Ohm Kyvik

MD, Ph.d. Thomas Heiberg Brix
email: thomas.brix@ouh.regionsyddanmark.dk

Pia Skov Hansen

Kirsten Ohm Kyvik

Laszlo Hegedüs

Kirsten Ohm Kyvik

The pituitary-thyroid axis set point in women is uninfluenced by X chromosome inactivation pattern? A twin study

Keywords: Thyroid hormones, X chromosome inactivation, Epigenetics, Twins Thyroid hormones, X chromosome inactivation, Epigenetics, Twins

The pituitary-thyroid axis set point in women is uninfluenced by X chromosome inactivation pattern.

Introduction

In a population of healthy euthyroid subjects, serum levels of thyrotropin (TSH) and the thyroid hormones (free thyroxine, FT4 and free triiodothyronine, FT3) show substantial interindividual variation, whereas the intraindividual variation is generally within a narrow range [START_REF] Andersen | Narrow individual variations in serum T(4) and T(3) in normal subjects: a clue to the understanding of subclinical thyroid disease[END_REF] . It appears therefore that each individual has a unique set point of the pituitary-thyroid axis (PTA). Recently, two large twin studies have provided indisputable evidence for the contribution of both genetic and environmental factors in the regulation of these parameters [START_REF] Hansen | Genetic and environmental interrelations between measures of thyroid function in a healthy Danish twin population[END_REF][START_REF] Panicker | Heritability of serum TSH, free T4 and free T3 concentrations: a study of a large UK twin cohort[END_REF] . A number of genetic loci [START_REF] Panicker | Genetic loci linked to pituitarythyroid axis set points: A genome-wide scan of a large twin cohort[END_REF] as well as polymorphisms in different thyroid hormone pathway genes, such as phosphodiesterase 8B [START_REF] Arnaud-Lopez | Phosphodiesterase 8B gene variants are associated with serum TSH levels and thyroid function[END_REF] , the deiodinases [START_REF] Peeters | A new polymorphism in the type II deiodinase gene is associated with circulating thyroid hormone parameters[END_REF][START_REF] Van Der Deure | The effect of genetic variation in the type 1 deiodinase gene on the interindividual variation in serum thyroid hormone levels: an investigation in healthy Danish twins[END_REF][START_REF] Panicker | A common variation in deiodinase 1 gene DIO1 is associated with the relative levels of free thyroxine and triiodothyronine[END_REF] , the TSH receptor [START_REF] Peeters | Polymorphisms in thyroid hormone pathway genes are associated with plasma TSH and iodothyronine levels in healthy subjects[END_REF][START_REF] Hansen | The impact of a TSH receptor gene polymorphism on thyroid related phenotypes in a healthy Danish twin population[END_REF] , the thyroid hormone receptors [START_REF] Sorensen | Identification and consequences of polymorphisms in the thyroid hormone receptor alpha and beta genes[END_REF] , and thyroid hormone transporters [START_REF] Van Der Deure | Thyroid hormone transport and metabolism by organic anion transporter 1C1 and consequences of genetic variation[END_REF][START_REF] Friesema | Genetics and phenomics of thyroid hormone transport by MCT8[END_REF] have been associated with serum levels of TSH, FT3 and FT4. So far, however, their contribution to phenotypic variance is week to modest, indicating that there is no single gene with a major regulatory influence on the PTA set point. The same is certainly true for suggested non-genetic variables such as body mass index (BMI) [START_REF] Knudsen | Small differences in thyroid function may be important for body mass index and the occurrence of obesity in the population[END_REF][START_REF] Manji | Lack of association between serum TSH or free T4 and body mass index in euthyroid subjects[END_REF] , iodine intake [START_REF] Knudsen | Comparative study of thyroid function and types of thyroid dysfunction in two areas in denmark with slightly different iodine status[END_REF] and smoking habits [START_REF] Vejbjerg | The impact of smoking on thyroid volume and function in relation to a shift towards iodine sufficiency[END_REF][START_REF] Soldin | Thyroid hormone levels associated with active and passive cigarette smoking[END_REF] .

In the last several years, there has been considerable discussion and disagreement regarding gender specific differences in the PTA set point [START_REF] Franklyn | The influence of age and sex on tests of thyroid function[END_REF][START_REF] Jensen | Establishment of a serum thyroid stimulating hormone (TSH) reference interval in healthy adults. The importance of environmental factors, including thyroid antibodies[END_REF][START_REF] Volzke | Reference intervals of serum thyroid function tests in a previously iodine-deficient area[END_REF][START_REF] Hollowell | Serum TSH, T(4), and thyroid antibodies in the United States population (1988 to 1994): National Health and Nutrition Examination Survey (NHANES III)[END_REF][START_REF] Hamilton | Thyrotropin levels in a population with no clinical, autoantibody, or ultrasonographic evidence of thyroid disease: implications for the diagnosis of subclinical hypothyroidism[END_REF][START_REF] Estaquio | Serum thyrotropin and free thyroxine reference ranges as defined in a disease-free sample of French middleaged adults[END_REF] . However, in most studies -irrespective of significant gender differences in median TSH [START_REF] Volzke | Reference intervals of serum thyroid function tests in a previously iodine-deficient area[END_REF] , presence of thyroid autoantibodies [START_REF] Hollowell | Serum TSH, T(4), and thyroid antibodies in the United States population (1988 to 1994): National Health and Nutrition Examination Survey (NHANES III)[END_REF] and/or thyroid ultrasound abnormalities [START_REF] Hamilton | Thyrotropin levels in a population with no clinical, autoantibody, or ultrasonographic evidence of thyroid disease: implications for the diagnosis of subclinical hypothyroidism[END_REF][START_REF] Estaquio | Serum thyrotropin and free thyroxine reference ranges as defined in a disease-free sample of French middleaged adults[END_REF] -the variation of the PTA variables is greater (reflected by wider reference limits) in women than in men. At present, the reason for this phenomenon is unknown.

Recent evidence indicates that some of the observed gender specific differences in a number of common traits and diseases might, at least partially, be due to sexspecific differences in genetic architecture [START_REF] Ober | Sex-specific genetic architecture of human disease[END_REF] [START_REF] Brix | High frequency of skewed Xchromosome inactivation in females with autoimmune thyroid disease: A possible explanation for the female predisposition to thyroid autoimmunity[END_REF][START_REF] Brix | Preliminary evidence of a noncausal association between the X-chromosome inactivation pattern and thyroid autoimmunity: a twin study[END_REF][START_REF] Ozcelik | Evidence from autoimmune thyroiditis of skewed X-chromosome inactivation in female predisposition to autoimmunity[END_REF] , the pattern of X chromosome inactivation (XCI) could offer an explanation for the observed increased variability of the PTA set point variables in women. In female cells, one of the two X chromosomes is inactivated in early embryonic life in order to equalize the dosage of X linked genes between females and males [START_REF] Brown | The causes and consequences of random and non-random X chromosome inactivation in humans[END_REF] . Consequently, females are mosaics for two cell lines, cells with the paternal and cells with the maternal X chromosome as the active X. In young to middle aged females the distribution of the two cell lines is close to normal (50:50 ratio) [START_REF] Brown | The causes and consequences of random and non-random X chromosome inactivation in humans[END_REF][START_REF] Kristiansen | Twin study of genetic and aging effects on X chromosome inactivation[END_REF] . A skewed XCI pattern is a marked deviation from the 50:50 ratio (80:20 or more) and is present in approximately 15% of females below 55 years [START_REF] Brown | The causes and consequences of random and non-random X chromosome inactivation in humans[END_REF][START_REF] Kristiansen | Twin study of genetic and aging effects on X chromosome inactivation[END_REF] .

The XCI process is under genetic control [START_REF] Kristiansen | Twin study of genetic and aging effects on X chromosome inactivation[END_REF] and it has been linked to loci on the X chromosome [START_REF] Naumova | Genetic mapping of X-linked loci involved in skewing of X chromosome inactivation in the human[END_REF] . The X chromosome may also be involved in the regulation of the PTA set point because variables with impact on the serum levels of TSH, FT3 and FT4 such as monocarboxylate transporter 8 (thyroid hormone transporter) [START_REF] Friesema | Genetics and phenomics of thyroid hormone transport by MCT8[END_REF] and thyroxine-binding globulin 32 are X linked. Additionally, the X chromosome may also be involved in the development of thyroid diseases biochemically characterized by skewed values of TSH, FT3 and FT4 [START_REF] Barbesino | Linkage analysis of candidate genes in autoimmune thyroid disease. ll. Selected gender-related genes and the X-chromosome[END_REF] .

It follows that variation in XCI pattern may allow female monozygotic (MZ) twins to express discordance for traits that are influenced by X linked genes [START_REF] Ober | Sex-specific genetic architecture of human disease[END_REF] . This has been demonstrated in X linked single gene disorders such as Duchenne muscular dystrophy and X linked immunodeficiencies [START_REF] Brown | The causes and consequences of random and non-random X chromosome inactivation in humans[END_REF] . Recently, the potential contribution of polymorphic X linked quantitative trait loci to complex phenotypes has been evaluated by comparing correlations between female and male MZ pairs [START_REF] Loat | X inactivation as a source of behavioural differences in monozygotic female twins[END_REF] . If the phenotype is influenced by polymorphic X linked loci, female MZ pairs should be more discordant than male MZ pairs as a result of skewed XCI [START_REF] Loat | X inactivation as a source of behavioural differences in monozygotic female twins[END_REF] . Recently, we have Given the potential gender specific differences in the PTA set point variables together with the possible involvement of X linked genes, the aim of our study was to test whether, or not, variation in the PTA set point variables in women is influenced by XCI pattern.

