regulation strategies, and implementing these self-regulation skills in daily-life. Two training protocolstheta / beta training and training of slow cortical potentials (SCPs) -are typically used for children with ADHD [START_REF] Heinrich | Annotation: Neurofeedbacktrain your brain to train behaviour[END_REF].

In theta / beta training, children learn to reduce activity in the theta band of the EEG [START_REF] Birbaumer | Slow potentials of the cerebral cortex and behavior[END_REF][START_REF] Boutron | Extending the CONSORT statement to randomized trials of nonpharmacologic treatment: explanation and elaboration[END_REF][START_REF] Döpfner | Psychopathologisches Befund-System für Kinder und Jugendliche (CASCAP-D)[END_REF][START_REF] Döpfner | DISYPS-KJ -Diagnostik-System für psychische Störungen im Kindes-und Jugendalter[END_REF][START_REF] Döpfner | Therapieprogramm für Kinder mit hyperkinetischem und oppositionellem Problemverhalten (THOP)[END_REF] and to increase activity in the beta band [START_REF] Gevensleben | Distinct EEG effects related to neurofeedback training in children with ADHD: a randomized controlled trial[END_REF][START_REF] Heinrich | Training of slow cortical potentials in attention-deficit/hyperactivity disorder: Evidence for positive behavioral and neurophysiological effects[END_REF][START_REF] Heinrich | Annotation: Neurofeedbacktrain your brain to train behaviour[END_REF][START_REF] Kotchoubey | A new method for selfregulation of slow cortical potentials in a timed paradigm[END_REF][START_REF] Leins | Neurofeedback for children with ADHD: a comparison of SCP-and theta/beta-protocols[END_REF][START_REF] Lütcke | Brain networks involved in early versus late response anticipation and their relation to conflict processing[END_REF][START_REF] Mash | Treatment of childhood disorders[END_REF][START_REF] Molina | The MTA at 8 years: prospective follow-up of children treated for combined-type ADHD in a multisite study[END_REF]. In the resting EEG, increased slow wave (theta) activity and/or reduced relative alpha (8-13 Hz) and beta activity was reported in several studies on children with ADHD (for review see [START_REF] Banaschewski | Annotation: what electrical brain activity tells us about brain function that other techniques cannot tell usa child psychiatric perspective[END_REF] and [START_REF] Barry | A review of electrophysiology in attentiondeficit/hyperactivity disorder: I. Qualitative and quantitative electroencephalography[END_REF]). Thus, theta / beta training may address an underlying neuronal dysfunction. On the other hand, NF may simply be seen as a tool for enhancing specific cognitive or attentional states (an alert and focused but relaxed state in theta / beta training), irrespective of supposed neurophysiological deviations [START_REF] Heinrich | Annotation: Neurofeedbacktrain your brain to train behaviour[END_REF]. SCPs are changes of cortical electrical activity lasting from several hundred milliseconds to several seconds. They are thought to represent task-dependent short-term mobilizations of cortical processing resources. While negative SCPs reflect increased excitation (e.g., during states of behavioural or cognitive preparation), positive SCPs indicate reduction of cortical excitation of the underlying neural networks (e.g., during behavioural inhibition) [START_REF] Birbaumer | Slow potentials of the cerebral cortex and behavior[END_REF].

The contingent negative variation (CNV) is an SCP that reflects anticipation and / or preparation [START_REF] Lütcke | Brain networks involved in early versus late response anticipation and their relation to conflict processing[END_REF]. It is for example elicited in cue trials of a continuous performance test. In event-related potential studies, the CNV was found to be reduced in children with ADHD (for a review see [START_REF] Banaschewski | Annotation: what electrical brain activity tells us about brain function that other techniques cannot tell usa child psychiatric perspective[END_REF]). Training of slow cortical potentials leads to an increase of the CNV [START_REF] Heinrich | Training of slow cortical potentials in attention-deficit/hyperactivity disorder: Evidence for positive behavioral and neurophysiological effects[END_REF].

Thus, SCP training, in which surface-negative and surface-positive SCPs have to be generated over the sensorimotor cortex, could help children with ADHD to improve their assumed dysfunctional regulation of energetical resources [START_REF] Sergeant | Information processing and energetic factors in attention-deficit/hyperactivity disorder[END_REF].

