M Boutahar
email: boutahar@univmed.fr

B Ghattas
email: ghattas@univmed.fr

D Pommeret
email: pommeret@univmed.fr

Nonparametric test for detecting change in distribution with panel data

This paper considers the problem of comparing two processes with panel data. A nonparametric test is proposed for detecting a monotone change in the link between the two process distributions. The test statistic is of CUSUM type, based on the empirical distribution functions. The asymptotic distribution of the proposed statistic is derived and its finite sample property is examined by bootstrap procedures through Monte Carlo simulations.

keywordsnonparametric estimation panel data process

Introduction

Many situations lead to the comparison of two random processes. In a parametric case, the problem of change detection has been widely studied in the time series literature. A common problem is to test a change in the mean or in the variance of the time series by using a parametric model (see for instance [START_REF] Gombay | Change detection in autoregressive time series[END_REF] or [START_REF] Galeano | Covariance changes detection in multivariate time series[END_REF], and references therein). In the Gaussian case comparisons of processes are considered through their covariance structures (see [START_REF] Gupta | Distribution of likelihood ratio statistic for testing equality of covariance matrices of multivariate Gaussian models[END_REF], [START_REF] Panaretos | Second-Order Comparison of Gaussian Random Functions and the Geometry of DNA Minicircles[END_REF]). These distribution assumptions can be relaxed when the study concerns processes observed through panel data. This situation is frequently encountered in medical follow-up studies when two groups of patients are observed and compared. Each subject in the study gives rise to a random process (X t ) denoting the measurement of the patient up to time t (such data are referred to as panel data). In this context, [START_REF] Balakrishnan | A class of multi-sample nonparametric tests for panel count data[END_REF][START_REF] Balakrishnan | New multi-sample nonparametric tests for panel count data[END_REF][START_REF] Balakrishnan | A nonparametric test for the equality of counting processes with panel count data[END_REF] considered the problem of testing the equality of mean functions and proposed new multi-sample tests for panel count data.

In this paper we consider the general problem of comparison of two processes which may differ by a transformation of their distributions. Our purpose is to test whether this transformation changes over time. For this, two panels are considered: (X i,t ) 1≤i≤Nx;1≤t≤n and (Y i,t ) 1≤i≤Ny;1≤t≤n , not necessarily independent; that is, we can have i.i.d. paired observations (X i , Y i ) i=1,••• ,N with dependence between X i and Y i . It is assumed that for each t, the X i,t , 1 ≤ i ≤ N x (resp. Y i,t , 1 ≤ i ≤ N y ) are i.i.d. random variables with common distribution function F t (resp. G t ) and with support X (resp. Y). Also we assume that for all 1 ≤ t ≤ n there exists monotone transformations h t such that the following equality in distribution holds: X t = d h t (Y t ). Without loss of generality we consider that the functions h t (.) are increasing. Note that if F t is invertible then there exists a trivial transformation h t given by h t = F -1 t • G t . We are interested in testing whenever this transformation is time independent; that is, for all t, the equality h t = h occurs. A simple illustration is the case where X t and Y t are Gaussian processes with mean m X and m Y and variance tσ 2

X and tσ 2 Y , respectively. In that case the function h is linear. More generally, observing both processes X t and Y t with panel data we want to test

H 0 : ∀t, h t = h against H 1 : ∃t 1 = t 2 , h t1 = h t2 .
It is clear that H 0 coincides with the equality in distribution: X t = d (h(Y t )), for all t. Following [START_REF] Gombay | Change detection in autoregressive time series[END_REF] (see also [START_REF] Galeano | Covariance changes detection in multivariate time series[END_REF]), we construct a non parametric test statistic based on the empirical estimator of h t , denoted by h t . We show that h t is proportional to a Brownian bridge under H 0 .

When H 0 is not rejected, it is of interest to estimate h and to interpret its estimator h. Then this test can be viewed as a first step permitting to legitimate estimation and interpretation of a constant transformation h between the distributions of two samples, possibly paired.

The paper is organized as follows: In Section 2 we construct the test statistic. In Section 3 we perform a simulation study using a bootstrap procedure to evaluate the finite sample property of the test. The power is evaluated against alternatives where there are smooth scale or position time changes in the process distribution. Section 4 contains brief concluding remarks.

The test statistic

A natural nonparametric estimator of h t is given by

h t (•) = X (Nx Gt(•)),t ,
where X (i),t denotes the ith order statistic and G t is the empirical distribution function of (Y i,t ) 1≤i≤Ny , that is

G t (x) = 1 N y Ny i=1 1 {Yi,t≤x} .
A nonparametric test is considered to test the variation of h t . For τ ∈ (0, 1), x ∈ Y, write

B n (τ, x) = 1 √ n σ n   [nτ ] t=1 h t (x) - [nτ ] n n t=1 h t (x)   , (1)
where

σ 2 n = 1 n n t=1 ( h t (x) -h(x)) 2 , h(x) = 1 n n t=1 h t (x).
For a given square integrable function w we define the following test statistic

S n (w) = R w(x) sup 1≤τ ≤1
|B n (τ, x)| dx.

To establish the limiting distribution of the statistic S n (w) under the null, we need the following assumptions:

• Assumption 1. There exists a < ∞ such that N x /(N x + N y ) → a.

• Assumption 2. There exist γ 1 > 0 and γ 2 > 0 such that f t (x) ≥ γ 1 and g t (y) ≥ γ 2 for all (x, y) ∈ X × Y, where f t and g t are the density functions of X t and Y t .

• Assumption 3. For all x ∈ X , there exist 0

< σ 2 2 (x) < ∞ such that 1 n n t=1 σ 2 1,t (x) → σ 2 2 (x), as n → ∞,
where

σ 2 1,t (x) = σ 2 t (x) N x + N y N x N y , and
σ 2 t (x) = G t (x)(1 -G t (x)) f 2 t (h t (x))
.

• Assumption 4.

n(N x + N y ) N x N y → 0.
Remark 2.1 Assumptions 1 and 2 are standard. Assumption 3 states that the second moments converge on average.

If Assumption 1 is satisfied, Assumption 4 is equivalent to n = o(N x ) or n = o(N y ).
Theorem 2.1 Let assumptions 1-4 hold. Then under the null H 0 we have the following convergence in distribution

S n (w) → d S(w) = B ∞ R w(x)dx, as n → ∞, N x → ∞ and N y → ∞, (3)
where B ∞ = sup 0≤τ ≤1 |B(τ )|, and B is a Brownian bridge.

Remark 2.2

The cumulative distribution function of B ∞ is given by (see [START_REF] Billingslley | Convergence of probability measures[END_REF])

F B∞ (z) = 1 + 2 ∞ k=1 (-1) k exp{-2k 2 z 2 }.
Before proving Theorem 1, we state three lemmas.

Lemma 2.1 Under Assumption 1 we have

N x N y N x + N y 1/2 ( h t (x) -h t (x)) → d N (0, σ 2 t (x)), as N x → ∞, N y → ∞, (4)
where σ 2 t (x) is given by [START_REF] Balakrishnan | New multi-sample nonparametric tests for panel count data[END_REF].

Proof (1 {Yi,t≤x} ) is an i.i.d sequence with mean G t (x) and variance G t (x)(1 - G t (x))
, hence an immediate application of the central limit theorem yields

N 1/2 y G t (x) -G t (x) → d N (0, G t (x)(1 -G t (x))). ( 5
)
By the delta-method the last convergence implies that

N 1/2 y F -1 G t (x) -F -1 (G t (x)) → d N (0, σ 2 t (x)). (6)
For

p ∈]0; 1[ fixed, denote by F -1 t (p) the sample p-quantile; that is, F -1 t (p) = X (r),t , where r = [N x p] + 1. By Theorem 3 of [13] we obtain N 1/2 x ( F -1 t (p) -F -1 t (p)) → d N 0, p(1 -p) f 2 t (F -1 t (p))
, ∀p ∈ (0, 1).