Subjects

A detailed description of the ascertainment procedure has been published elsewhere 35 . In brief, in 1997 a representative sample of self-reported healthy twin pairs born between 1931 and 1982 was recruited from the Danish Twin Register on the basis of nationwide questionnaire surveys concerning health and health related behaviour performed in 1994 and 1996. In all, 1512 individuals (756 twin pairs) were examined from 1997 to 2000. Blood samples were available from 736 twin pairs. Twin pairs with self-reported thyroid disease (28 twin pairs) or overt biochemical thyroid disease (18 pairs) were excluded. Moreover, all males (688 subjects) and females from opposite sex pairs (120 subjects) were also excluded leaving 572 female subjects (286 pairs). Of these, 318 subjects (159 twin pairs, distributed in 82 MZ and 77 dizygotic (DZ) pairs) were informative regarding TSH, thyroid hormones and XCI pattern and hence suitable for data analysis. Informed consent was obtained from all participants, and the study was approved by all the Regional Scientific-Ethical Committees in Denmark.

Statistical methods

Group frequencies were compared with the Pearson x 2 test, whereas group medians were compared with a two-sample Wilcoxon rank-sum (Mann-Whitney) test using Brunner's adjustment for clustering within twin pairs [START_REF] Brunner | A nonparametric estimator of the shift effect for repeated observations[END_REF] . Because of deviation from normality, logarithmic transformation of TSH, TPOAb, XCI and BMI was carried out.

The relationship between the thyroid traits (TSH, FT4 and FT3) and the logged values of XCI (within cohort analysis) as well as the relationship between the within twin pair differences in the thyroid traits and within pair differences in logged XCI (within twin pair analysis) were assessed by linear regression.

In the within-cohort analysis, the paired nature of the twin data was taken into account by using the cluster option in STATA (StataCorp, College Station, Texas, USA). Subsequently, the data were analysed with the thyroid traits (TSH, FT4, FT3)

as the response variable and XCI, TPOAb, age, BMI, smoking and zygosity as the explanatory variables.

In the within twin-pair analyses, the regression line was constrained to pass through the origin so that the results were independent of the labelling of the twin as the first or the second. Subsequently, we stratified according to zygosity and adjusted for TPOAb, BMI and smoking by using the within pair differences of these variables as explanatory variables in the within twin-pair regression model.

Results

Descriptive characteristics and distribution of thyroid variables and XCI

measurements in the twin cohort, as a whole and stratified by zygosity, are summarized in table 1.

In the overall study population (within cohort), no significant correlations were found between TSH [regression coefficient (β) = -0.28 (95% confidence intervals, -0.66-0.11), p = 0.158], FT3 [β= -0.25 (-0.85-0.34), p = 0.403], FT4 [β= 0.08 (-0.91-1.07), p = 0.876] and XCI pattern. Controlling for potential confounders (age, TPOAb, BMI, smoking and zygosity) did not change the findings of nonsignificant regression coefficients (Table 2).

The analyses relating the within twin pair differences in TSH, FT3 and FT4 to the within pair differences in XCI pattern are outlined in table 2. Overall, irrespective of zygosity, no significant associations were found. Stratifying for zygosity, and controlling for TPOAb, BMI and smoking did not change the results (Table 2).

However, the within-pair difference in the logged value of XCI pattern is rather small.

Theoretically, this small variability could mask a genuine correlation. To overcome this potential limitation, we restricted the analyses to twin pairs with the highest degree of within-pair variation in lnXCI (difference of at least 0. Finally, since mutations in the MCT8 gene results in an increase in FT3 and a decrease in FT4, the FT3/FT4 ratio could be a more sensitive parameter for variation in XCI pattern. However, neither in the overall study population (within cohort) nor in the within-pair analyses did we find significant correlations between the FT3/FT4 ratio and XCI pattern (β = -0.22 (-0.71-0.03), p = 0.389 and β= -0.02 (-0.08-0.04), p = 0.499, respectively). Adjusting for confounders as well as stratifying for zygosity did not change the finding of non-significant correlations between FT3/FT4 ratio and XCI pattern (data not shown).

Discussion

The present study was initiated to test the hypothesis that a non-hormonal sex specific genetic mechanism related to the X chromosome could influence the set point of the PTA in women. In our sample of euthyroid Danish female twins, we did not observe a significant correlation between the serum concentrations of TSH, FT3 and FT4 and the XCI pattern. To minimize the effect of genetic and environmental confounding we also analysed the association between TSH, FT3 and FT4 and XCI within MZ and DZ twin pairs, which did not change the finding of a non-significant correlation. Essentially similar results were obtained when the analyses were restricted to twin pairs with the most pronounced within pair differences in the XCI pattern. Thus, neither in the within cohort nor in the within twin pair analysis could we demonstrate a statistically significant correlation between thyroid variables and the XCI pattern.

The strengths of this study include: a relatively large sample size (n = 159 twin pairs), ascertainment of participants from a nation-wide population based register, use of standardized and validated procedures for examination and measurement of Our findings should be interpreted cautiously in the light of some methodological limitations. First, the XCI pattern was tested in the leukocytes of peripheral blood, but not in the hypothalamus, pituitary or thyroid gland, the primary tissues involved in determining the PTA set point. Clearly, there are fundamental ethical and practical limitations in obtaining brain and thyroid biopsies from otherwise healthy individuals.

However, from the few studies available, it appears that the correlations of XCI profiles between peripheral blood and different human tissues, including brain tissue, are reasonably high and that XCI in blood can be extrapolated to the XCI profile in other less accessible tissues [START_REF] Brown | The causes and consequences of random and non-random X chromosome inactivation in humans[END_REF][START_REF] Bittel | Comparison of X-chromosome inactivation patterns in multiple tissues from human females[END_REF] . Second, it is important to notice that variations in TBG has a much more pronounced effect on the levels of total T3 and T4 than on FT3 and FT4 levels, making a XCI dependent variation of total T3 and T4 possible.

In the present study we measured FT3 and FT4. Thus, we are not able to investigate this issue further. Finally, all the participants were twins, but there is no reason to suspect that the XCI process and the factors influencing the PTA differ between twins and singletons. Available studies have clearly shown that Danish twins are representative of the general background population as for a number of thyroid traits including distribution of thyroid hormones 2 and the XCI pattern [START_REF] Kristiansen | Twin study of genetic and aging effects on X chromosome inactivation[END_REF] . Epidemiological surveys have repeatedly shown that the spectrum of thyroid phenotypes, including the values of TSH, FT3 and FT4, in a community changes with variations in the iodine intake [START_REF] Laurberg | The Danish investigation on iodine intake and thyroid disease, DanThyr: status and perspectives[END_REF] . In the present study, all participants were Caucasian women living in Denmark, an area with borderline iodine deficiency and with only minor regional

F o r P e e r R e v i e w

Table 2 Regression coefficients for correlation between different thyroid variables and X chromosome inactivation pattern.

Regression coefficients (95% confidence intervals) Unadjusted Adjusted a Analysis TSH Free T3 Free T4 TSH Free T3 Free T4 Within cohort -0.28 (-0.66-0.11) -0.25 (-0.85-0.34) 0.08 (-0.91-1.07) -0.25 (-0.63-0.12) -0.24 (-0.84-0.37) 0.21 (-0.83-1.24) Within twin pairs 0.11 (-0.29-0.50) -0.18 (-0.90-0.54) 0.08 (-1.32-1.48) 0.04 (-0.36-0.44) -0.22 (-0.95-0.52) 0.35 (-1.10-1.79) MZ pairs -0.17 (-0.88-0.54) 0.03 (-1.05-1.11) -0.81 (-3.10-1.48) -0.22 (-0.93-0.49) 0.04 (-1.05-1.12) -0.76 (-3.09-1.56) DZ pairs 0.21 (-0.29-0.71) -0.25 (-1.27-0.77) 0.41 (-1.49-2.30) 0.14 (-0.39-0.67) -0.35 (-1.43-0.73) 0.87 (-1.12-2.87)

a Adjusted for age, TPOAb, BMI, smoking and zygosity in the within cohort analysis. In the within twin pair analysis there is adjusted for TPOAb, BMI and smoking.

  lower correlations (reflecting more discordance) in the PTA set point variables in female MZ pairs compared with male MZ pairs 2 .

7

 7 hormones and thyroid autoantibodies Serum concentrations of TSH and thyroid peroxidase autoantibodies (TPOAb) were measured using solid-phase time-resolved fluoroimmunometric assays(AutoDELFIA; Perkin Elmer/Wallac, Turku, Finland). Serum free T4 and free T3 were determined using the AutoDELFIA FT4 and FT3 kits (Perkin Elmer/Wallac, Turku, Finland), respectively. The reference range for TSH, FT4, FT3 and TPOAb is 0.30-4.00 mU/l, 9.9-17.7 pmol/l, 4.3-7.4 pmol/l and 2-10 KIU/l, respectively[START_REF] Jensen | Establishment of a serum thyroid stimulating hormone (TSH) reference interval in healthy adults. The importance of environmental factors, including thyroid antibodies[END_REF][START_REF] Jensen | Establishment of reference distributions and decision values for the antibodies against thyroid peroxidase (TPOAb), thyroglobulin (TgAb) and the thyrotropin receptor (TRAb)[END_REF] . All blood samples were drawn between 8 and 9 a.m. hour after a 12 hour fast.X-chromosome inactivation analysisDNA was extracted from peripheral blood cells. The X chromosome phenotype was determined by polymerase chain reaction (PCR) analysis of a polymorphic (CAG)n repeat in the first exon of the androgen receptor gene. After digestion of the DNA with the methylation sensitive enzyme Hpall, a PCR product is obtained from the inactive X chromosome only. The PCR products were separated on an ABI 3100 automated sequencer, and analysed by GeneScan software (Applied Biosystems, Foster City, California, USA). Each sample was analysed in duplicate and blinded as to the result in the corresponding co-twin. XCI was calculated as the percentage of the predominantly inactive allele to the sum of both alleles and varies between 50 and 100, where 50 reflects random XCI and 100 reflects a completely skewed XCI.Zygosity was established by DNA fingerprinting using a PE Applied Biosystems AmpFISTER Profiles Plus Kit (Foster City, California, USA).