In the last decade, several NF studies in children with ADHD have been published which manage to overcome the methodological shortcomings of earlier studies [START_REF] Drechsler | Controlled evaluation of a neurofeedback training of slow cortical potentials in children with attentiondeficit/hyperactivity disorder (ADHD)[END_REF][START_REF] Fuchs | Neurofeedback treatment for attention-deficit/hyperactivity disorder in children: A comparison with methylphenidate[END_REF][START_REF] Heinrich | Training of slow cortical potentials in attention-deficit/hyperactivity disorder: Evidence for positive behavioral and neurophysiological effects[END_REF][START_REF] Monastra | The effects of stimulant therapy, EEG-biofeedback, and parenting style on the primary symptoms of attention-deficit/hyperactivity disorder[END_REF][START_REF] Strehl | Self-regulation of slow cortical potentials: a new treatment for children with attention-deficit/hyperactivity disorder[END_REF]. In all of these studies, positive behavioural, cognitive and / or neurophysiological effects were described. Our group conducted a randomised controlled trial encompassing 102 children with ADHD. In this trial behavioural and neurophysiological effects of NF, which included one training block of theta / beta training and one block of SCP training, were analyzed in comparison to a computerised attention skills training [12,[START_REF] Gevensleben | Distinct EEG effects related to neurofeedback training in children with ADHD: a randomized controlled trial[END_REF][START_REF] Wangler | Neurofeedback in children with ADHD: Specific event-related potential findings of a randomized controlled trial[END_REF]. According to parent and teacher ratings, children of the NF group showed larger behavioural improvements than those of the control group (medium effect size of 0.6 for the primary outcome measure, total score of the German ADHD rating scale, FBB-HKS [START_REF] Döpfner | DISYPS-KJ -Diagnostik-System für psychische Störungen im Kindes-und Jugendalter[END_REF]). Due to comparable settings and demands for NF and the control training, superiority of NF was first and foremost ascribed to specific factors. A tendency for larger improvements was observed if theta / beta training preceded SCP training [12].

At the neurophyiological level (resting EEG, event-related potentials), specific associations with behavioural improvements could be revealed for theta / beta and SCP training (e.g., association between decrease of theta activity and reduction of ADHD symptomatology) [START_REF] Gevensleben | Distinct EEG effects related to neurofeedback training in children with ADHD: a randomized controlled trial[END_REF][START_REF] Wangler | Neurofeedback in children with ADHD: Specific event-related potential findings of a randomized controlled trial[END_REF]. These neurophysiological effects contribute to a better understanding of the mechanisms underlying a successful training and indicate specificity of NF training effects.

Both behavioural and neurophysiological findings of our trial indicate that NF may be considered as a clinically efficacious module in the treatment of children with ADHD.

One of the questions which has not been studied under controlled conditions is whether NF training effects remain stable after completing the training. Leins et al. reported that children with ADHD, who had participated in either a theta / beta training or an SCP training, were able to learn cortical self-regulation accompanied by significant improvements in behaviour and cognition [START_REF] Leins | Neurofeedback for children with ADHD: a comparison of SCP-and theta/beta-protocols[END_REF]. These effects remained constant after 6 months. For a subgroup of 23 (from initially 47) children, two-year follow-up data could also be assessed [START_REF] Gani | Long term effects after feedback of slow cortical potentials and of theta-beta-amplitudes in children with attention-deficit/hyperactivity disorder[END_REF].

Neuroregulation skills were still preserved. Behavioural and cognitive effects were reported to be stable or even further enhanced. However, due to the lack of a control group, the effects cannot be differentiated from the natural course. This paper reports follow-up behavioural data assessed 6 months after completion of the training (either neurofeedback training or attention skills training) for the children with ADHD of our previous paper [12]. We hypothesized that behavioural improvements in the NF group remain stable and superior to those of the control group.