Let φ X (t) = E(exp(itX)) denotes the characteristic function of the random variable X and let φ X|Y (t) = E(exp(itX) | Y ) denotes the conditional characteristic function of the random variable X conditional on Y . We have

H t = N x N y N x + N y 1/2 ( h t (x) -h t (x)) = N x N y N x + N y 1/2 F -1 t ( G t (x)) -F -1 t (G t (x)) = H 1,t + H 2,t ,
where

H 1,t = N y N x + N y 1/2 N 1/2 x F -1 t ( G t (x)) -F -1 t ( G t (x)) H 2,t = N x N x + N y 1/2 N 1/2 y F -1 t ( G t (x)) -F -1 t (G t (x)) .
Then we get

φ Ht (u) = E(exp(iu H t )) = E E exp(iu H t ) | Y t = E exp(iu H 2,t ) E exp(iu H 1,t ) | Y t . Moreover E exp(iu H 1,t ) | Y t = φ H 1,t|Y t (u) (8) = φ N 1/2 x ( F -1 t ( Gt(x))-F -1 t ( Gt(x)))|Yt (N y /(N x + N y )) 1/2 u
From ( 7) it follows that, ∀v ∈ R,

φ N 1/2 x ( F -1 t ( Gt(x))-F -1 t ( Gt(x)))|Yt (v) -→ exp - 1 2 v 2 σ 2 t (x) , (9)
as N x → ∞, where

σ 2 t (x) = G t (x)(1 -G t (x)) f 2 t F -1 t ( G t (x))
.

The convergence (5) yields G t (x) P --→ G t (x), as N y → ∞, which implies, combined with ( 8)-( 9), Assumption 1 and

h t (x) = F -1 t (G t (x)), that E exp(iu H 1,t ) | Y t d -→ exp - 1 2 (1 -a)u 2 σ 2 t (x) , (10)
as N x → ∞ and N y → ∞. Moreover we have

exp(iu H 2,t ) = exp iu N x N x + N y 1/2 N 1/2 y F -1 t ( G t (x)) -F -1 t (G t (x)) .
Since the function x → exp(iux) is continuous, then the convergence (6) and Assumption 1 yield

exp(iu H 2,t ) → d exp(iua 1/2 H 2,t ), as N x → ∞, N y → ∞, (11)
where H 2,t is centered Gaussian distributed with variance equal to σ 2 t (x)). From [START_REF] Hawkins | Testing a sequence of observations for a shift in location[END_REF] and [START_REF] James | Tests for a change point[END_REF] it follows that, as

N x → ∞ and N y → ∞, exp(iu H 2,t )E exp(iu H 1,t ) | Y t → d exp(iua 1/2 H 2,t ) exp - 1 2 (1 -a)u 2 σ 2 t (x) . (12)
Since E exp(iu H

φ Ht (u) = E exp(iu H 2,t )E exp(iu H 1,t ) | Y t → E exp iua 1/2 H 2,t exp - 1 2 (1 -a)u 2 σ 2 t (x) , as N x → ∞, N y → ∞ = exp - 1 2 au 2 σ 2 t (x) exp - 1 2 (1 -a)u 2 σ 2 t (x) = exp - 1 2 u 2 σ 2 t (x) ,
therefore the desired conclusion (4) holds.

Lemma 2.1 implies that

h t (x) = h t (x) + σ 1,t (x)ε t + r t , (13)
where σ 2 1,t (x) is given by ( 2), (ε t ) is a standard Gaussian white noise and the remainder term r t is such that

r t = O P {(N x + N x )/N x N y } 1/2 . (14)
Let D = D[0, 1] be the space of random functions that are right-continuous and have left limits, endowed with the Skorohod topology. The weak convergence of a sequence of random elements X n in D to a random element X in D will be denoted by X n =⇒ X. Let

W n (τ ) = 1 √ n [nτ ] t=1 σ 1,t (x)ε t , τ ∈ [0, 1]. ( 15
)
Lemma 2.2 Under Assumptions 1-3 we have

W n =⇒ σ 2 (x) W, (16)
where W stands for the standard Brownian motion.

Proof Assumption 2 implies that

σ 2 1,t (x) ≤ 1 γ 2 1 N x + N y N x N y ≤ C,
for some positive constant C and N x and N y large enough. Hence σ 2 1,t (x) is a bounded deterministic sequence, therefore the weak convergence (16) follows from Theorem A.1 of [START_REF] Boutahar | Testing for change in the mean of heteroskedastic time series[END_REF].

Lemma 2.3 Under the null H 0 , as n → ∞, N x → ∞ and N y → ∞ we have

σ 2 n = 1 n n t=1 ( h t (x) -h(x)) 2 d --→ σ 2 2 (x). ( 17
)
Proof Under the null H 0 : h t (x) = h(x) the equality (13) becomes

h t (x) = h(x) + σ 1,t (x)ε t + r t .
Let y t = h(x) + σ 1,t (x)ε t , y = n t=1 y t /n, then by using the same argument as in Theorem 1 of [START_REF] Boutahar | Testing for change in the mean of heteroskedastic time series[END_REF] we obtain

1 n n t=1 (y t -y) 2 d -→ σ 2 2 (x). ( 18
)
We have

1 n n t=1 ( h t (x) -h(x)) 2 = 1 n n t=1 (y t -y) 2 + 1 n n t=1 (r t -r) 2 + 2 1 n n t=1 (y t -y)(r t -r), (19)
where r = n t=1 r t /n. From ( 14) it follows that

r = O P (((N x + N y )/N x N y ) 1/2 ) = o p (1), as N x → ∞, N y → ∞, which implies that 1 n n t=1 (r t -r) 2 = o p (1), as N x → ∞, N y → ∞. (20)
By using the Cauchy Shwartz inequality, we have

1 n n t=1 (y t -y)(r t -r) ≤ 1 n n t=1 (y t -y) 2 1/2 1 n n t=1 (r t -r) 2 1/2 .
Hence by using ( 18) and (20) we get

1 n n t=1 (y t -y)(r t -r) = o p (1), as N x → ∞, N y → ∞. (21)
The desired conclusion (17) holds by combining (18)-(21).

Proof of Theorem 1 Under the null, the process B n (τ, x) in (1) can be rewritten as

B n (τ, x) = 1 √ n σ n   [nτ ] t=1 σ 1,t (x)ε t - [nτ ] n n t=1 σ 1,t (x)ε t   + R n (τ, x) = 1 σ n W n (τ ) - [nτ ] n W n (1) + R n (τ, x),
where the remainder term R n (τ, x) is given by

R n (τ, x) = 1 √ n σ n   [nτ ] t=1 r t - [nτ ] n n t=1 r t   .

Now observe that

[nτ ] t=1

r t = O P [nτ ] ((N X + N Y )/N X N Y ) 1/2 ,
which together with (17) implies that

R n (τ, x) = O P {n(N x + N y )/N x N y } 1/2 , = o p (1) under assumption 4. Hence R n (τ, x) = 1 σ n W n (τ ) - [nτ ] n W n (1) + o p (1),
which combined with ( 16) and (17) yields

B n (., x) =⇒ B,
where

B(τ ) = W (τ ) -τ W (1) is a Brownian bridge. Therefore sup 1≤τ ≤1 |B n (τ, x)| → d sup 1≤τ ≤1 |B(τ )| . (22)
Let F (R, R) be the space of square integrable functions endowed with the uniform norm . ∞ . For a given square integrable function w, the functional G w :

(F (R, R), . ∞ ) → (R, |.|) defined by G w (g) = R w(x)g(x)dx,
is continuous. To obtain the convergence (3) it is sufficient to apply (22) and the continuous mapping theorem.

Empirical study

For simplicity we consider N x = N y = N . Data are generated from three models: first, Y t is normally distributed with mean 0 and variance 1, and X t is generated independently by the transformation X t = h t (Z t ), where Z t is another Gaussian process with mean 0 and variance 1. Second, Y t is an autoregressive process of order 1 (AR1) with correlation coefficient equal to 0.5, and X t is generated independently by the transformation X t = h t (Z t ), where Z t is another AR1 process. For the last model random variables are paired: Y t are independent Gaussian variables with mean 0 and variance 1, and

X t = h t (Y t ),
that is, the time transformation is on the random variables. It is clear that this implies the same transformation for the corresponding distributions.