  Significant differences were defined as a P-value less than 0.05 using two tailed tests. All analyses were carried out using version 11 of the STATA statistical package (StataCorp, College Station, Texas, USA).

  2, n = 46 pairs). The regression coefficients were, however still nonsignificant [TSH; β = 0.25 (-0.24-0.73), p = 0.312. FT3; β = -0.17 (-1.32-0.97), p = 0.761. FT4; β = 0.42 (-1.56-2.40), p = 0.670].

  and exclusion of subjects with evidence of thyroid disease. In addition, our study is very robust, because the twin design allows optimal control for genetic and environmental factors affecting both the pituitary-thyroid axis set point and the XCI pattern[START_REF] Macgregor | Twins. Novel uses to study complex traits and genetic diseases[END_REF] .

  . Theoretically, as shown for

	autoimmune thyroid disease
	F o r
	P
	e
	e r
	R
	e
	v i e
	w
	4

  (10.1-14.8) 12.4 (10.4-14.5) 12.3 (9.8-15.0) All values, except for smoking, are presented as medians with 5th and 95th percentiles in parentheses. MZ = monozygotic and DZ = dizygotic.

	differences in iodine intake. Moreover, cultural background and living conditions are Table 1
	generally quite homogeneous in Denmark. Consequently, our results cannot Basic characteristics
	uncritically be extrapolated to other populations. Study population
	Variable	MZ + DZ (n=318)	MZ (n=164)	DZ (n=154)
	In conclusion, in a sample of healthy euthyroid Danish female twins, we found no Smoking (%) 52 48 56
	Age (year)	36 (19-51)	35 (19-51) a	38 (21-52)
	evidence of a relationship between XCI pattern and pituitary-thyroid axis set point. BMI (kg/m 2 ) 23 (19-31) 23 (19-31) 23 (19-31)
	Degree of skewing	63 (51-87)	63 (51-87)	63 (51-88)
	TPOAb (kIU/liter)	4.7 (2.2-93.0)	4.6 (2.3-79.6)	4.9 (2.8-156.3)
	TSH (mU/liter)	1.6 (0.7-3.3)	1.7 (0.7-3.6) b	1.4 (0.6-3.2)
	Free T3 (pmol/l)	5.8 (4.6-7.3)	5.9 (4.8-7.1)	5.8 (4.5-7.3)
	Free T4 (pmol/l)	12.4
		F o r F o r F o r F o r F o r F o r
		P P P P P P
		e e e e e e
		e r e r e r e r e r e r
			R R R R R R
			e e e e e e
			v i e v i e v i e v i e v i e v i e
				w w w w w w
				11

a MZ vs DZ, p = 0.022 b MZ vs DZ, p = 0.017

Clinical Endocrinology

This study was supported in part by grants from the Agnes and Knut Mørks foundation, The Danish Research Agency, The Foundation of 17-12-1981, The Novo Nordisk Foundation, The Danish Medical Research Council, The Danish Diabetes Association, and The Danish Hearth Foundation.


MD MSc Wishal D Ramdas

Najaf Amin Dsc

MD Leonieke M E Van Koolwijk

A Cecile

PhD J W Janssens

MSc Aisa Demirkan

MD Paulus T V M De Jong

PhD Yurii S Aulchenko

MD Roger C W Wolfs

MD Albert Hofman

MD Fernando Rivadeneira

PhD Andre G Uitterlinden

PhD Ben A Oostra

MD Hans G Lemij

MD Caroline C W Klaver

MD Johannes R Vingerling
email: j.vingerling@erasmusmc.nl

MD Nomdo M Jansonius

PhD Cornelia M Van Duijn

Genetic architecture of open-angle glaucoma and related determinants

Keywords: Genetics, Vertical cup-disc ratio, Intraocular pressure, Open-angle laucoma, Polygenomic, Genetic epidemiology

Background -Although the vertical cup-disc ratio (VCDR) and intraocular pressure (IOP) are important determinants of open-angle glaucoma (OAG), it is unclear to what extent the genetic origin of these traits overlap with those of OAG. We evaluated whether the same genes that determine VCDR and IOP also predict OAG.

Methods -Genetic risk scores were constructed from single nucleotide polymorphisms (SNPs) using genome-wide association data of 9,326 participants from the Rotterdam Study cohorts (mean±standard deviation age: 64.6±9.1). These risk scores were used to calculate the explained variance of VCDR and IOP in an independent cohort (Erasmus Rucphen Family study) consisting of 1,646 participants (mean±standard deviation age: 46.8±14.1) and the OAG risk in a subset of the Rotterdam Study cohorts.

To evaluate false positive findings, we generated two new variables containing randomly sampled values to serve as a negative control.

Results -The explained variance of VCDR increased when increasing the number of SNPs included in the risk score, suggesting a polygenic model. We found no clear evidence for a similar model for IOP, suggesting that a small number of SNPs determine the susceptibility to IOP. The SNPs related to IOP in terms of p-values contributed little to VCDR. The risk scores associated with VCDR were also associated significantly with OAG. This suggests a common polygenic background for VCDR and OAG Conclusions -We found evidence for a polygenic model underlying one of the major traits of OAG, VCDR, and OAG itself. The IOP did not show any evidence for such a model.

INTRODUCTION

Open-angle glaucoma (OAG) is a neurodegenerative disease that leads to progressive damage to the retinal ganglion cells and nerve fibers, resulting in glaucomatous visual field loss. It is one of the leading causes of irreversible blindness in the world, affecting over 60 million persons worldwide. [START_REF] Quigley | The number of people with glaucoma worldwide in 2010 and 2020[END_REF] Although the etiology of OAG is still obscure, it has been recognized for long that genetic factors play a role in its pathogenesis. Relatives of patients with OAG have an increased risk of OAG. [START_REF] Wolfs | Genetic risk of primary open-angle glaucoma. Population-based familial aggregation study[END_REF] Several rare genetic variants associated with a high risk of disease have been implicated in familial forms of OAG, but these variants only explain a small percentage of OAG patients. [START_REF] Allingham | The genetics of primary open-angle glaucoma: a review[END_REF] This raises the question whether the genetic etiology of OAG is determined by a large number of rare variants with major effects on the disease risk (rare variant, common disease hypothesis) or whether there are multiple common variants underlying the disease (common variant-common disease hypothesis).

Genome-wide association studies (GWAS) have identified a large number of common variants (single nucleotide polymorphisms [SNPs]) with a low risk for a large number of common diseases (e.g. type 2 diabetes, Alzheimer's disease, and various forms of cancer), but also for diseases that are less common such as multiple sclerosis and Crohn's disease. [START_REF] Manolio | Finding the missing heritability of complex diseases[END_REF] GWAS have also been powerful in studies on quantitative risk factors of diseases such as blood pressure, glucose levels and height. [START_REF] Manolio | Finding the missing heritability of complex diseases[END_REF] More recently, GWAS were used to identify polygenic forms of inheritance for schizophrenia, implicating ten thousands of genetic variants covering the whole genome rather than a distinct number of variants. [START_REF] Purcell | Common polygenic variation contributes to risk of schizophrenia and bipolar disorder[END_REF] The essence of the latter approach is that a gene discovery sample is used to define risk scores, and the risk is subsequently predicted in an independent sample. In this approach, not only the variants that are genomewide significant are used but also those who do not reach this threshold. This method can also be applied to test for overlapping of genes between diseases and their underlying liability.

If there is a polygenic model that explains part of the susceptibility to a disease, the classical theory of Fisher predicts that the number of risk alleles is distributed according to the Gaussian distribution in the population. [START_REF] Fisher | The correlation between relatives on the supposition of Mendelian inheritance[END_REF] It is easy to show that if there are >30 genetic variants, no person in the population carries zero risk alleles but everyone carries a gradient number of risk alleles. [START_REF] Janssens | Genome-based prediction of common diseases: advances and prospects[END_REF] Thus, a polygenic model implies that individuals in a population always carry risk alleles for a disease and that the liability to disease increases with the number risk alleles one carries. It also follows from this model that underlying a disease that is defined as present or absent, there is probably a quantitative trait. The persons in the tail of the distribution of this trait are considered to be "diseased". If the quantitative trait (or risk factor) is related to the genotypic distribution and increases with the number of risk alleles, the risk of disease will behave accordingly.