Material and Methods

Subjects

102 children with ADHD (8 to 12 years) participated in a NF training or an attention skills training (AST). Subjects were randomly assigned to one of the two study groups (ratio NF : control training = 3:2; see also Fig. 1). Eight children (NF: n=5, AST: n=3) discontinued the study due to immediate need for medical treatment (n=3), organizational problems of the parents (n=2), loss of motivation (n=1) or protocol violation (n=2). Sample size had been estimated a-priori to be large enough to detect a medium effect size of about 0.5 with a power of 0.8 (one-sided, 0.05-level test).

-Table 1 about here Table 1 summarizes inter alia demographic, psychological and clinical variables of the children completing their training. Concerning these variables, there were no significant differences between NF group (n=59) and control group (n=35) 1 .

All patients fulfilled DSM-IV criteria for ADHD [START_REF]Diagnostic and Statistical Manual of Mental Disorders[END_REF]. Diagnoses were based on a semistructured clinical interview (CASCAP-D, [START_REF] Döpfner | Psychopathologisches Befund-System für Kinder und Jugendliche (CASCAP-D)[END_REF]) and confirmed using the Diagnostic Checklist for Hyperkinetic Disorders/ADHD [START_REF] Döpfner | DISYPS-KJ -Diagnostik-System für psychische Störungen im Kindes-und Jugendalter[END_REF] by a child and adolescent psychiatrist or a clinical psychologist, supervised by a board-certified child and adolescent psychiatrist. With mean total FBB-HKS scores of about 1.5 (range 0 to 3; see The study follows the CONSORT guidelines for randomised trials [START_REF] Boutron | Extending the CONSORT statement to randomized trials of nonpharmacologic treatment: explanation and elaboration[END_REF]. It was approved by the local ethics committees of the participating clinics and conducted according to the declaration of Helsinki. Assent was obtained from the children and written informed consent from their parents.

Design of the study

-Figure 1 about here The design of the study is illustrated in Fig. 1. Neurofeedback and attention skills training both consisted of 36 units of 50 minutes each. Both treatments were divided in two blocks of 18 units. These 18 units were combined in nine sessions. These sessions took place two to three times a week. The NF training consisted of one block of 18 units of theta / beta training and one block of 18 units of SCP training (balanced order). For both NF and AST training, there was a break of 2-3 weeks between the two treatment blocks. The NF and the AST training were designed as similarly as possible concerning the setting and the demands upon the participants, e.g., both treatments encompassed attention demanding tasks on a computer (to a comparable amount, 25-30 minutes per training unit), acquirement of strategies for focussing attention, and efforts to transfer learned strategies into daily life [12]. Parents were not explicitly informed about the treatment condition of their child (NF vs. AST). In contrast to the other assessment points, neuropsychological / -physiological data were not measured at follow-up.

Training programs

Neurofeedback

The neurofeedback system SAM ("Self regulation and Attention Management") which was developed by our study group was used for neurofeedback training.

In theta / beta training, the task was to reduce theta and enhance beta activity. A bar on the left of the screen (representing theta activity) had to be reduced while simultaneously a bar on the right (representing beta activity) had to be increased. In each unit, about 5 or 6 trials of 5 minute each, or up to 3 trials of 10 minutes each, were performed. Baseline values of theta and beta activity were determined at the beginning of each session (3 minutes). Children were instructed to reach a relaxed but attentive state and to find individual strategies to control the bars.

In SCP training, the children had to generate negative or positive slow cortical potentials.

They had to find appropriate strategies to direct a ball upwards (negativity trials) or downwards (positivity trials). Negativity (50%) and positivity trials (50%) were presented in random order. A trial lasted for 8 seconds (baseline period: 2 sec, feedback period: 6 sec).

Children were instructed to reach an attentive (negativity trials) or relaxed state (positivity 3 These ratings will not be considered for the evaluation of follow-up results in this paper. trials). In each SCP training unit, approximately 120 trials were performed, divided into 2 to 3 blocks of 40 to 60 trials in each treatment unit.