Alternatives. The following five alternatives are considered

First alternative: A1

Change in the mean.

h 1,t (x) = 2t 2 1 + t 2 + x.

Second alternative: A2

Change in the variance. h 2,t (x) = 2t 2 1 + t 2 x.

Third alternative: A3

Jump. h 3,t (x) = x + 0.05tI t<n/2 + 0.005(nt)I t≥n/2 , where I t≥n/2 = 1 if t ≥ n/2 and 0 otherwise.

Fourth alternative: A4

Smooth change in the mean. h 4,t (x) = x + (1 + exp(-0.01(t -1))) -1 Fifth alternative: A5 Smooth change in the mean. h 5,t (x) = x + (1 + exp(-0.05(t -1))) -1 All alternatives are smooth and are less rough than classical rupture on the mean or on the variance, except A3 which coincides with a jump on the mean. The first two alternatives A1-A2 tend quickly to the null model under H 0 when the length n increases. Figure 1 illustrates the proximity of h t to a constant for large times length in the case of alternative A1. In opposition, alternatives A4-A5 are very smooth and converge slowly to the null model. Figure 2 illustrates this smooth convergence under alternative A4. Bootstrap procedure. To evaluate the power of our testing procedure we first consider a Monte Carlo statistic.

Given M points x 1 , • • • , x M in Y we consider S M (w) = 1 M M i=1 w(x i )A(x i ), (23)
where

A(x i ) = max 1≤k≤n 1 σ n (x i ) √ n k t=1 h t (x i ) -k h(x i ) , with          σ 2 n (x) = 1 n n t=1 ( h t (x) -h(x)) 2 h(x) = 1 n n t=1 h t (x).
The convergence of the statistic S M (w) is not guaranteed since the A(x i ) are dependent. To carry out this problem, a bootstrap procedure is proposed. We construct a naive bootstrap statistic; that is, the test statistic S M (w) given in ( 23) is compared to the empirical bootstrapped distribution obtained from (S M * b ) b=1,••• ,B , with S * b M constructed from the bootstraped sample drawn randomly with replacement and satisfying the size equalities N * x = N x and N * y = N y . We fix w as a constant. Note that if X and Y are paired, the bootstrap procedure consists in drawing randomly with replacement N pairs (X, Y ) from the data. We fix B = 200 bootstrap replications.

Powers. For each alternative, the test statistic is computed, based on sample sizes N = 50, 100, for a theoretical level α = 5%. The lengths of time's intervals are n = 20, 100 and 200; that is, the function h t is observed N times for each t varying in [0; 20], or [0; 100], or [0; 200], with a step equal to one. The empirical power of the test is defined as the percentage of rejection of the null hypothesis over 10000 replications of the test statistic under the alternative.

Figure 3 presents empirical powers of the bootstrap test for all alternatives, in the case where X t are independent standard Gaussian variables. Solid lines and dotted lines correspond to N = 50 and 100 respectively. It can be observed that the power decreases with the length for alternatives A1 and A2. It is in accordance with the previous remark: h t is close to the null hypothesis for relatively large values of n. Then passing from a time length equal 20 to a time length equal to 200 corresponds to adding variables with nearly constant transformation in distribution (see Figure 1).

Alternatives A4-A5 have similar behaviors, with a power increasing with n. It can be explained by the very slow convergence to the null model. Here, passing from a time length equal 20 to a time length equal to 200 corresponds to adding new observations with a time depending transformation (see Figure 2).

It is also observed that power associated to alternative A3 increases with n.

In Figure 4 empirical powers are presented in the case where Y t follows an AR1 process with a correlation coefficient equal to 0.5. Here powers are slightly better and more stable with respect to the length. This is due to the correlation inducing more stability of the process Y t and permitting a better estimation of h t .

Figure 5 presents results in the case of paired data, with Y t normally distributed. Powers are good, due to the fact that transformations occur not randomly since we have considered X t = h t (Y t ). Then h t can be efficiency estimated and its variations are well detected.

Concluding remarks

The proposed method concerns the comparison of two processes when panel data are available. The test permits to detect a change in the relation between the two process distributions. Therefore it can detect a change in a higher moments (not only in the mean and/or in the variance as almost tests do in this framework). The asymptotic distribution of the proposed statistic was derived under the null of no change in the relation between the two process distributions. The Monte Carlo simulations show that our test performs well in finite sample and has a good power against either abrupt or smooth changes. It is also valid for paired processes and then it can be used to detect a change in h t in the relation X t = h t (Y t ) (see the paired case in our simulations). The test can also be used as a first step permitting to legitimate estimation and interpretation of a constant transformation h between two panel data, as for instance in a medical follow-up study.

A direction for future research is to consider a d-sample comparison of distributions, for d > 2, in the way of [START_REF] Balakrishnan | A class of multi-sample nonparametric tests for panel count data[END_REF][START_REF] Balakrishnan | New multi-sample nonparametric tests for panel count data[END_REF]. Another direction should consider multivariate distributions.

Figure 1 :

 1 Figure 1: Representation under alternative A1 of h t = 2t 2 /(1 + t 2 ) for time length = 20 (a) and time length = 200 (b)

Figure 2 :

 2 Figure 2: Representation under alternative A4 of h t = (1+exp(-0.01(t-1) 2 )) -1 for time length = 20 (a) and time length = 200 (b)

Figure 3 :Figure 4 :Figure 5 :

 345 Figure 3: Empirical powers for alternatives A1 (•) and A2 (•) on the left, A3 (⋄), A4 (△) and A5 (▽) on the right, with X t distributed as N (0, 1). Solid lines correspond to N = 50 and dotted lines correspond to N = 100. The lengths of time's intervals are n = 20, 100, 200

  1,t ) | Y t and exp(iu H 2,t ) are bounded almost surely, it follows from (12) that


Laura Ae Hughes
email: laura.hughes@epid.unimaas.nl

Piet A Van Den Brandt

R Alexandra Goldbohm

Anton Fpm De Goeij

Adriaan P De Bruine

Manon Van Engeland

Matty P Weijenberg

Adriaan P De Bruïne

Childhood and adolescent energy restriction and subsequent colorectal cancer risk: results from the Netherlands Cohort Study

Keywords: colorectal neoplasms, cohort studies, adolescence, caloric restriction, starvation colorectal neoplasms, caloric restriction, starvation, adolescence, cohort studies