If we translate this to OAG, there are two clinical measures consistently associated with this disease and may thus determine its (polygenic) liability to OAG. First, OAG is characterized by damage to the optic nerve head, which is visible as an (increased) excavation (cupping) upon ophthalmological examination. The extent of the excavation is commonly quantified as the vertical cup-to-disc ratio (VCDR), ranging from 0 to 1. [START_REF] Kwon | Primary open-angle glaucoma[END_REF] Both an increase in VCDR and an unusually large VCDR at a single observation may indicate glaucomatous changes of the optic nerve head. [START_REF] Quigley | Open-angle glaucoma[END_REF] The second clinical measure that may determine the (polygenic) liability to OAG is the intraocular pressure (IOP). Current treatment for OAG is based on lowering the IOP. VCDR as well as IOP are highly heritable, making these two variables potentially important endophenotypes for OAG. [START_REF] Van Koolwijk | Genetic contributions to glaucoma: heritability of intraocular pressure, retinal nerve fiber layer thickness, and optic disc morphology[END_REF] Until now it is unknown to which extent the genes involved in VCDR overlap with those involved in IOP, and to which extent they actually predict OAG.

The aim of this study was to investigate whether there is a polygenic model in which many variants apply to VCDR and IOP and whether these genes also predict the risk of OAG. For this study, we used the Rotterdam Study I, Rotterdam Study II and Rotterdam Study III (RS-I, RS-II and RS-III, respectively) as discovery cohorts to derive genetic risk scores that capture the genes involved in VCDR and IOP. Next, we assessed whether these risk scores predicted VCDR and IOP in an independent target cohort (the Erasmus Rucphen Family [ERF] study). For the prediction of OAG, RS-I served as the target cohort, because no OAG cases were included in ERF.

METHODS

Study Populations

The RS-I is a prospective population-based cohort study of 7,983 residents aged 55 years and older living in Ommoord, a suburb of Rotterdam, the Netherlands. Baseline examination for the ophthalmic part took place between 1991 and 1993; follow-up examinations were performed from 1997 to 1999 and from 2002 to 2006. [START_REF] Hofman | The Rotterdam Study: objectives and design update[END_REF][START_REF] Stricker | Analysis of individual drug use as a time-varying determinant of exposure in prospective population-based cohort studies[END_REF] RS-I included 188 OAG cases that either already had OAG at baseline or developed OAG during follow-up.

The RS-II and RS-III are two other prospective population-based cohort studies of respectively 3,011 residents aged 55 years and older and 3,392 residents aged 45 years and older. The rationale and study design are similar to that of the RS-I. [START_REF] Hofman | The Rotterdam Study: objectives and design update[END_REF] The baseline examination of RS-II took place between 2000 and 2002; the follow-up examination was performed from 2004 to 2005. Baseline examination of RS-III took place between 2006 and 2009. [START_REF] Hofman | The Rotterdam Study: 2010 objectives and design update[END_REF] The ERF Study is a family-based cohort in a genetically isolated population in the southwest of the Netherlands with over 3,000 participants aged between 18 and 86 years. Cross-sectional examinations took place between 2002 and 2005. The rationale and study design of this study have been described elsewhere. [START_REF] Aulchenko | Linkage disequilibrium in young genetically isolated Dutch population[END_REF][START_REF] Pardo | The effect of genetic drift in a young genetically isolated population[END_REF] All measurements in these studies were conducted after the Medical Ethics Committee of the Erasmus University had approved the study protocols and all participants had given a written informed consent in accordance with the Declaration of Helsinki.

Ophthalmic examination

The ophthalmic assessment in RS-I and RS-II, both for baseline and follow-up, included a medical history, autorefraction, keratometry, IOP measurement, visual field testing and optic nerve head imaging with simultaneous stereoscopic photography (ImageNet; Topcon Corporation, Tokyo, Japan) of both eyes after mydriasis with tropicamide 0.5% and phenylephrine 2.5% eye drops.

RS-III was similar to RS-I except for optic nerve head imaging with Heidelberg Retina Tomograph 2 (HRT; Heidelberg Engineering, Dossenheim, Germany). The ophthalmic assessment in ERF included a medical history, autorefraction, keratometry, IOP measurement, and optic nerve head imaging with HRT of both eyes after pharmacologic mydriasis.

In all cohorts we measured IOP at baseline and at every follow-up round with Goldmann applanation tonometry (Haag Streit, Bern, Switzerland) after applying oxybuprocaine 0.4% eye drops. The median value of three successive measurements was recorded. [START_REF] Dielemans | Reliability of intraocular pressure measurement with the Goldmann applanation tonometer in epidemiological studies[END_REF] The OAG definition was based on the presence of glaucomatous visual field loss. Details have been described elsewhere. Neither VCDR nor IOP were part of the diagnosic criteria of OAG. [START_REF] Wolfs | Changing views on open-angle glaucoma: definitions and prevalences--The Rotterdam Study[END_REF][START_REF] Czudowska | Incidence of Glaucomatous Visual Field Loss: A Ten-Year Follow-up from the Rotterdam Study[END_REF] ImageNet, which was used for optic nerve head imaging in RS-I and RS-II, takes simultaneous stereoscopic images of the optic disc at a fixed angle of 20°, using a simultaneous stereoscopic fundus camera (Topcon TRC-SS2; Tokyo Optical Co., Tokyo, Japan). Images were analyzed using the ImageNet retinal nerve fiber layer height module. On each stereoscopic pair of optic disc images four points were marked on the disc margin, defined as the inner border of the peripapillary ring or the outer border of the neural rim, if a scleral ring was visible. Next, the software drew an ellipse using these points to outline the disc margin and to determine the cup. The amount of correspondence between the marked points on the two images of the stereoscopic pair is expressed as a "bad points" percentage, which indicates the percentage of points lacking correspondence. This percentage can be used as an indicator of image quality.

Images with 25% or more bad points were excluded. [START_REF] Rolando | Measurement variability in digital analysis of optic discs[END_REF] HRT 2, used for optic nerve head imaging in RS-III and ERF, uses a focused 670-nm diode laser light beam to acquire scans of the optic nerve head region, using the confocal principle. The HRT obtains, during one scan, three series of 16 to 64 confocal frontal slices. From each of these series, a 3-dimensional image of the optic nerve head is reconstructed, from which the software calculates several optic disc parameters. To define the cup, the HRT places a reference plane 50 μm below the peripapillary retinal surface in the region of the papillomacular bundle. Imaging was performed after entering the participant's keratometry data into the software and after adjusting the settings in accordance with the refractive error.

In RS-III all HRT 2 data were converted to HRT 3. As an indicator of image quality we used the topographic standard deviation of the scan, which is a measure of the variability among the three series of a single HRT scan. Scans with a topographic standard deviation exceeding 50 μm were excluded. The inter-observer variability and agreement for both systems have been described elsewhere. [START_REF] Ikram | Comparing ophthalmoscopy, slide viewing, and semiautomated systems in optic disc morphometry[END_REF][Ramdas, et al.

unpublished data]

Genotyping

In the RS-I, RS-II and RS-III cohorts, DNA was genotyped using the Illumina Infinium II HumanHap arrays according to the manufacturer's protocols. After exclusion of participants for reasons of low-quality DNA, a total of 5,974 participants were available with genotyping data from RS-I (HH550 v3.0®), 2,157 participants were from RS-II and 2,082 from RS-III (HH550Duo and HH610Quad®). Details are described elsewhere. [START_REF] Rivadeneira | Twenty bone-mineral-density loci identified by large-scale meta-analysis of genome-wide association studies[END_REF] In ERF, DNA was genotyped on four different platforms (Illumina 6k, Illumina 318K, Illumina 370K and Affymetrix 250K), which were then merged and imputed to 2.5 million SNPs hapmap using build 36 HapMap (release 22) CEU population as a reference cohort. After exclusion of participants for whom genotyping data were unavailable, 2,385 had genotyping data.

Extensive quality control analyses have been performed in each cohort.

Statistical analysis

Statistical analysis within the discovery cohorts

If data of both eyes were available, we randomly chose one. In case of missing or unreliable baseline data of both eyes, we used follow-up data whenever possible. For each of the discovery cohorts (comprising RS-I, RS-II and RS-III) linear regression models were used to examine the associations between the risk scores and VCDR and IOP, adjusted for age and gender. For VCDR we also adjusted for optic disc area.

For IOP the mean value of both eyes was included and adjusted for IOP lowering treatment. This adjustment was done by adding 30% upon the measured IOP for medical treatment and by fixing the IOP at 30 mmHg for surgical/laser treatment in the past.

To summarize the results through the three discovery cohorts, we performed a meta-analysis with Metal for Linux (www.sph.umich.edu/csg/abecasis/metal) using the inverse variance method of each effect size estimate. [START_REF] Sanchez-Meca | Weighting by Inverse Variance or by Sample Size in Meta-Analysis: A Simulation Study[END_REF] All statistical analyses were performed using SPSS version 15.0.0 for Windows (SPSS inc., Chicago, IL, USA; 2006), PLINK [START_REF] Purcell | PLINK: a tool set for whole-genome association and population-based linkage analyses[END_REF] and R statistical package version 2.10.1 for Mac (www.r-project.org).

Risk scores within the independent cohort

The risk score profiling method tests the association of a genetic score variable that reflects a combined effect of a number of selected SNPs with a trait. Details of this method have been described elsewhere. [START_REF] Purcell | Common polygenic variation contributes to risk of schizophrenia and bipolar disorder[END_REF] The results of the SNPs of the meta-analyzed discovery cohorts were categorized according to p-value and examined in an independent cohort (ERF). The p-value thresholds were as follow: p<10 -10 , p<10 -9 , p<10 - 8 , p<10 -7 , p<10 -6 , p<10 -5 , p<10 -4 , p<10 -3 , p<10 -2 , p<0.1, p<0.2, p<0.3, p<0.4, p<0.5, p<0.6, p<0.7, p<0.8, p<0.9, p<1.0. For each category the effect (beta) of each SNP was multiplied by the corresponding number of alleles of each participant to calculate a score for that particular SNP. The mean of all these scores for the respective participant was the risk score. In case of missing SNP data from a participant of the replication cohort the allele frequencies were used and multiplied by the betas. Next, we ran linear regression analyses with the VCDR as the outcome measure and the risk scores adjusted for age and gender as the determinant in order to calculate the proportion of explained variance (PEV) of each group of SNPs (i.e. p-value threshold) as determined in the discovery cohort. A p-value of 0.05 or smaller was considered as statistically significantly associated with the trait.