For both NF protocols, feedback was calculated from Cz (reference: mastoids, bandwidth: 1-30 Hz for theta / beta training and 0.01-30 Hz for SCP training, respectively, sampling rate: 250Hz). Vertical eye movements, which were recorded with electrodes above and below the left eye, were corrected online using slightly different regression-based algorithms for theta / beta training [START_REF] Semlitsch | A solution for reliable and valid reduction of ocular artifacts, applied to the P300 ERP[END_REF] and SCP training [START_REF] Kotchoubey | A new method for selfregulation of slow cortical potentials in a timed paradigm[END_REF]. For segments containing artefacts exceeding ±100 V in the EEG channel and ±200 V in the EOG channel, no feedback was calculated.

Transfer trials, i.e. trials without contingent feedback, were conducted about 1/3 at the beginning of a training block and about 2/3 at the end of a training block. These transfer trials, as well as homework, were intended to improve generalisation of treatment effects.

Attention skills training

The attention skills training was based on "Skillies" (Auer-Verlag, Donauwörth, Germany), a German learning software programme which primarily exercises visual and auditory perception, vigilance, sustained attention, and reactivity. In "Skillies", the children had to sail to several islands. On each island, a clearly defined taskeach requiring different attentionbased skillshad to be solved (for further information see [12]). The training was complemented by some self-directed interventions from cognitive therapy to assure comparability to NF, i.e., the children were to compile (meta-)cognitive strategies such as focusing attention, careful processing of tasks and impulse control. Corresponding to the NF group, children of the AST group should practice their compiled strategies in daily-life situations.

Behavioural assessment

The following questionnaires (assessed at pre-training, post-training and follow-up) were completed by parents to evaluate the follow-up results:

-German ADHD rating scale (FBB-HKS) [START_REF] Döpfner | DISYPS-KJ -Diagnostik-System für psychische Störungen im Kindes-und Jugendalter[END_REF]: The FBB-HKS is a 20-item questionnaire related to DSM-IV and ICD-10 criteria for ADHD and hyperkinetic disorders, frequently used in Germany in the evaluation of medical and cognitive behavioural treatment of ADHD (e.g. [START_REF] Sevecke | The effectiveness of stimulants of retard forms in children and adolescents with ADHD -a systematic overview[END_REF]). The severity of each item is rated from 0 to 3. Outcome measures were the main FBB-HKS total score, i.e., the mean value of all items as well as subscores for inattention and hyperactivity/impulsivity. The FBB-HKS total score constituted the primary outcome measure of the study.

-German Rating Scale for Oppositional Defiant/Conduct Disorders (FBB-SSV) [START_REF] Döpfner | DISYPS-KJ -Diagnostik-System für psychische Störungen im Kindes-und Jugendalter[END_REF]: It is comprised of 25 items. The severity of each item is rated from 0 to 3. Outcome measures were the subscales oppositional behaviour (mean value of the first 9 items) and delinquent and physical aggression (mean value of the remaining 16 items).

-The Strength and Difficulties Questionnaire (SDQ, German version) [START_REF] Rothenberger | Psychometric properties of the parent strengths and difficulties questionnaire in the general population of German children and adolescents: results of the BELLA study[END_REF][START_REF] Woerner | Normative data and scale properties of the German parent SDQ[END_REF] is comprised of 25 items which address both positive and negative attributes. Each item is rated from 0 to 2. Outcome measures were the total difficulties score as well as the 5 subscales (emotional symptoms, conduct problems, hyperactivity, peer problems, prosocial behaviour).

-The Home Situations Questionnaire (HSQ, German version) [START_REF] Döpfner | Therapieprogramm für Kinder mit hyperkinetischem und oppositionellem Problemverhalten (THOP)[END_REF] was used to assess behaviour problems of the child in specific home situations. The HSQ consists of 16 situations in which problematic child behaviour can occur. Parents rate whether the problem behaviour is present in that setting; if so, they rate its severity on a 9-point scale.

-Problem behaviour during homework was assessed using the Homework Problem Checklist (HPC, German version) [START_REF] Döpfner | Therapieprogramm für Kinder mit hyperkinetischem und oppositionellem Problemverhalten (THOP)[END_REF]. This checklist consists of 20 items, rated on a 4-point frequency scale.