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

Cancers of the colon and rectum are the third most common type of cancer worldwide. [START_REF]Food, 390 Nutrition, Physical Activity, and the Prevention of Cancer: a Global Perspective[END_REF] According to the World Cancer Research Fund (WCRF), there is convincing evidence that components of energy balance such as body and abdominal fatness, and factors leading to a greater adult attained height increase the risk of colorectal cancer 50 (CRC). [START_REF]Food, 390 Nutrition, Physical Activity, and the Prevention of Cancer: a Global Perspective[END_REF] Numerous animal studies suggest that energy restriction may lead to the development of CRC, [START_REF] Albanes | The effect of early caloric restriction on colonic cellular growth in rats[END_REF][START_REF] Kritchevsky | Colorectal cancer: the role of dietary fat and caloric restriction[END_REF][START_REF] Kritchevsky | Influence of caloric intake on experimental carcinogenesis: a review[END_REF][START_REF] Kritchevsky | Interaction of fiber and energy registration in 400 experimental colon carcinogens[END_REF][START_REF] Ross | Lasting influence of early caloric restriction on prevalence of neoplasms in the rat[END_REF] and observational research suggests that energy restriction during childhood and adolescence may especially be protective against CRC cancer development and or mortality later in life. [START_REF] Dirx | Energy restriction early in life and colon carcinoma risk: results of The Netherlands Cohort Study after 7.3 years 405 of follow-up[END_REF][START_REF] Frankel | Childhood energy intake and adult mortality from cancer: the Boyd Orr Cohort Study[END_REF][START_REF] Svensson | Trends in 410 colorectal cancer incidence in Norway by gender and anatomic site: an age-period-cohort analysis[END_REF][START_REF] Svensson | Early life events and later risk of colorectal cancer: age-period-cohort modelling in the Nordic countries and Estonia[END_REF] For ethical and practical reasons, it is difficult to initiate a study to test the 55 hypothesis of childhood energy restriction and future cancer risk. However, information derived from a unique situation in the Netherlands has enabled this hypothesis to be During the Economic Depression, there was a high rate of unemployment in the Netherlands. Surveys document that while the amount of total calories available to families with an unemployed father was sufficient, they consumed fewer calories (3000 vs. 3400 kcal/day) and had less variation in their food patterns than families with an 65 employed father. The daily menu of the unemployed families consisted primarily of boiled potatoes and some fat, and lacked cheese, meat and confectionary. [START_REF] Dirx | Energy restriction early in life and colon carcinoma risk: results of The Netherlands Cohort Study after 7.3 years 405 of follow-up[END_REF]11 During WW2, food rationing was introduced and because of poor food availability in large cities, nutritional differences emerged between people living in urban versus rural areas. Individuals living in cities consumed fewer calories daily; the average allowance was 70 about 1600 kcal, which was maintained with difficulty. Furthermore, urban diets consisted of a higher percentage of carbohydrates (70% vs 65%) and a lower percentage of fat (10% vs 15%). [START_REF] Dirx | Energy restriction early in life and colon carcinoma risk: results of The Netherlands Cohort Study after 7.3 years 405 of follow-up[END_REF][START_REF] Dols | De voedselvoorziening in Nederlands tijdens en onmiddelijk na den tweeden wereldoorlog 1940-1945 (Food supply during and after the second World War 1940-1945[END_REF] The southern part of The Netherlands was liberated from German occupation in September 1944, whereas the western part of the country remained under German rule. The Hunger Winter occurred primarily in the western cities of the 75 Netherlands as the end result of a German imposed embargo on food transport into the major Dutch cities. While the diet remained nutritionally balanced, individuals living in western urban areas experienced rationing of less than 700 kcal per day. [START_REF] Elias | Long term consequences of the 1944-1945 Dutch famine on the insulin-like growth factor axis[END_REF][START_REF] Burger | Malnutrition and starvation in Western Netherlands[END_REF] The famine was relatively short; it abruptly ended in May 1945 when the western and northern part of the Netherlands was liberated as well. [START_REF] Elias | Long term consequences of the 1944-1945 Dutch famine on the insulin-like growth factor axis[END_REF][START_REF] Burger | Malnutrition and starvation in Western Netherlands[END_REF] This situation provides a rare opportunity to 80 study the association between severe energy restriction during childhood and adolescence, and CRC risk later in life.

The Netherlands Cohort Study on diet and cancer (NLCS) is comprised of individuals who were children or going through the adolescent growth spurt during the years spanning the Economic Depression, WW2, and the Hunger Winter. Although 85 individual food intake data is not available for these time periods, a number of proxy measures have been collected from cohort members that reflect their exposure to energy restriction. While the association between energy restriction during childhood and colon carcinomas was studied in the NLCS at an earlier follow-up point, [START_REF] Dirx | Energy restriction early in life and colon carcinoma risk: results of The Netherlands Cohort Study after 7.3 years 405 of follow-up[END_REF]

Methods

Study population and design 95

The NLCS is a prospective cohort study that was initiated in 1986 with the purpose of investigating the association between diet and the development of cancer. The cohort included 58,279 men and 62,573 women between the ages of 55-69 years at baseline who completed a self-administered questionnaire on dietary habits and lifestyle, health, and demographic information. The questionnaire requested information about the 100 residence(s) of cohort members, including their residence during WW2 and the winter of 1944-45, and also asked for their father's employment status during the Economic Depression. Municipal registries from throughout the Netherlands were used to constitute an efficient sampling frame. [START_REF] Goldbohm | Validation of a dietary questionnaire used in a large-scale prospective cohort study on diet and cancer[END_REF][START_REF] Goldbohm | Reproducibility of a food 430 frequency questionnaire and stability of dietary habits determined from five annually repeated measurements[END_REF][START_REF] Van Den Brandt | A large-scale prospective cohort study on diet and cancer in The Netherlands[END_REF] A case -cohort approach (12) was used for data processing and analysis; case subjects were derived from the entire cohort, and the 105 number of person-years at risk for the entire cohort was estimated from a subcohort of 5000 men and women who were randomly sampled from the full cohort at baseline. Further details of the design of the NLCS have been described in previous publications. 15- 17 Incident CRC cases from the entire cohort were identified by annual record 110 linkage to nine regional cancer registries and a national pathology database (PALGA). This method of record linkage has been described elsewhere. [START_REF] Van Den Brandt | Development of a record linkage protocol for use in the Dutch Cancer Registry for Epidemiological Research[END_REF] The completeness of cancer follow-up has been estimated to be almost 100% . [START_REF] Goldbohm | Estimation of the coverage of municipalities by cancer registries and PALGA using hospital discharge data[END_REF]

Assessment of Energy Restriction

Individual food intake data of the cohort members was not available for the three 125 periods of energy restriction. Therefore, proxy variables were used to describe exposure to energy restriction.

With respect to the Economic Depression years, the best proxy available was the employment status of the cohort member's father during this time frame. On the baseline questionnaire, individuals were asked 'was your father employed during the crisis of 130 1932-1940?' Cohort members could either answer yes, along with the years that he was employed, or no. For analyses purposes, answers were categorised into 'father was employed during the economic depression' or 'father was not employed during the economic depression'.

For the years spanning both WW2 and the Hunger Winter, place of residence was 135 used to approximate the exposure to energy restriction. Place of residence in 1942 (midpoint of the war years) was used as a proxy for energy restriction during WW2. On

Data Analyses 150

Data were analysed using Stata (version 9.2, Statacorp, College Station, TX, USA). Because the etiology of CRC may differ according to sex and sub-site of the cancer, men and women were analysed separately for overall colorectal (including rectosigmoid), colon, proximal colon, distal colon, rectosigmoid, and rectal cancers.

Cox proportional hazard analysis using the case-cohort approach was used to 155 obtain incidence rate ratios (RR) and 95% confidence intervals (CI) for the association between the three proxy measures of energy restriction and the incidence of CRC overall, and for each sub-site. The proportional hazards assumption was tested using the scaled Schoenfeld residuals and by visual inspection of the hazard curves. When the proportional hazards test appeared to be violated, analyses were done for short and long 160 RRs were estimated using age-adjusted and multivariate adjusted models.

Potential confounders that introduced a more than 10% change in RRs or contributed 165 significantly to the prediction were included as covariates in the multivariate model. These ultimately included age, family history of CRC, height, body mass index, baseline alcohol and energy intake, recreational physical activity, level of education, and smoking history. [START_REF] Dirx | Energy restriction early in life and colon carcinoma risk: results of The Netherlands Cohort Study after 7.3 years 405 of follow-up[END_REF] RRs were expressed using 'father was employed during the economic depression', 'lived in a rural area in 1942', and 'lived in a non-western area of the 170 Netherlands during the Hunger Winter' as the reference categories for the different proxies of energy restriction.