The same analysis was also done with IOP as the outcome. To examine whether a "non-ophthalmic" variable reveals the same results we performed the same analysis for a negative control. For this variable we used a sampling method without replacement. We sampled the VCDR respectively IOP in the target cohort, and thus, the distribution remained the same. In addition, we created a second normal distributed variable by assigning a random number (with mean: 10; standard deviation: 1) to all participants.

For the analysis of the family-based data (ERF) we used SOLAR version 4.1.5 for Linux to adjust for the pedigree structure. [START_REF] Almasy | Multipoint quantitative-trait linkage analysis in general pedigrees[END_REF] Finally, we applied the same approach for the OAG cases of RS-I by calculating the Nagelkerke R-square of OAG for the VCDR and IOP risk scores. This was done using logistic regression analyses adjusted for age and gender. Since, as a matter of course, individuals with OAG may have an increased VCDR and/or IOP, we performed a secondary analysis in which we excluded RS-I from the meta-analysis of the discovery cohorts to avoid possible biased results. Differences in general characteristics between cases and controls were analyzed with independent t-tests and chi-square statistics.

Only autosomal SNPs that were available in all four cohorts (~520.000 SNPs) were included. To have less overlap of linkage disequilibrium blocks we did not use imputed data. SNPs that deviated significantly from Hardy-Weinberg equilibrium (p<0.0001) or that had a minor allele frequency less than 0.05 were excluded from the present study.

RESULTS

Study samples

Of the 5,974 participants that were genotyped in the RS-I cohort 5,312 had valid VCDR data. The remaining 662 were excluded because of missing or unreliable data. From the RS-II cohort 2,048 from 2,157 genotyped participants were included in this study. For 109 persons we did not have (reliable) data.

From the RS-III cohort a total of 1,966 participants were included. The target cohort for the quantitative analysis (ERF) included 1,646 participants. Table 1 summarizes the general characteristics for the discovery cohorts and the replication cohort. The participants of the target set were significantly younger (p<0.001), and had lower VCDR values (p<0.001). The latter difference can be explained by the use of different measurement techniques for optic disc imaging (since we analyzed the cohorts separately and subsequently meta-analyzed the findings, this difference will not influence our findings). The distributions of VCDR and IOP for the four cohorts are shown in Supplementary Figure S1. Genetic outliers of non-European ancestry were excluded. No institutional heterogeneity between the cohorts or residual population sub-stratification was noticed after inspecting the genotype data. [START_REF] Liu | Digital quantification of human eye color highlights genetic association of three new loci[END_REF] Inflation factors for the included cohorts for VCDR and IOP ranged from 1.024 to 1.061 and from 1.006 to 1.037, respectively.

Risk score analysis

For VCDR, age and gender explained 0.3% of the total variance. Figure 1A presents the PEV attributable to the risk scores when risk scores from the VCDR analysis in the discovery sample were used to predict VCDR in the target sample ERF. Figure 1B shows the PEV when the same risk scores were used to predict a random variable (negative control). For VCDR, the risk score based on the p-value threshold of p<10 -10 (first bar) consisted of 9 SNPs (Table 2) and explained only 0.1% (in addition to age and gender; region above the dotted line) of the variance in VCDR (Figure 1A) in the target population. Increasing the number of SNPs included in the risk scores (i.e. increasing the p-value thresholds for the SNPs to be included) resulted in a gradual increase in the PEV until the p<10 -2 threshold was used (consisting of 7,260 SNPs; p=8.68x10 -4 ). The PEV increased up to 1.0% (p=1.91x10 -4 ) at the threshold of p<0.2 for the SNPs to be included in the risk score and then stabilized. This suggests that there is a polygenic model underlying the genetics of VCDR. No similar pattern was observed for the negative controls, i.e., the random variable used to evaluate false positive associations other than due to confounding by admixture (lowest observed p = 0.204). For the second negative control, based on sampling without replacement, increasing the p-value thresholds for the SNPs did not have any effect on the PEV, neither for the negative control based on random generated numbers (Figure 1B; Supplementary Figure S2).

Figures 1C and1D present the corresponding graphs for IOP. For IOP, age and gender explained 2.5% of the total variance, while the first risk score based on a threshold of p<10 -8 explained an additional 0.2% (region above the dotted line) of the variation in IOP in the target sample ERF. This score included only one SNP and did not reach nominal significance (p=0.093). However, the addition of more SNPs did not yield any increase in PEV. None of the more extended risk scores were even marginally significant.

To evaluate whether there is a shared genetic component between VCDR and OAG and IOP and OAG, we tested the association of risk scores based on VCDR and IOP in the discovery sample with OAG cases and controls in RS-I. Table 3 shows the descriptive data of the OAG cases and controls in RS-I. A total of 5,304 participants of RS-I had complete data for optic nerve head measurements, IOP and reliable visual fields. Of these, 171 were classified as having OAG. This classification was based on the presence or absence of glaucomatous visual field loss. OAG cases were significantly older and more often men compared to controls. Not surprisingly, OAG cases had a larger VCDR and an increased IOP compared to controls, although these measures were not part of the classification process.

Figure 2A displays the PEV (Nagelkerke R-square) of OAG in RS-I attributable to VCDR genetic risk scores. Age and gender explained 4.0% of the variance of OAG and the first risk score based on the pvalue threshold of p<10 -10 (first bar) explained only an additional 0.3% of the OAG variance. However, when more SNPs with higher p-values were added to the score, the PEV increased sharply from p<10 -2 and kept on increasing up to the inclusion of SNPs in the risk score with p<0.3 in the discovery set. The latter score explained up to 4.7% of the variance of OAG above the attribution of age and gender, which is more than what is explained by age and gender together. The pattern of Figure 2A suggests a polygenic model for OAG as well and also a large genetic component shared by both VCDR and OAG. In a secondary analysis we excluded RS-I from the meta-analysis of the discovery cohorts and found a similar trend (Figure 3A). Nevertheless, as a consequence the PEV was much lower due to a major reduction in power. Results for IOP were similar to VCDR if we included RS-I, in that the risk scores were significantly associated with OAG (Figure 2B). However, in contrast to VCDR the evidence for a polygenic model underlying IOP disappeared in the secondary analyses (i.e. after excluding RS-I from the meta-analysis of the discovery cohorts; Figure 3B).

DISCUSSION

In the present study of GWAS data from Caucasian participants, we found evidence for a polygenic model underlying VCDR, one of the major known measures for OAG. In contrast, IOP did not show significant evidence for a polygenic model in the current study. Of these two traits, VCDR is the most relevant trait related to the diagnosis of OAG, while IOP is the most relevant trait for OAG treatment in patients with elevated IOP. Of interest is the finding that the risk scores based on the VCDR also predicted OAG.

Although both VCDR and elevated IOP are putative endophenotypes for OAG, only VCDR showed evidence for a joint polygenic origin with OAG.

In the approach of the present study a large discovery cohort is crucial, while the size of the target cohort is less important. Therefore, we combined data of three independent cohorts from the Rotterdam Study, and used a single target cohort (ERF). We also repeated our analysis in which we used RS-III as a second replication cohort, rather than as one of the discovery cohorts. The results were similar, further supporting the evidence for a polygenic model of VCDR (data not shown).

At first sight, it seems unexpected that we did not find a similar model for IOP as for VCDR, because an elevated IOP has been implicated as the most important determinants of both glaucomatous optic neuropathy (often referred to as an increased VCDR) and glaucomatous visual field loss. In some patients with OAG the IOP is not elevated. This form of disease is referred to as normal tension glaucoma, which represents about 15-40% of all patients with OAG. [START_REF] Dielemans | The prevalence of primary open-angle glaucoma in a population-based study in The Netherlands. The Rotterdam Study[END_REF] In these patients, however, IOP lowering also causes a decrease in the rate of disease progression, suggesting that IOP matters in normal tension glaucoma as well. Due to the relatively low number of cases in our study, we did not separately analyze patients with normal tension glaucoma. For both traits, IOP and VCDR, strong evidence of genetic correlation with OAG susceptibility has been found. These two traits also correlate with each other in our population (Pearson's p<0.001) and in other populations. [START_REF] Charlesworth | The path to open-angle glaucoma gene discovery: endophenotypic status of intraocular pressure, cup-to-disc ratio, and central corneal thickness[END_REF] Nonetheless, when we tested whether the risk scores based on IOP predicted VCDR in the target cohort we found 0.54% as the highest significant PEV for VCDR of IOP-related SNPs (at p<10 -8 ; data not shown). This finding suggests there is little overlap in the genes determining IOP and VCDR. Combined with the current findings this suggests that there are other, possibly rare variants with strong effects or yet unidentified environmental risk factors that determine IOP. However, a drawback of our analysis of IOP is that we did not adjust for the central corneal thickness, while the measurement of IOP may also be affected by central corneal thickness. [START_REF] Wolfs | Distribution of central corneal thickness and its association with intraocular pressure: The Rotterdam Study[END_REF] All OAG cases were only derived from RS-I. A potential problem in the interpretation of the findings on OAG might be that the OAG cases derived from RS-I were also included in the discovery cohorts for the VCDR genetic risk scores. Although this may create an autocorrelation between the VCDR risk scores defined in the discovery set and OAG prediction conducted in the same set, this problem also occurred for the IOP scores. As the IOP score did not associate with the risk of OAG, it appears unlikely that any autocorrelation bias explained the overlap in genes involved in VCDR and OAG. Furthermore, it is important to realize that the diagnosis for OAG in RS-I was primarily based on glaucomatous visual field loss, thus not on VCDR or IOP. Moreover, the results did not alter when we excluded RS-I from the discovery cohorts.