Data analysis

Per-protocol analysis was conducted to avoid confounding the treatment effects with additional treatment. Children were classified as dropouts and excluded if they had started another treatment (e.g., medication, psychotherapy) or if questionnaires were not returned. Since there were about 35% dropouts (see Results section), we also tested for differences in the clinical (behavioural) data between the dropouts and the remaining children. We computed ANOVAs with between-subject factors GROUP (NF vs. control training) and DROPOUT (dropout vs. follow-up data available) and within-subject factor TIME (pretraining, post-training). SPSS (v.16) was used for statistical analysis. For all statistical procedures significance was assumed if p < 0.05. 2 (already reported in [12]). Only effect sizes 0.3 are reported.

Results

Dropouts

For the FBB-HKS total score (primary outcome measure), statistics revealed a significant For the SDQ subscale Hyperactivity, a significant GROUP effect (F(1,57) = 5.49; p < 0.05) indicated larger improvements for the NF group in comparison to the control group still evident at follow-up. While medium effect sizes were observed for this subscale, small effect sizes resulted for the SDQ total score. For the remaining SDQ subscales (due to lack of space not reported in Table 2), effect sizes were smaller than 0.3.

Concerning homework problems (HPC-D), the GROUP x TIME interaction turned out to be significant (F(1,54) = 4.18; p < 0.05). Post-hoc, we computed an ANOVA with the follow-up score as dependent variable, GROUP as between-subject factor and the pre-training score as covariate. For this analysis, a significant GROUP effect was found (F( 1

Order of NF protocols

For the follow-up sample, improvements in the FBB-HKS total score at post-training were non-significantly higher when theta / beta training preceded SCP training as compared to the reversed order of protocols (t(35)=-0.75, p = 0.46; d = 0.25). The effect size for the posttraining sample had been 0.43 [12]. At follow-up, the improvements for both orders were nearly identical (t(35)=-0.04, p = 0.97; d = 0.01)

Discussion

The impact of a treatment significantly relies on the generalization of treatment effects, which can be conceptualized as occurring across settings, behaviour variables and time [START_REF] Mash | Treatment of childhood disorders[END_REF]. This underlines the necessity to consider multiple indicators as well as to assess follow-up measurements concerning the stability of effects. In previous papers we reported the immediate treatment effects of a NF training (theta / beta training and SCP training) compared to an attention skills training on different outcome levels, encompassing behavioural and neurophysiological measures [12,[START_REF] Gevensleben | Distinct EEG effects related to neurofeedback training in children with ADHD: a randomized controlled trial[END_REF][START_REF] Wangler | Neurofeedback in children with ADHD: Specific event-related potential findings of a randomized controlled trial[END_REF]. This paper deals with the 6-month follow-up analyses of the behavioural outcome. Since some children started medication, we conducted a per-protocol analysis in order to avoid confounding the treatment effect with medication effect. For 61 of the 94 children (ca. 65%) of this sample, 6-month follow-up data (parent ratings) could be analysed. On average, effects were sustained at follow-up and the effects in the NF group were still superior to those of the control group. For the total score of the German ADHD rating scale (primary outcome measure), a medium effect size was obtained. Further, effects were not restricted to core ADHD symptoms but could also be observed in other domains (homework situation, conduct disorder; small to medium effect sizes).

Regarding order effects for the NF protocols, the tendency for larger improvements when theta/beta training preceded SCP training could not be confirmed in the follow-up sample.

Since settings and demands for NF and the control training were comparable, these findings indicate that mainly specific effects accounted for the superiority of NF compared to the attention skills trainings. Specificity of effects is further supported by associations between neurophysiological patterns and the outcome at the clinical (behavioural) level as reported in [START_REF] Gevensleben | Distinct EEG effects related to neurofeedback training in children with ADHD: a randomized controlled trial[END_REF] and [START_REF] Wangler | Neurofeedback in children with ADHD: Specific event-related potential findings of a randomized controlled trial[END_REF]. For example, in theta / beta training the decrease of theta activity in the resting EEG was associated with a decrease of the FBB-HKS total score [START_REF] Gevensleben | Distinct EEG effects related to neurofeedback training in children with ADHD: a randomized controlled trial[END_REF]. Concerning SCP training, children with a higher CNV in an attention test at baseline showed larger improvements after the relatively short training block [START_REF] Wangler | Neurofeedback in children with ADHD: Specific event-related potential findings of a randomized controlled trial[END_REF].