Previously in the NLCS, sub-group analysis was conducted after 7.3 years of follow-up to assess if timing of exposure modified the effect of energy restriction on colon carcinoma cancer risk. [START_REF] Dirx | Energy restriction early in life and colon carcinoma risk: results of The Netherlands Cohort Study after 7.3 years 405 of follow-up[END_REF] In that study there were too few cases in each sub-group to 175 reach definitive conclusions; therefore, we repeated this sub-group analysis in terms of total CRC to include the maximum number of cases. For each of the three exposure periods, individuals were classified into a sub-group depending on if they were exposed to energy restriction before, during, or after the adolescent growth spurt. Men and women were analysed separately. For men, the growth spurt was defined as occurring between 180 the ages of 12 and 15. For women, the growth spurt was defined as 2 years before the reported age of menarche to 1 year after the reported year of menarche (mean age Economic Depression: 13.1 years, WW2: 14.3 years, Hunger Winter: 14.8 years). The Finally, tests for heterogeneity were done to evaluate differences between subtypes of tumors (e.g., colon vs. rectum) using the competing risks procedure in STATA. However, the SE for the difference of the log-hazard ratios from this procedure 195 assumes independence of both estimated hazard ratios, which would overestimate that SE and thus overestimate the P values for their difference. Therefore, these P values and the associated confidence intervals were estimated based on a bootstrapping method that was developed for the case-cohort design. [START_REF] De Vogel | Associations of dietary methyl donor intake with MLH1 promoter hypermethylation and related molecular phenotypes in 445 sporadic colorectal cancer[END_REF] Each bootstrap analysis was based on 1,000 replications. 200

Results

Baseline characteristics of the sub-cohort are presented in table 1 and table 2 for men and women, respectively. Men who lived in a western rural area during the Hunger Winter had a higher level of occupational physical activity and a higher level of education compared to men who lived in a non-western area or a western city. With 205 respect to the war years, men who lived in a western city in 1942 had a lower BMI and a F o r R e v i e w O n l y 10 lower daily intake of calories at baseline compared to men who did not. Additionally, these men had a higher level of education and a lower level of occupational physical activity. Men who had an employed father during the Economic Depression had a higher level of education and had a lower level of occupational physical activity than men whose 210 father was not employed. Women who lived in a western city during the Hunger Winter had a lower energy intake and a higher intake of alcohol at baseline compared to women who lived in a non-western or western rural area, and a higher level of education. Women who lived in a western rural area had the lowest proportion of CRC family history.

Women who lived in an urban area in 1942 were shorter, had a lower energy intake and a 215 higher alcohol intake at baseline, had a higher level of education, exercised more, and smoked less than women who lived in a rural area. Women whose father was employed during the Economic Depression were younger, taller, had a lower BMI, a higher intake of alcohol and energy at baseline, were better educated, and smoked less than women whose father did not have a job. 220

Associations between the three proxy measures of energy restriction and CRC risk are presented in table 3 for men and table 4 for women. Men who reported living in a western city during the winter of 1944-1945 had a lower risk of CRC compared to men who lived in a non-western area (RR 0.77, 95% CI: 0.63-0.94). With respect to tumor sub-site, living in a western city during the Hunger Winter was protective against risk of 225 tumors of the total colon (RR: 0.76, 95% CI: 0.60-0.96), proximal colon (RR 0.68, 95% CI: 0.49-0.94), and rectum (RR: 0.66, 95% CI: 0.47-0.92). Living in a city in 1942 and having a father without a job during the economic depression were not associated with

F o r R e v i e w O n l y
CRC, or the specific CRC sub-sites. Tests for heterogeneity suggest that there is no difference in risk between the tumor subsites. 230

In women, living in a western city during the Hunger Winter was not associated with total CRC risk (RR: 0.99, 95% CI: 0.83-1.18). Analyses according to tumor sub-site were heterogeneous. Living in a western city was inversely associated with tumors of the distal colon, rectosigmoid, and rectum, while an increased risk was observed for proximal colon tumors (RR: 1.23, 95% CI: 0.97-1.56). Residing in a city in 1942 was inversely 235 associated with risk of rectal cancer (RR 0.69, 95%CI: 0.47-0.93), but was not associated with tumors at other sub-sites. Having a father without a job during the economic depression was not associated with CRC risk or the risk of cancer at each sub-site. Tests for heterogeneity indicate that there is no difference in risk between the tumor subsites.

Results from the sub-group analyses can be seen in table 5, for both men and 240 women. Our cohort has no men who were exposed to the Hunger Winter before their growth spurt. In men, exposure to energy restriction during or after the growth spurt was protective against CRC for those who resided in a western city during the Hunger Winter compared to those who resided in non-western area (RR: 0.56, 95% CI: 0.31-1.03 and RR: 0.76, 95% CI: 0.61-0.94, respectively). Residing in a western rural area during the 245 Hunger Winter also seemed to be protective of CRC in men as opposed to residing in a non-western area a that time (RR: 0.52, 95% CI: 0.26-1.02). In women, too few cases were exposed to the Hunger Winter before the growth spurt to reach definitive conclusions. There were no clear associations or differences between sub-groups during the War Years or Economic Depression for both men and women. 250

Discussion F o r R e v i e w O n l y 12
In this large prospective cohort study, we examined the association between energy restriction in childhood and adolescence and CRC risk at a number of tumor subsites. After 16.3 years of follow-up in the NLCS, almost 3000 CRC cases were available for analysis. In men, we observed that living in a city classified as having experienced the 255 greatest degree of caloric rationing during the Hunger Winter 22 was inversely associated with the risk of developing CRC. While inverse non-statistically significant associations were also observed in women, it appears that risk may differ according to sub-site.

Prior to discussing these results further, it is important to address an important limitation in our study. Because information on individual food for the cohort was not 260 available for the exposure periods, three proxy measures of energy restriction were used to describe energy restriction during the Economic Depression, WW2, and the Hunger Winter. It is possible that using the father's employment status, place of residence during WW2, and place of residence during the Hunger Winter has resulted in some misclassification of exposure; however, in a follow-up study in the NLCS, female sub-265 cohort members were asked if they really had experienced hunger during the winter of 1944-1945. Of the women who reported severe hunger, 80% lived in a western city during this winter. [START_REF] Dirx | Diet in adolescence and the risk of breast cancer: results of the Netherlands Cohort Study[END_REF] These results indicate that place of residence during the Hunger Winter is a reasonably adequate proxy of exposure, although unavoidable misclassification may have caused attenuation of the association. While it has been 270

proposed that individual famine data may be more accurate than area-exposure data, [START_REF] Elias | Breast cancer risk after caloric 450 restriction during the 1944-1945 Dutch famine[END_REF] other studies in the Netherlands have used place of residence during the Hunger Winter as a measure of energy restriction, [START_REF] Lumey | Offspring birth weights after maternal intrauterine undernutrition: a comparison within sibships[END_REF][START_REF] Van Noord | The effect of wartime conditions and the 1944-45 'Dutch 455 famine' on recalled menarcheal age in participants of the DOM breast cancer screening project[END_REF] and their results indicate that it is an adequate proxy. Winter, especially for those who lived in a western city, suggest that in men, it may be caloric restriction as opposed to specific nutrient restriction that modulates the risk of CRC. This observation is in line with evidence from animal studies [START_REF] Albanes | Total calories, body weight, and tumor incidence in mice[END_REF][START_REF] Shimokawa | Influence of diet on fatal neoplastic disease in 460 male Fischer 344 rats[END_REF][START_REF] Weindruch | The role of calories and caloric restriction in carcinogenesis[END_REF] and could explain why we observed weaker, non-statistically significant associations during the other two periods of exposure when caloric restriction was assumed to be less severe. 280

Alternatively, new insights reported by Trienekens et al [START_REF] Trienekens | The food supply in the Netherlands during the Second World War, in Food, science, policy and regulation in the twentieth century[END_REF] suggest that while there was heavy rationing in the years prior to the Hunger Winter, food distribution was generally satisfactory in the Netherlands, regardless of geographical area. However, this was only true up until the period of the Hunger Winter (1944-1945). It could therefore be argued that differences are not to be expected when using 'place of residence during the war 285 years (1940-44)' as a proxy for exposure to energy restriction as the contrast in exposures is too small.

In the female population, our observations were more heterogeneous. It was interesting to observe that energy restriction during the Hunger Winter was inversely associated with tumors of the distal colon, rectosigmoid, and rectum, but a slightly 290 elevated risk was observed with tumors of the proximal colon. Also, energy restriction during WW2 was significantly protective against rectal tumors, but again, positively associated with tumors of the proximal colon, although not statistically significant.