As far as we know, the current study is the first to describe the phenomenon of a polygenic model for optic disc cupping (expressed in the VCDR). We further showed that the same risk scores predicted OAG. This finding sheds new light on the etiology of OAG. The amount of variance explained by the VCDR risk scores exceeded that of age and gender. Nevertheless the PEV of both was still too low to allow the prediction of OAG in persons at risk. Rare variants with large effects have also been implicated in the etiology of OAG. However, the current data suggest that many SNPs (probably with very small effects) may collectively account for a substantial proportion of variation in VCDR. This implies that many common variants have not been identified yet, because of low effect estimates of risks associated with single SNPs. Larger GWAS are needed to detect those variants and to further replicate our findings.

In conclusion, the present study has three major implications. First, in our large epidemiological study there is little overlap between the genes involved in VCDR and IOP. Second, the current study provides evidence for a polygenic model in VCDR involving many common SNPs that are not shared by a random variable, but specific to VCDR itself. This polygenic basis is not shared by the IOP, which may suggest that either the trait IOP has underlying rare variants, or the variants involved in IOP have such small effects that they were not well detected in the discovery set because of lack of power. Third, the genetics of VCDR overlap convincingly with that of OAG.

LICENCE STATEMENT

"The Corresponding Author has the right to grant on behalf of all authors and does grant on behalf of all authors, an exclusive licence (or non exclusive for government employees) on a worldwide basis to the BMJ Publishing Group Ltd to permit this article (if accepted) to be published in JMG and any other BMJPGL products and sublicences such use and exploit all subsidiary rights, as set out in our licence (http://group.bmj.com/products/journals/instructions-for-authors/licence-forms)."

Figure 1 .

 1 Figure 1. The explained variance of risk scores of the vertical cup-disc ratio (VCDR) and intraocular pressure (IOP) for each p-value threshold for their respective trait in the target cohort ERF (A and C, respectively); and the explained variance of the risk scores of VCDR and IOP for each p-value threshold for the negative control (B and D, respectively). The y1-axis (left; histogram) depicts the explained variance (in %) against different p-value thresholds on the x-axis that were used to construct the risk scores. The y2-axis (right; line) depicts the p-values from the association analysis of the risk scores with the trait. Dotted line represents the attribution of age and gender to the explained variance.

Figure 2 .

 2 Figure 2. The Nagelkerke R-square of risk scores of the vertical cup-disc ratio (VCDR) and intraocular pressure (IOP), adjusted for age and gender, for each p-value threshold on open-angle glaucoma (OAG) in RS-I (A and B, respectively).The y1-axis (left; histogram) depicts the explained variance (in %) against different p-value thresholds on the x-axis, which were used to construct risk scores. The y2-axis (right; line) depicts the p-values from the association analysis of the risk scores with the trait.

Figure 3 .

 3 Figure 3. The Nagelkerke R-square of risk scores of the vertical cup-disc ratio (VCDR) and intraocular pressure (IOP), adjusted for age and gender, for each p-value threshold on open-angle glaucoma (OAG) in RS-I (A and B, respectively) after excluding RS-I from the discovery cohort. To display the pattern more clear the attribution of age and gender has already been subtracted. The y1-axis (left; histogram) depicts the explained variance (in %) against different p-value thresholds on the x-axis, which were used to construct risk scores. The y2-axis (right; line) depicts the p-values from the association analysis of the risk scores with the trait.

ACKNOWLEDGEMENTS

Financial support: The Rotterdam Study is funded by Erasmus Medical Center and Erasmus University, Rotterdam, Netherlands Organization for the Health Research and Development (ZonMw), the Research Institute for Diseases in the Elderly (RIDE), the Ministry of Education, Culture and Science, the Ministry for Health, Welfare and Sports, the European Commission (DG XII), and the Municipality of Rotterdam. The generation and management of genome-wide association studies (GWAS) genotype data for the Rotterdam Study is supported by the Netherlands Organisation of Scientific Research NWO Investments (nr. 175.010.2005.011, 911-03-012). This study is funded by the Research Institute for Diseases in the Elderly (014-93-015; RIDE2), the Netherlands Genomics Initiative (NGI)/Netherlands Organisation for Scientific Research (NWO)/Netherlands Consortium for Healthy Aging (NCHA) project nr. 050-060-810. The genetic study in the Erasmus Rucphen (ERF) Study was supported by the Center for Medical Systems Biology (CMSB) of NGI. The ophthalmic part of the Rotterdam Study and ERF are supported by the Netherlands Organization for Health Research and Development (ZonMw) grant 2200.0035; Lijf en Leven, Krimpen a/d Lek; MD Fonds, Utrecht. Oogfonds Nederland, Utrecht; Stichting Nederlands Oogheelkundig Onderzoek, Nijmegen/Rotterdam; Swart van Essen, Rotterdam; Netherlands Organisation for Scientific Research (NWO); Bevordering van Volkskracht, Rotterdam; Blindenhulp, The Hague; Landelijke Stichting voor Blinden en Slechtzienden (LSBS), Utrecht; Rotterdamse Vereniging voor Blindenbelangen, Rotterdam; OOG, The Hague; Algemene Nederlandse Vereniging ter Voorkoming van Blindheid (ANVVB), Doorn; The Rotterdam Eye Hospital Research Foundation (Stichting Wetenschappelijk Onderzoek Het Oogziekenhuis [SWOO] Prof. Dr. H.J. Flieringa, Rotterdam); Laméris Ootech BV, Nieuwegein; Topcon Europe BV, Capelle aan de IJssel, all in the Netherlands, and Heidelberg Engineering, Dossenheim, Germany.

The sponsors or funding organizations had no role in the design, conduct, analysis or publication of this research.

We thank Pascal Arp, Mila Jhamai, Dr. Michael Moorhouse, Jeannette Vergeer, Marijn Verkerk, and Sander Bervoets for their help in creating the GWAS database, and Dolinda Pottuit and Raph de Haas for their help in analyzing the optic disc images. The authors are grateful to the study participants, the staff

from the Rotterdam and ERF Study and the participating general practitioners and pharmacists.

TABLES


Helle-Brit Fiebrich

Gerrit Van Den Berg

Ido P Kema

Thera P Links

Jan H Kleibeuker

Andre Van Beek

Annemiek Me

Wim J Sluiter

Elisabeth Ge De Vries
email: e.g.e.de.vries@int.umcg.nl

Kleibeuker

André Van Beek

Annemiek M E Walenkamp

Elisabeth G E Vries

MD, PhD E G E De Vries

Deficiencies in fat-soluble vitamins in long-term somatostatin analogue users

Keywords: Small intestine < Organ-based, Cancer -biology < Topics, Clinical pharmacology < Topics, Nutrition < carcinoid tumour, acromegaly, fat-soluble vitamin deficiency, somatostatin 22 analogues

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Long-term somatostatin analogue therapy is important to control symptoms of hypersecretion 3 of biologically active amines and peptides in diseases such as acromegaly or metastatic 4 carcinoid tumours. Other indications for long-term use include certain pancreatic islet cell 5 tumours and the dumping syndrome. Side effects of somatostatin analogue treatment are 6 usually acceptable and often passing. The most common adverse effects are diarrhoea, 7 abdominal discomfort and nausea. Gallstones, usually asymptomatic, occur in 10-50% of the 8 treated patients. [START_REF] Oberg | Consensus report on the use of somatostatin analogs 3 for the management of neuroendocrine tumors of the gastroenteropancreatic system[END_REF][2][3] Steatorrhoea is an adverse effect experienced by up to 29% of patients on 9 somatostatin analogue treatment. 4 When patients with acromegaly or a carcinoid tumour 10 cannot be cured by complete surgical tumour resection, they often require long-term 11 somatostin analogue treatment for symptom control. 12 Two somatostatin analogues are currently available for clinical use. Octreotide is 13 registered since 1989, and lanreotide in the Netherlands since 1999. With the passing of time, 14 numerous patients have now received a significantly long-term treatment with these drugs. 15

Given the fat loss in the stool following somatostatin treatment, this will go together with loss 16 of fat-soluble vitamins A, D, E and K1 in the stool. A likely risk factor is the duration of 17 somatostatin analogue treatment. Studies performed thusfar have investigated patients after a 18 relative short treatment period and may therefore well underestimate the frequency of vitamin 19 deficiencies during long-term somatostatin analogue treatment. The only evidence pointing 20 towards an increased risk of a deficiency of fat-soluble vitamins in patients receiving long-21 term somatostatin analogue treatment comes from a study in acromegaly patients. In this 22 study 103 patients were treated for 3-30 months with a mean duration 24 months. They 23 reported a slight but significant fall of carotene, although values remained in the normal 24 range. In addition, one patient in their study developed a prolonged prothrombin time (PT), 25 The frequent resection of a part of the small intestine, as performed in midgut 4 carcinoid patients, could be an additional reason for malabsorption. The severity of the 5 malabsorption after an intestinal resection depends on the total of digestive surface area and 6 the portion of small intestine remaining. 7 Carcinoid patients, that have undergone a partial 7 small bowel resection, may therefore be at greater risk to develop vitamin deficiency than 8 carcinoid or acromegaly patients without a bowel resection. 9