On the other hand, partly due to the non-blind design, it can not be ruled out, that unspecific effects might have also contributed to the behavioural effects 5 .

There was a relatively large number of dropouts, i.e., children who either started a medication or for whom no questionnaires were received, at follow-up (about 35%). However, dropouts were not characterized by a worse training outcome at the end of the training. These children (mainly in the control group) had slightly (but non-significantly) higher scores on the German ADHD rating scale already at the beginning of the training. In general, children for whom follow-up data were available could not be differentiated from dropouts with respect to behavioural or demographic characteristics. So, it seems unlikely that the follow-up results were strongly biased by the large portion of dropouts.

5

For a more detailed discussion concerning the control condition, see [12].

Findings are based on parent ratings only. At the post-training assessment, for only about 70% of the children, ratings of the same teacher who had completed the pre-training questionnaires were available. Expecting further high dropout rates due to change of teachers and loss of motivation to complete the questionnaires, we decided not to include teacher ratings in the follow-up analysis. It can be questioned whether teacher ratings would have supported the follow-up results obtained from parent ratings. However, in [START_REF] Leins | Neurofeedback for children with ADHD: a comparison of SCP-and theta/beta-protocols[END_REF] parent and teacher ratings did not develop differentially from post-training to follow-up. In our study, comparable effects resulted for parent and teacher ratings at the post-training assessment [12]. 50% of the children completing the training were categorized as non-responders, according to a criterion of 25% reduction in the primary outcome measure. 11 out of 59 children of the NF group started a medication during the follow-up interval. Our study was not designed to achieve maximum NF training effects but had an arguably artificial scientific setting (e.g. separating theta / beta and SCP training in two separate, non-coordinated blocks).

Nevertheless, the low responder rate and the portion of children starting a medication in our study argue against neurofeedback as a stand-alone intervention for children with ADHD. The results indicate that not every child with ADHD may improve after NF treatment. In our opinion, NF should rather be seen as a treatment module for children with ADHD which can be embedded in a multimodal treatment program tailored to the individual needs of a child.

Stability of training effects at follow-up refers to the mean scores of the NF group and naturally does not apply to all individuals within the group. In our design, we used a fixed number of training sessions. This was intended to standardize number of treatment sessions across all individuals. However, qualitative analyses of our data suggest that children vary in their abilities and speed to learn and apply NF techniques. One block of 18 units for a single protocol might have been too short at least in some children to build up stable regulation capability and to establish transfer into daily life sufficiently. Further research therefore would be required to determine the optimal NF protocol (or combination of protocols) and the adequate number of treatment sessions for a particular child.

Coming back to the long term outcome, it could be helpful at least for some of the children to conduct further training sessions with longer intervals between the training session to sustain and consolidate regulation capabilities and the transfer into daily life, just as it is usually practiced in conventional cognitive-behaviour therapy. In this respect, the possible benefit of such booster sessions should also be investigated in further studies.

Conclusions

Table and figure legends

2 2

 2 At the post-training assessment, about 40 % of the parents could not reliably quote treatment assignment of their child. The attitude of the parents in the two groups towards the treatment of their child and post hoc evaluation of the training did not differ (rated via "placebo scales"). Parent ratings were assessed in the week before the training course started (pre-training), about one week after the last session of the first (intermediate 3 ) and second training block (post-training), respectively, and 6-months after the end of the training (follow-up).

  Behavioural data were analyzed in repeated-measures ANOVAs with between-subject factor GROUP (NF vs. control training), within-subject factor TIME (post-training, follow-up) using the baseline (pre-training) measure as a covariate. If NF training effects are still superior to the control training at follow-up, the ANOVA is expected to reveal a significant GROUP effect. If the difference between the two trainings becomes significantly greater or smaller, this effect is expected to be indicated by a significant GROUP x TIME interaction. Effect sizes (Cohen's d) were calculated as the difference of the change of a measure from pre-training to an assessment point (post-training and follow-up, respectively) and the corresponding change score in the control group divided by the pooled standard deviations of these change scores. To compare the ratio of responders ( 25% reduction of the primary outcome measure) in the NF group and the control group, the odds ratio was calculated. Though limited by a smaller sample size, possible effects of the order of the NF training blocks were tested by comparing improvements in the FBB-HKS obtained at post-training and at follow-up (t-tests).