Further research is needed to elucidate the association between energy restriction and risk of CRC at different sub-sites. We can only speculate about why we observed differences 295 between men and women in terms of CRC risk. The majority of female cases were exposed to the Hunger Winter at least one year following their first menstrual period. For women in this sub-group, circulating estrogen levels would have been much higher compared to the women exposed before and during the adolescent growth spurt. It has been suggested that the beneficial effect of caloric restriction on CRC risk is mediated 300 through its influence on IGF (especially IGF-1). [START_REF] Okasha | Childhood growth and adult cancer[END_REF] However, IGF is also influenced by estrogen. [START_REF] Yee | Crosstalk between the insulin-like growth factors and estrogens 470 in breast cancer[END_REF] Therefore, some interaction between these two hormones may have offset a positive influence of caloric restriction, and could explain why we did not observe a reduced RR for CRC in women exposed to the Hunger Winter.

Our findings are generally consistent with other epidemiological studies. The 305 association between energy restriction in childhood and colon carcinoma risk was previously investigated in the NLCS after 7.3 years of follow-up. While limited in terms of size, a weak, inverse association was reported. [START_REF] Dirx | Energy restriction early in life and colon carcinoma risk: results of The Netherlands Cohort Study after 7.3 years 405 of follow-up[END_REF] After 16.3 years, it appears that the protective association previously observed for women residing in a Western city during the Hunger Winter and colon carcinoma risk has been attenuated. Stratifying our data by 310 early and late follow-up time (not shown) suggests that at both early and late follow-up there was an increased risk of proximal colon cancer, and a decreased risk of distal colon cancer. Furthermore, the difference in risk between sub-sites becomes greater with the later follow-up time. This may help explain why different RR for total colon cancer were observed in women at 7.3 vs. 16.3 years of follow-up, and reiterates the importance of 315 assessing risk by colorectal sub-site. As of yet, there is no clear biological explanation for this observation, but as none of the RR calculated at 7.3 years of follow-up or in the present study were statistically significant, it is possible that these the associations observed when only considering later follow-up are due to chance alone. WW2 has also been collected in several Nordic countries. In Norway, a country that experienced a 20% reduction in energy intake during WW2, it has been observed that individuals born during or shortly after the War have a decreased risk of CRC compared to individuals who were not born around this period. [START_REF] Svensson | Trends in 410 colorectal cancer incidence in Norway by gender and anatomic site: an age-period-cohort analysis[END_REF] It is interesting to observe that this trend extends to Estonia, but not to Finland; two countries that both experienced severe 325 restriction. [START_REF] Svensson | Early life events and later risk of colorectal cancer: age-period-cohort modelling in the Nordic countries and Estonia[END_REF] It has been proposed that the effects of caloric restriction may have been counteracted in the Finnish population because approximately 70,000-80,000 Finnish children were sent to Sweden during WW2, a country that experienced less severe rationing. [START_REF] Svensson | Early life events and later risk of colorectal cancer: age-period-cohort modelling in the Nordic countries and Estonia[END_REF] Furthermore, observations from the Boyd Orr cohort study in the United Kingdom suggest that there is a positive association between childhood energy intake and 330 adult mortality from cancer, including gastrointestinal cancers. [START_REF] Frankel | Childhood energy intake and adult mortality from cancer: the Boyd Orr Cohort Study[END_REF] It is important to determine the mechanisms through which energy restriction in childhood is associated with a decreased risk of CRC and how this might differ by subsite. Adult attained height is marker of an aggregated fetal and childhood experience, and can be considered a proxy measure for important nutritional exposures, which impact on 335 several hormonal and metabolic axes. [START_REF]Food, 390 Nutrition, Physical Activity, and the Prevention of Cancer: a Global Perspective[END_REF] In the present study, height was not acting as an intermediate variable in the risk between exposure to energy restriction and CRC. This was not surprising, as adult height may be most influenced early in childhood, and most individuals in our study were exposed to the Hunger Winter in the later years of childhood and puberty. Long term effects on insulin, insulin like growth factors 340 (especially IGF-1), and adipokines such as leptin have also been proposed as possible mechanisms, [START_REF] Kritchevsky | Influence of caloric intake on experimental carcinogenesis: a review[END_REF][START_REF] Svensson | Trends in 410 colorectal cancer incidence in Norway by gender and anatomic site: an age-period-cohort analysis[END_REF][START_REF] Svensson | Early life events and later risk of colorectal cancer: age-period-cohort modelling in the Nordic countries and Estonia[END_REF][START_REF] Elias | Long term consequences of the 1944-1945 Dutch famine on the insulin-like growth factor axis[END_REF][START_REF] Weindruch | The role of calories and caloric restriction in carcinogenesis[END_REF] but how exposure during childhood and adolescence translates [START_REF] Trienekens | The food supply in the Netherlands during the Second World War, in Food, science, policy and regulation in the twentieth century[END_REF] While not directly related to the strong one-time population starvation 345 studies considered here, it has been argued that acute periods of energy restriction are dangerous for humans because it can lead to the phenomenon of weight cycling, that is, repetitive periods of weight loss and weight gain that may result in a modest acceleration of the carcinogenic response. [START_REF] Elias | Transient caloric restriction and cancer risk (The Netherlands)[END_REF][START_REF] Thompson | Protection against cancer by energy restriction: all experimental approaches are not equal[END_REF] However, research also suggests that subtle nutritional stimuli at specific critical periods of development can induce persistent changes in gene 350 expression and metabolism through modulation of genetic and epigenetic mechanisms. [START_REF] Waterland | Epigenetic mechanisms and gastrointestinal development[END_REF][START_REF] Heijmans | Persistent epigenetic differences associated with prenatal exposure to famine in humans[END_REF] This idea is supported by the observation that being overweight during adolescence may be a more powerful predictor of CRC risk than being overweight during adulthood. [START_REF] Must | Long-term morbidity and mortality of overweight adolescents. A follow-up of the Harvard Growth Study of 1922 to 1935[END_REF] We have observed that exposure to the Hunger Winter during adolescence is associated with a lower risk of developing a tumour characterized by the CpG island 355 methylator phenotype (CIMP) after 7.3 years of follow-up. [START_REF] Hughes | Early life exposure to famine and colorectal cancer risk: a role for epigenetic mechanisms[END_REF] CIMP is a distinct characteristic of epigenetic instability in CRC development, [START_REF] Weisenberger | CpG island methylator phenotype underlies sporadic microsatellite instability and is tightly associated with BRAF mutation in colorectal cancer[END_REF] and our observations support the hypothesis that adolescence may be a critical period of development for epigenetic changes that influence CRC risk. Evidence suggests that CIMP is highly prevalent in the proximal colon, and in females. [START_REF] Weisenberger | CpG island methylator phenotype underlies sporadic microsatellite instability and is tightly associated with BRAF mutation in colorectal cancer[END_REF] While the present study suggests a 360 positive association between exposure to the Hunger Winter and risk of tumours at the proximal colon in women, this may be a finding of chance. Although we did not observe statistically significant differences with respect to risk at tumor sub-sites, focusing on associations between energy restriction and molecular characteristics of CRC may shed light on potential differences in risk 365 a reference category b model adjusted for age c model adjusted for age, family history of colorectal cancer, BMI (kg/m 2 ), energy intake at baseline (kcal/day), alcohol intake at baseline (g/day), recreational physical activity (minutes/day), level of education, and smoking status (yes, ex, never)

F o r R e v i e w O n l y

F o r R e v i e w O n l y

  tested. During the early to mid 20 th century, a large part of the Dutch population experienced three subsequent periods of impaired nutrition: the Economic Depression of 1932-40, World War 2 (WW2) (1940-44), and a severe famine: the Hunger Winter of 60 1944-45.

  that study lacked the power to consider risk at multiple tumor sub-sites. The present study investigated the 90 association between population based energy restriction in childhood and adolescence risk of total CRC, and for tumors of the proximal and distal colon, rectosigmoid, and rectum.