No previous study has systematically investigated long-term somatostatin users and 10 has done so for levels of the complete set of fat-soluble vitamins. Therefore, the extent and 11 severity of fat-soluble vitamin deficiency in patients with acromegaly or a carcinoid tumour 12 are unknown. We hypothesise that deficiencies may be more frequent than currently reported. 13

This study aims to determine the frequency and severity of fat-soluble vitamin deficiency in 14 these patients with a treatment duration of at least 18 months. 15 Patients younger than 18 years and those unable to give informed consent were excluded. 1 Patient charts were checked for diagnosis, reports of previous intestinal resection and length 2 of resection, dose and length of treatment with somatostatin analogues and possible signs and 3 symptoms of a deficiency of fat-soluble vitamins. In addition, patients were asked if they took 4 vitamin supplements, whether they experienced steatorrhoea (defined as loose, greasy stools 5 that are difficult to flush), had special eating habits and if they had complaints that could be 6 due to a vitamin deficiency, such as night blindness or an increased propensity for bleeding. 7

The study was approved by the local medical ethics committee (approved on the 16 th 8 December 2008) and all patients gave written informed consent. The study was registered at 9 the Nederlands (Dutch) trial register (registry), trial code 1730. 10 11

Biochemical Analysis 12

To estimate the extent of side effects of somatostatin analogues on the uptake of fat-13 soluble vitamins we determined plasma concentrations of the fat-soluble vitamins A (retinol), 14 D, E, K1 as described earlier. [8][9][10][11] Vitamin E was also measured in the membrane of 15 erythrocytes with an HPLC method, as vitamin E levels in erythrocytes provide a better 16 measure of long-term vitamin E status than plasma levels. 12, 13 After liquid/liquid extraction 17 with hexane, using tocol and retinol acetate as internal standards, a 35 µL sample was injected 18 on a Waters Symmetry C18 column (Waters Corporation, Milford, MA) and analysed, using a 19 ASI-100 autosampler (Dionex, Sunneyvale, CA), a P680 HPLC pump (Dionex, Sunneyvale, 20 CA) and a Thermo-Finnigan PDA (photodiode array) detector (Thermo Fisher Scientific Inc., 21 Waltham, MA). ATLAS Chromatography Data System (Thermo Fisher Scientific Inc., 22 Waltham, MA) was used for recording and calculating the results. Lower limits of normal in 23 plasma were 0.8 µmol/L for vitamin A, 50 nmol/L for vitamin D 14 , 19.2 µmol/L for vitamin E 24 and 0.8 nmol/L for vitamin K1. Lower limits of normal for vitamin E in erythrocytes was 4.58 25 To measure effects of a possible fat-soluble vitamin deficiency, vitamin-dependent 5 parameters were included. For vitamin D parathyroid hormone (PTH) was measured, while 6 for vitamin K1, PT and activated partial thromboplastin time (aPTT) were evaluated. 7 8

Statistical Analysis 9

Normal values were those determined for healthy controls in our laboratory. In a 10 sample size of 19 acromegaly patients we can demonstrate a significant difference between 11 vitamin levels if 3 abnormal values are measured (15%). In 35 carcinoid patients it is possible 12 to show a significant difference between vitamin levels if 4 abnormal values are measured 13 (12%). 14

The chi-square test was used to test for differences between patients and normal values 15 and the Mann-Whitney U test to test for differences between groups of carcinoid and 16 acromegaly patients. In a linear univariate regression analysis age, sex, length of treatment, 17 treatment dose, type of somatostatin analogue, presence of steatorrhoea, pancreatic enzyme 18 supplementation, diagnosis and bowel resection were tested for associations with plasma 19 vitamin levels. For the analysis, bowel resection was divided into four categories based on the 20 risk of developing malabsorption: no resection, less than 100 cm of bowel resected, between 21 100 and 200cm resected and more than 200 cm resected. 16 Factors with a P level < 0.15 were 22 tested in a multivariate regression analysis, using the reverse stepwise method. Significance 23 level was .05, two-sided. Statistical tests were performed using the SPSS package version 24 16.0. 25 Abnormally low plasma levels for one or more fat-soluble vitamins were detected in 7 78% (n = 42). A deficiency of multiple vitamins was present in 32% (n = 17); in 26% (n = 14) 8 of two, in 4% (n = 2) of three and in 2% (n = 1) of all fat-soluble vitamins. The three patients 9 with a deficiency of three or four vitamins were all carcinoid patients. When vitamin E levels 10 in erythrocytes, which reflects long-term vitamin E status, were considered for vitamin E 11 status instead of plasma vitamin E levels more patients (n = 51, 91%) had a deficiency. 12

The most frequently deficient vitamin overall was vitamin K1 (n = 34, 63%). Low plasma 13 levels of vitamin A were detected in 6%, of vitamin D in 28%, of vitamin E in 15% and low 14 levels of vitamin E in erythrocytes in 58% of patients. The number of patients with a 15 deficiency for vitamin D, E and K1 in plasma and for vitamin E in erythrocytes was 16 significantly higher compared to what can be expected based on the normal values of our 17 laboratory (P < 0.001). This was not the case for vitamin A (P = 0.15). 18 19

Subgroup Analysis 20

In the group of acromegaly patients 74% (n = 14) had abnormally low plasma levels 21 for at least one vitamin and 47% for two vitamins. Based on vitamin E levels in erythrocytes, 22 89% had abnormally low levels for at least one vitamin. In this group all patients had normal 23 levels of vitamin A. Vitamin D levels below 50 nmol/L were found in four acromegaly 24 Lowered vitamin E plasma levels were present in three patients (16%), while 1 vitamin K1 was deficient in 10 patients (53%). Vitamin E levels in erythrocytes were lowered 2 in 14 (74%). 3

Of the carcinoid patients 80% (n = 28) had abnormally low plasma levels of at least 4 one fat-soluble vitamin, while 32% had more than one deficiency. When vitamin E levels in 5 erythrocytes were considered for vitamin E status, 91% had abnormally low levels for at least 6 one vitamin. Vitamin A deficiency was detected in three patients (9%). Vitamin D levels 7 below 50 nmol/L were found in 11 patients (31%). Low vitamin E plasma levels were found 8 in five patients (14%), while vitamin K1 was deficient in 24 patients (69%). Vitamin E in 9 erythrocytes was low in 15 (43%). Vitamin plasma levels for both patient groups are shown in 10 Figure 1. Frequency or severity of deficiencies for vitamin D, E and K1 did not differ 11 significantly between patients with acromegaly or a carcinoid tumour, while vitamin A 12 deficiency was only seen in carcinoid patients. 13 14

Vitamin-dependent parameters 15

In 60% of patients (n = 9) with vitamin D levels below 50 nmol/L PTH was elevated; 16 these included 3 acromegaly patients and 6 patients with a carcinoid tumour. Four (12%) out 17 of 34 patients with low vitamin K1 level, all with a carcinoid tumour, showed a prolonged PT, 18 one patient also had a prolonged aPTT. This last patient was also on a vitamin K antagonist, 19 which explains the prolonged PT and aPTT. 20 21

Multivariate regression analysis 22

Neither duration of treatment nor dose of either somatostatin analogue was associated 23 with the plasma level of the various fat-soluble vitamins or vitamin E in erythrocyte levels. 24 Increased stool frequency was not associated with lower vitamin levels in plasma or 1 erythrocytes. Resection of a longer portion of small bowel showed an association with lower 2 plasma vitamin A levels (P = 0.004). In addition, higher vitamin A levels were negatively 3 associated with older age (P = 0.03). Plasma levels of vitamin D, E, K or levels of vitamin E 4 in erythrocytes were not associated with any of the investigated factors. 5 6 DISCUSSION 7 8

This study shows that 78% of acromegaly and patients with metastasised midgut 9 carcinoids who received a somatostatin analogue for longer than 18 months have deficiencies 10 of one or more fat-soluble vitamins. 11

This high level of vitamin deficiency was until now unknown. Two earlier studies 12 addressing vitamin levels in patients treated with a somatostatin analogue 5, 6 found a lowering 13 of carotene, but values remained in the normal range. The major difference of our study 14 versus these studies is a median treatment duration which was much longer. Interestingly, we 15 did not observe in our patients an association between length and dose of somatostatin 16 analogue treatment with the incidence and severity of vitamin deficiencies. This may indicate 17 that once a certain threshold in length or dose of treatment is reached, additional length of 18 treatment or higher doses of somatostatin analogues do not affect incidence and severity of 19 vitamin deficiencies. 20

Our study indicates that over the coming years vitamin deficiencies will be seen more 21 frequently in the increasingly growing group of long-term somatostatin analogue users. 22 Acromegaly patients precluded from surgical cure can receive long-term somatostatin 23 analogue treatment. 17 In addition, the life expectancy of patients with a metastasised carcinoid 24 The most impressive symptom we observed was night blindness in two patients which 3 showed a quick resolution of their complaints after vitamin A supplementation. Vitamin K1 4 was the most frequently observed deficient vitamin in our study. This deficiency resulted only 5 in few patients in prolonged clotting times. However the plasma values indicate that these 6 patients should be closely followed, in case of surgery vitamin K1 should be supplemented to 7 prevent bleeding. 8