  17 children (NF group: n = 11, 18.6%4 ; control group n = 6, 17.1%) started a medication during the follow-up interval. None of the children started any other treatment. Parents of 16 children (NF group: n = 10, 16.9%; control group n = 6, 17.1%) did not return the questionnaires. Thus, there were 33 dropouts with percentages of dropouts not differing between NF and control group ( 2 = 0.034, df = 2, n.s.).Analysis of the training effects at 6-month follow-up encompassed 61 of the 94 children completing the training (NF group: n=38, control group n=23; see also Fig.2).-Figure2about here Comparing the dropouts and the remaining children, dropouts were not characterized by significantly smaller training effects, i.e., no significant effect containing the factor TIME (pre-vs. post-training) was obtained in the repeated-measures ANOVAs. Dropouts tended to have higher FBB-HKS scores (FBB-HKS total score: factor DROPOUT: F(1,90) = 3.22; p< 0.1; FBB-HKS Inattention subscale (F(1,90) = 3.18; p < 0.1), mainly in the control group as indicated by a trend for GROUP x DROPOUT interaction. For all other rating scales, no significant effect or trend containing the factor DROPOUT was obtained. Neurofeedback vs. control training -Table 2 about here 4 5 (of these 11) children of the NF group starting a medication had been classified as responders at the end of the training. Results are summarized in Table 2. Ratings of the children for whom follow-up data were available ("follow-up sample") are presented in the upper part. To facilitate comparison, the pre-training and post-training measures of all children who completed the treatment ("posttraining sample") are shown in the lower part of Table

  GROUP effect (F(1,58) = 10.10; p < 0.005) but no significant TIME (post-training vs. followup) (F(1,58) = 0.69; n.s.) or GROUP x TIME interaction effect (F(1,58) = 0.01; n.s.). Hence, NF training effects were still superior to the control training at follow-up (medium effect size of 0.71 for change measure from pre-training to follow-up). Correspondingly, a significant GROUP effect for the FBB-HKS Inattention subscale (F(1,58) = 4.72; p < 0.05) and a trend for the FBB-HKS Hyperactivity / Impulsivity subscale (F(1,58) = 3.45; p < 0.1) were obtained. Reductions of inattention and hyperactivity / impulsivity at follow-up (compared to pre-training) were about 25 -30% in the NF group compared to about 10-15% in the control group. Concerning the FBB-SSV subscale Delinquent and Physical Aggression, the ANOVA yielded a trend for the factor GROUP (F(1,56) = 3.64; p < 0.1) due to larger decreases in the NF group. Small to medium effect sizes were obtained at post-training and follow-up, respectively.

  ,54) = 5.48, p < 0.05) indicating a larger positive effect concerning homework after NF training (compared to the control training) at follow-up (medium effect size of 0.60). Neither the decrease in NF group (F(1,32) = 1.56, n.s.) nor the increase in the control group from post-training to follow-up (F(1,32) = 0.02, n.s.) reached significance. For the problem situations in family (HSQ-D) questionnaire, no significant effects were obtained. Responder rate: In the follow-up sample, 50% (19 of 38) of the children of the NF group showed a reduction of 25% or more in the primary outcome measure at post-training and also 50% at follow-up. According to this criterion, 26.1% (6 of 23) of the children of the control group were responders at post-training and 30.4% (7 of 23) at follow-up. Odds ratios were 2.83 (post-training) and 2.29 (follow-up) and, thus, in the same range as in the post-training sample (odds ratio: 2.68 [12]) but failed to reach significance due to the smaller sample size (Fisher's exact test, one-sided: post-training: p = 0.06; follow-up: p = 0.11).

  Behavioural effects of NF training were maintained in children with ADHD at 6-month follow-up, further supporting clinical efficacy of this neurobehavioral training. NF may be recommended as a treatment module for children with ADHD besides conventional behavioural trainings and medication. Future studies should systematically address how to optimize / individualize NF training and how to embed it in a multimodal treatment in children with ADHD.