  questionnaire, cohort members were asked to list the last four residences they lived in, the province where these residences were located, and the years that they lived in these locations. The location of residence during the mid-point of WW2 was 140 dichotomised to 'lived in a city in 1942', or 'lived in a rural area in 1942'. Additionally, cohort members were specifically asked where they lived during the winter of 1944-45.Residing in a western city during the Hunger Winter was considered an indicator for severe energy restriction. Eleven cities in the west of the Netherlands were considered famine cities based on the definition by Stein et al.[START_REF] Stein | Famine and human development, the Dutch Hunger winter of 1944-1945[END_REF] : Amsterdam, Rotterdam, The Hague, 145 Utrecht, Zaandam, Hilversum, Amersfoort, Dordrecht, Vlaardingen/Schiedam, Delft, and Leiden. Answers to this question were classified into three categories: lived in a western city, lived in a western rural area, and lived in a non-western area of the Netherlands during the winter of1944-1945.

  by splitting up the follow-up time at 8 years. To account for the additional variance introduced by sampling the sub-cohort from the entire cohort, standard errors were estimated using the robust option.

  of the Economic Depression and WW2 years were restricted to those years when food shortages were reported to be the most severe: 1933-1934 and 185 1942-1943 respectively. Therefore, only individuals who met the definition of going through their growth spurt (i.e. males who were 12-15 years of age) during 1933-34 or 1942-43 were included in the sub-group. While many more CRC cases were available compared to the analysis at 7.3 years follow-up, the female sub-group 'exposed before growth spurt to the Hunger Winter' still had too few cases for the model to converge. Due 190 to an extremely low number of cases, associations for women exposed to the Hunger Winter before their adolescent growth spurt are not reported.

  associations we observed during the period of the Hunger 275

  exposure to energy restriction around the time period of 320

  adulthood remains elusive. The exposure time frames that we considered, especially the Hunger Winter, were short-term, transient periods of energy restriction.

  In conclusion, data from this unique period of Dutch history has allowed us to obtain rare observational evidence suggesting that severe energy restriction during adolescence may lower CRC risk at a number of tumor sub-sites, especially in men.These results give further insight into the role of energy intake during this period of life in CRC development. 370 Key Messages: Severe energy restriction during childhood and adolescence may lower colorectal cancer risk, especially in men. More research is warranted to establish the difference in risk between colorectal tumour sub-sites, as well as the association between energy restriction and molecular characteristics of colorectal cancer. 375

  CRCs were classified as proximal colon cancer (International Classification of Diseases for Oncology, first edition codes 153.0, 153.1, 153.4, 153.5 and 153.6), distal colon cancer (codes 153.2, 153.3 and 115 153.7), rectosigmoid (code 154.0) and rectal cancer (code 154.1). All prevalent cancer cases at baseline other than non-melanoma skin cancer and subjects with incomplete or

		inconsistent questionnaires were excluded. After additionally excluding individuals who
		were missing variables that were adjusted for in the multivariate analysis, 3981 sub-
	120	cohort members and 2971 CRC cases (Men: 484 proximal colon, 584 distal colon, 189
		rectosigmoid, 436 rectum, 48 not classified; Women: 514 proximal colon, 372 distal
		F colon, 95 rectosigmoid, 225 rectum, 30 not classified) were ultimately included in the o r analysis.
		R e v i e w
		O n
		l y

Table 1 .

 1 Distribution a of potential confounders according to time period of exposure to energy restriction in the male sub-cohort of the NLCS (1986) after 16.3 years of follow-up.

		Hunger Winter (1944-45) b	War Years (1940-44) b	Economic Depression (1932-40) b
		Lived in	Lived in	Lived in	Lived in Rural	Lived in Urban	Father had a job	Father did not
		Non-West	Western	Western City	area in 1942	area in 1942		have a job
			Rural
	Number of Individuals c	980	249	415
	Baseline and Population
	Characteristics
	Age	61.1 (4.2)	60.4 (4.2)	61.0 (4.4)
	Age range during period of exposure d	12-28 (19)	12-27 (18)	12-28 (19)
	Height (m)	176.2 (6.4)	178.2 (6.8)	176.8 (6.4)
	Weight (kg) Body Mass Index (kg/m 2 )	78.2 (9.2) 25.1 (2.5)	77.5 (9.4) 24.4 (2.6)	77.3 (9.3) 24.7 (2.5)
	Alcohol Intake (g/day)	14.8 (16.5)	14.7 (16.7)	14.9 (17.6)
	Energy Intake (kcal)	2194 (520)	2197 (504)	2107 (471)
	Family history of CRC (%)
	No	95.3	96.0	94.7
	Yes	4.7	4.0	5.3
	Level of Education (%)
	Low	49.0	45.4	33.7 e
	Medium	33.1	34.9	40.2
	High	17.8	19.7	26.0
	Recreational Physical Activity(%)
	<30 mins/day	16.9	20.5	17.8
	30-60 mins/day	32.2	33.5	27.0
	60-90 mins/day	18.7	21.7	22.4
	>90 mins/day	32.1	24.5	32.8
	Smoking Status (%)
	Current smoker	35.6	35.3	32.8
	Ex-smoker	50.8	49.8	54.2
	Never smoked	13.6	14.9	13.0

Table 3 .

 3 Age adjusted and multivariate adjusted hazard rate ratios (RR) for colorectal cancers according to three time periods of energy restriction in the male population of the Netherlands Cohort Study on diet and cancer(1986-2002). model adjusted for age, family history of colorectal cancer, BMI (kg/m 2 ), energy intake at baseline (kcal/day), alcohol intake at baseline (g/day), recreational physical activity (min/day), level of education, and smoking status (yes, ex, never)

			Hunger Winter (1944-45)	War Years (1940-44)	Economic Depression (1932-40)
		Lived in non-	Lived in a	Lived in a	Lived in a	Lived in an urban	Father had a	Father did not have
		western area a	western rural	western city	rural area in	area in 1942	job a	a job
			area		1942 a
	Person years at risk	13, 798	3646	5774	10,336	10,445	24, 098	2850
	Total Colorectal
	Number of cases	905	218	314	666	654	1484	184
	RR b (95% CI)	1.00	0.95 (0.77-1.16)	0.83 (0.69-0.99)	1.00	0.96 (0.82-1.12)	1.00	1.03 (0.83-1.28)
	RR c (95 % CI)	1.00	0.96 (0.78-1.19)	0.81 (0.68-0.98)	1.00	0.95 (0.81-1.12)	1.00	0.92 (0.72-1.18)
	Proximal Colon
	Number of cases	255	67	78	201	169	422	45
	RR b (95% CI)	1.00	1.04 (0.77-1.41)	0.73 (0.55-0.97)	1.00	0.82 (0.65-1.03)	1.00	0.88 (0.62-1.24)
	RR c (95% CI)	1.00	1.03 (0.76-1.41)	0.72 (0.54-0.96)	1.00	0.78 (0.61-1.00)	1.00	0.79 (0.53-1.18)
	Distal Colon
	Number of cases	297	68	118	195	226	502	63
	RR b (95% CI)	1.00	0.90 (0.66-1.22)	0.95 (0.74-1.21)	1.00	1.13 (0.91-1.41)	1.00	1.04 (0.77-1.41)
	RR c (95% CI)	1.00	0.92 (0.67-1.26)	0.89 (0.69-1.15)	1.00	1.07 (0.84-1.36)	1.00	0.95 (0.66-1.37)
	Rectosigmoid
	Number of cases	87	30	31	76	63	153	21
	RR b (95% CI)	1.00	1.35 (0.87-2.10)	0.85 (0.55-1.30)	1.00	0.81 (0.57-1.15)	1.00	1.14 (0.71-1.84)
	RR c (95% CI)	1.00	1.46 (0.92-2.32)	0.85 (0.55-1.31)	1.00	0.82 (0.58-1.17)	1.00	1.12 (0.65-1.95)
	Rectum
	Number of cases	247	50	71	186	169	369	49
	RR b (95% CI)	1.00	0.78 (0.56-1.10)	0.69 (0.51-0.92)	1.00	0.89 (0.71-1.13)	1.00	1.11 (0.80-1.55)
	RR c (95 % CI)	1.00	0.81 (0.57-1.15)	0.71 (0.43-0.96)	1.00	0.95 (0.74-1.23)	1.00	0.94 (0.64-1.38)
	a reference category
	b model adjusted for age

c

Table 4 .