In our study bowel resection also influenced plasma levels of vitamin A, but 9 interestingly not to plasma levels of the other fat-soluble vitamins. Patients with a less than 10 200 cm residual bowel after resection run the highest risk to develop vitamin deficiencies. 11

Both the guidelines of the British Society of Gastroenterology and of the American 12 Gastroenterology Association Institute mention that fat-soluble vitamins may need to be 13 supplemented if less than 200 cm of short bowel remains after resection, but do not suggest a 14 specific screening regimen. 16,19 Early studies in small numbers of patients who had 15 undergone resection of varying lengths of intestine showed that resection of more than 100 16 cm of the terminal ileum leads to insufficient intra-intestinal bile salt concentrations, which 17 might in turn lead to fat and fat-soluble vitamin malabsorption. 20,21 However, for patients 18 with a resection of more than 100 cm no regular monitoring of these vitamins is 19 recommended by any of the existing guidelines. In our study in only two carcinoid patients 20 this was the case. In most carcinoid patients only short segments of the small bowel have been 21

resected. 22

Current guidelines of various national and international consensus groups for the 23 treatment of acromegaly or carcinoid tumours do not recommend the assessment of fecal fat 24 malabsorption in patients treated with a somatostatin analogue. [START_REF] Oberg | Consensus report on the use of somatostatin analogs 3 for the management of neuroendocrine tumors of the gastroenteropancreatic system[END_REF]17,[22][23][24][25][26] Furthermore, only the 25 neuroendocrine tumour group mention that pancreatic enzyme replacement can be given to 2 alleviate steatorrhoea. 22,25 Deficiencies of fat-soluble vitamins may be less frequent in 3 patients, who take pancreatic enzyme supplementation to improve fat uptake and uptake of 4 fat-soluble vitamins and consequently steatorrhoea. In our population five patients took such 5 medication. These five patients did however still show deficiencies of the fat-soluble 6 vitamins. 7

Intestinal perfusion studies in humans have shown that somatostatin analogues 8 interfere with the absorption of nutrients. 27,28 Administration of somatostatin analogues 9 significantly decreases the duodenal absorption of carbohydrates and triglycerides. 28 10 Therefore, in addition to their effect on pancreatic enzyme secretion, direct antiabsorptive 11 effects of somatostatin analogues may also well play a role in explaining vitamin deficiencies 12 in these patients. 13 Carcinoid patients may have another risk factor for the development of deficiencies of 14 fat-soluble vitamins, as they frequently experience diarrhoea as part of the carcinoid 15 syndrome. Transit times in the small bowel and the colon are 2-6 times faster in carcinoid 16 patients compared to healthy controls. 29 A shorter transit time reduces the time available for 17 the absorption of water and nutrients. Consequently, less fat is absorbed with shorter transit 18 time. 30 In that case fat-soluble vitamins will also be absorbed less. In our study no association 19 between increased stool frequency and vitamin deficiency was found. 20

We determined plasma vitamin E as well as vitamin E levels in erythrocytes. Plasma 21 vitamin E half-life is subject to extensive interindividual variation because of the many 22 factors that influence the turnover of the various lipoproteins. 31 Vitamin E in erythrocytes, on 23 the other hand, has a longer half life than vitamin E in plasma and therefore better reflects 24 long-term vitamin E status. 12,13 Vitamin E in erythrocytes is located in the cell membrane 25 where it is essential to prevent free radical damage to tissues and specifically to unsaturated 1 lipids. [START_REF] Wang | Vitamin E and its function in membranes[END_REF] Vitamin E levels in erythrocytes were deficient in 58% patients illustrating a clear 2 effect on long-term vitamin E status in these patients while plasma vitamin E levels was only 3 lowered in 15%, likely due to recent dietary intake. 4

Decreased vitamin levels can also be the consequence of insufficient dietary intake. 5

We did not perform a detailed dietary history, but none of our patients reported an abnormal 6 eating behavior. The four patients who reported to take non-prescribed vitamin supplements 7 did still have vitamin deficiencies. Apparently, over-the-counter available vitamin 8 supplements do not contain doses high enough to prevent fat-soluble vitamin deficiencies in 9 these patients. 10

In conclusion, deficiencies of fat-soluble vitamins in patients receiving long-term 11 somatostatin analogue treatment are more frequent than previously known. Considering the 12 possible consequences, we recommend routine assessment of fat-soluble vitamin in plasma 13 and vitamin E in erythrocytes in these patients twice a year, especially in patients with an 14 intestinal resection, and earlier in case of signs or symptoms. Vitamin supplementation should 15 be initiated when symptoms are present or to prevent complications, such as a risk of 16 increased bleeding when surgery is planned (vitamin K deficiency) or osteoporosis in case of 17

1 vitamin

 1 administered to control hormone hypersecretion in acromegaly 4 and carcinoid patients. Somatostatin analogues can increase fat in the stools, which can lead 5 to loss of fat-soluble vitamins. The effect of long-term somatostatin analogue use on vitamin investigate the prevalence of fat-soluble vitamin deficiencies in long-term somatostatin 10 analogue users. 11 12 Methods 13 All acromegaly and carcinoid patients using somatostatin analogues for ≥ 18 months visiting 14 the UMCG, between December 2008 and April 2009 were eligible. Vitamin levels of fat-15 soluble vitamins in blood, clinical and vitamin-dependent laboratory parameters were 35 carcinoid patients were included. Twelve patients experienced 20 steatorrhoea, two carcinoid patients experienced night blindness. Forty-two (78%) were 21 deficient for ≥ one vitamins, 32% (n=17) for multiple. Deficiencies for vitamin A, D, E, K1 22and E in erythrocytes occurred in 6%, 28%, 15%, 63% and 58% of the patients. Prevalence of 23 vitamin D, E and K1 deficiencies were similar in both patients groups. Treatment duration did 24 levels. Length of intestinal resection and age correlated negatively with vitamins deficiencies are frequent during long-term somatostatin analogue 5 treatment. Therefore, fat-soluble vitamins should be monitored in these patients.

  vitamin K supplementation.5 Others observed no changes in serum 1 carotene, vitamin D, or vitamin K in eight acromegaly patients who were on octreotide for 2 four months. 6 3

  this cross-sectional study visited the UMCG between December 19 2008 and April 2009 for acromegaly or a metastasised midgut carcinoid tumour and were 20 treated with a somatostatin analogue for more than 18 months. Patient inclusion was limited 21 to the winter months in order to rule out seasonal influences on vitamin levels (especially 22 vitamin D, which levels are lowest during these months due to lack of sunlight-induced 23 vitamin D production) as an explanation for variation of vitamin levels between patients. 24

  per 10 13 erythrocytes. Reference interval verification, performed in 25 samples of 1 healthy individuals using the EP Evaluator software package release 8 (Data Innovations Inc., 2 South Burlington, VT), confirmed that the values used in our center for vitamin A and E were 3 correct. 15 4

5

  56 patients were asked to participate, one carcinoid patient and one 5 acromegaly patient declined participation. The remaining 54 patients were analyzed, 19 6 patients with acromegaly and 35 with a carcinoid tumour. Characteristics of these patients are 7shown in Table1. 8Median length of somatostatin analogue treatment was 48 months (range 18-155), and 9 did not differ between acromegaly and carcinoid patients (P = 0.18). Median dose of 10 somatostatin analogue was 30 mg per 4 weeks for octreotide and 75 mg per 4 weeks for 11 lanreotide. Median doses were the same for both patient groups. Of the carcinoid patients 25 12 had undergone a bowel resection in the past; in 23 less than 100 cm of bowel was resected, in 13 one between 100 and 200 cm and in one more than 200 cm. 14 15 Symptoms 16 Two carcinoid patients experienced night blindness, which coincided with vitamin A 17 deficiency. One patient was already referred to an ophthalmologist. Electroretinography 18 demonstrated abnormalities typical for vitamin A deficiency in both patients. Oral 19 supplementation of vitamin A resulted in a quick resolution of their symptoms. Twelve 20 patients (n = 4 acromegaly patients and n = 8 carcinoid patients) complained about 21 steatorrhoea. Twelve carcinoid patients experienced increased stool frequency (range 1-10 22 times daily), but most had normal stools. An additional five carcinoid patients received 23 supplementation of pancreatic enzymes for steatorrhoea. Despite this supplementation all five 24 vitamin levels. Four patients (one acromegaly patient and three 1 carcinoid patients) took non-prescribed vitamin supplements (which included vitamins A, D 2 and E), but three of these patients did show deficiencies for one (n = 2) or two (n = 1) of the 3 fat-soluble vitamins. None of the patients had symptoms of increased bleeding tendency. 4 Overall vitamin-deficiencies 6

  clearly during the last decades.18 Consequently, patients receive 1 somatostatin analogues for extended periods. 2

  European Neuroendocrine Tumour Society (ENETS) and of the Nordic 1

Figure 1 .

 1 Figure 1. Scatter plots showing the range of plasma levels (unless otherwise noted) of the

Figure 1 .

 1 Figure 1. Scatter plots showing the range of plasma levels (unless otherwise noted) of the different fat-soluble vitamins per patient. Panel A. Vitamin A, Panel B. Vitamin D, Panel C. Vitamin E, Panel D. Vitamin E in erythrocytes Panel E. Vitamin K. Horizontal bars represent the median value, dotted lines the lower and upper limits of normal. 96x52mm (600 x 600 DPI)

Table 1 .

 1 Patient characteristics

	Total group	Acromegaly	Carcinoid	P value
			tumour

Alimentary Pharmacology & Therapeutic