Table 1 :

 1 Demographic and clinical characteristics of the NF group and the control group. Data are presented for the children who had completed their training ("post-trainingsample") and, separately, for the children for whom follow-up data were available ("followup sample"). At the pre-training level, there were no significant differences between the groups (neither for the post-training sample nor for the follow-up sample).

Table 2 :

 2 Parent behaviour ratings (mean values standard deviation) assessed at pre-training, post-training and follow-up for the children with ADHD for whom follow-up data were available ("follow-up sample"). Only effect sizes (Cohen's d) 0.3 are reported. All effect sizes refer to comparisons of the change scores (from pre-to post-training or from pretraining to follow-up) between the training groups. For comparison purposes, the pre-training and post-training measures of all children who had completed the training ("post-training sample") are shown in the lower part (already reported in[12]).

Figure 1 :

 1 Figure 1: Schematic illustration of the design of the randomised trial in children with

Figure 2 :

 2 Figure 2: Patient flow diagram

  Figure 1

Figure1

  Figure1

Table 2

 2 were computed for age and IQ (|t(92)| < 1.54), for all other variables 2 -tests were applied ( 2 <2.19).

	), ADHD symptomatology was

1 t-tests

Table 1 NF

 1

		group		Control group
		Post-training	Follow-up	Post-training	Follow-up
		sample	sample	sample	sample
		n=59	n = 38	n=35	n = 23
	Age (years; month)	9;10 1;3	9;11 1;4	9;4 1;2	9;5 1;1
	Sex (boys / girls)	51 / 8 (86.4% /	32 / 6 (84.2% /	26 / 9 (74.3% /	16 / 7 (69.6% /
		13.6%)	15.8%)	25.7%)	30.4%)
	IQ (HAWIK-III, [30])	106.1 13.2	106.5 13.3	104.5 12.9	106.8 13.0
	DSM-IV subtype
	Combined type	39 (66.1%)	23 (60.5%)	27 (77.1%)	17 (73.9%)
	Inattentive type	20 (33.9%)	15 (40.5%)	8 (22.9%)	6 (26.1%)
	Drug-naive	54 (91.5%)	36 (94.7%)	33 (97.1%)	22 (95.7%)
	Associated disorders
	Conduct disorder	10 (16.9%)	5 (13.2%)	7 (20.0%)	3 (13.0%)
	Emotional disorder	3 (5.1%)	2 (5.2%)	3 (8.6%)	1 (4.3%)
	Tic disorder	3 (5.1%)	1 (2.6%)	0 (0.0%)	0 (0.0%)
	Dyslexia	12 (20.3%)	8 (21.1%)	10 (28.6%)	6 (26.1%)

Table 2 Behaviour Ratings Pre Post Follow-Up Pre Post Follow-Up Follow-up sample NF group (n=38) Control group (n=23)

 2

	FBB-HKS
	Total score	1.50 0.44	1.10 0.51	1.08 0.51	1.37 0.46	1.22 0.65	1.24 0.66
	Inattention	2.02 0.50	1.51 0.46	1.49 0.55	1.70 0.46	1.54 0.60	1.56 0.60
	Hyperactivity / impulsivity	1.10 0.67	0.79 0.69	0.76 0.68	1.18 0.68	1.08 0.79	1.00 0.78
	FBB-SSV
	Oppostional behaviour	1.16 0.70	0.89 0.70	0.86 0.74	1.13 0.70	0.99 0.74	0.97 0.71
	Delinquent and physical aggression	0.15 0.14	0.13 0.15	0.11 0.16	0.15 0.15	0.19 0.22	0.18 0.24
	SDQ
	Total score

Acknowledgments

The authors thank Christa Dahlmann, Martin Deinzer, Claudia Vogel, Susanne Wangler, Nicola Wöstmann and the student assistants for their valuable support as well as all the participating families for their contribution and effort. We thank Margaret and Simon Tiffin-Richards for language editing. This study was supported by the German Research Foundation (with a joint grant to H.H., G.H.M, and A.R.; HE 4536/2, MO 726/2, RO 698/4).