 4 Age adjusted and multivariate adjusted RR for colorectal cancers according to three time periods of energy restriction in the female population of the Netherlands Cohort Study on diet and cancer(1986-2002).

		Hunger Winter (1944-45)	War Years (1940-44)	Economic Depression (1932-40)
		Lived in a non-	Lived in a	Lived in a	Lived in a	Lived in an urban	Father had a	Father did not have
		western area a	western rural	western city	rural area in	area in 1942	job a	a job
			area		1942 a
	Person years at risk	16,070	4217	8117	10,425	3234	25,576	3234
	Total Colorectal
	Number of cases	658	178	327	451	497	1040	124
	RR b (95% CI)	1.00	1.05 (0.84-1.30)	0.98 (0.82-1.16)	1.00	0.94 (0.80-1.11)	1.00	0.90 (0.71-1.14)
	RR c (95 % CI)	1.00	1.08 (0.87-1.35)	0.99 (0.83-1.18)	1.00	0.94 (0.78-1.13)	1.00	0.91 (0.71-1.16)
	Proximal Colon
	Number of cases	254	75	157	176	237	442	54
	RR b (95% CI)	1.00	1.15 (0.86-1.55)	1.21 (0.97-1.52)	1.00	1.14 (0.91-1.43)	1.00	0.94 (0.68-1.29)
	RR c (95% CI)	1.00	1.20 (0.89-1.63)	1.23 (0.97-1.56)	1.00	1.15 (0.90-1.47)	1.00	0.90 (0.65-1.26)
	Distal Colon
	Number of cases	209	55	90	140	140	319	37
	RR b (95% CI)	1.00	1.02 (0.73-1.41)	0.85 (0.65-1.11)	1.00	0.86 (0.66-1.11)	1.00	0.90 (0.63-1.31)
	RR c (95% CI)	1.00	1.05 (0.75-1.48)	0.87 (0.66-1.15)	1.00	0.87 (0.66-1.15)	1.00	0.92 (0.63-1.34)
	Rectosigmoid
	Number of cases	54	12	20	37	31	85	7
	RR b (95% CI)	1.00	0.86 (0.45-1.62)	0.73 (0.43-1.22)	1.00	0.71 (0.44-1.16)	1.00	0.63 (0.29-1.39)
	RR c (95% CI)	1.00	0.86 (0.44-1.67)	0.67 (0.39-1.17)	1.00	0.71 (0.40-1.24)	1.00	0.63 (0.28-1.41)
	Rectum
	Number of cases	121	31	53	92	74	194	22
	RR b (95% CI)	1.00	0.96 (0.64-1.46)	0.84 (0.60-1.18)	1.00	0.69 (0.50-0.96)	1.00	0.88 (0.55-1.41)
	RR c (95 % CI)	1.00	0.99 (0.65-1.54)	0.85 (0.61-1.20)	1.00	0.66 (0.47-0.94)	1.00	0.90 (0.56-1.45)

Table 5 .

 5 RR of CRC for men and women in the NLCS(1986-2002) exposed to energy restriction before, during, and after the adolescent growth spurt a , according to three different time periods of energy restriction age of growth spurt defined as: Men (12-15 years old), Women (2 years before reported age of menarche -1 year after reported age of menarche ) b RR adjusted for age, family history of CRC, BMI, baseline alcohol intake, baseline energy intake, baseline recreational (women) or occupational (men) physical activity, level of education, and smoking status. c reference category d too few cases for model to converge e only age-adjusted model considered

			Before Growth Spurt		During Growth Spurt		After Growth Spurt
		#	Person	RR b (95% CI)	#	Person	RR b (95% CI)	#	Person	RR b (95% CI)
		cases	years at risk		cases	years at risk		cases	years at risk
	Exposure
	MEN
	Hunger Winter
	Lived in a non-west area c				87	2383	1.00	594	9796	1.00
	Lived in western rural area	No males exposed before growth spurt	16	819	0.52 (0.26-1.02)	145	2468	1.01 (0.78-1.30)
	Lived in western city				21	1042	0.56 (0.31-1.03)	208	4327	0.76 (0.61-0.94)
	War Years
	Lived in a rural area in 1942 c	25	836	1.00 d, e	90	2329	1.00	371	6014	1.00
	Lived in a city in 1942	24	795	0.91 (0.37-2.26)	92	2492	1.03 (0.68-1.55)	377	6480	0.85 (0.69-1.05)
	Economic Depression
	Father had a job c	783	16710	1.00	217	3546	1.00	113	1484	1.00
	Father had no job	92	1888	0.98 (0.73-1.31)	24	430	0.94 (0.51-1.70)	15	275	0.89 (0.34-2.29)
	WOMEN
	Hunger Winter
	Lived in non-west area	15	390	---d	88	3511	1.00	538	12169	1.00
	Lived in western rural area	1	81	---	21	1017	0.87 (0.50-1.53)	151	3119	1.14 (0.89-1.46)
	Lived in a western city	2	232	---	41	1840	0.89 (0.57-1.39)	277	6045	1.08 (0.89-1.32)
	War Years
	Lived in a rural area in 1942	28	1093	1.00	81	2701	1.00	336	6657	1.00
	Lived in city in 1942	32	1398	0.68 (0.30-1.53)	97	951	1.01 (0.66-1.54)	353	7560	0.94 (0.76-1.18)
	Economic Depression
	Father had a job	644	18290	1.00	296	5156	1.00	100	2131	1.00
	Father had no job	73	2189	0.90 (0.66-1.23)	30	659	0.77 (0.46-1.29)	17	385	0.92 (0.43-1.97)

a

Acknowledgements

We are indebted to the participants of this study and thank the cancer registries (IKA, 380 IKL, IKMN, IKN, IKO, IKR, IKST, IKW, IKZ, and VIKC) and the Netherlands Nationwide Registry Pathology (PALGA). We also thank S van de Crommert and J Nelissen for their assistance with data entry and data management; A Volovics and A Kester for statistical advice; L Schouten, C de Zwart, M Moll, W van Dijk, M Jansen, and A Pisters for assistance; and H van Montfort, T van Moergastel, L van den Bosch, and R 385 Schmeitz for programming assistance.

Funding

This work was supported by the World Cancer Research Fund [2007/54 to M.P.W]

F o r R e v i e w O n l y


Sue Povey

Aida I Al Aqeel

Anne Cambon-Thomsen

Raymond Dalgleish

Johan T Den Dunnen

Helen V Firth

Marc S Greenblatt

Carol Isaacson Barash

Michael Parker

George P Patrinos

Carol Isaacson Barash

Judith Savige

Maria-Jesus Sobrido

Ingrid Winship

Richard G H Cotton

Practical guidelines addressing ethical issues pertaining to the curation of human locus-specific variation databases (LSDBs)

Keywords: phenotype, LSDB, data-sharing, ethics, diagnostic test, consent, curators, guidelines

Introduction

This document is designed to assist a curator who intends to provide access to the information contained in a human Locus Specific variation Database (LSDB). For this purpose, an LSDB is defined as a listing of known sequence variants in a specific human gene together with some assessment of the effects of these variants on the phenotype. It may also highlight the frequency of both common and rare variants (e.g. single nucleotide polymorphisms) prevalent in particular populations groups. Although ethical issues arise in a database of any format, currently the access is nearly always provided via a web interface, usually available to everyone but occasionally restricted to selected professional groups. An example of a well-known LSDB which can be accessed by anyone is that describing the mutations in the gene DMD , deficient in Duchenne Muscular Dystrophy, curated by one of the authors (JdD) in Leiden (http://www.dmd.nl/nmdb2/home.php) . This format is available in open-source software and is now used in many other LSDBs. The need for these guidelines has been highlighted by the recognition by initiators of the Human Variome Project (HVP) of the immense unpublished and inaccessible resource of information existing in diagnostic laboratories and the significant clinical need to have access to this information [START_REF] Cotton | Recommendations of the 2006 Human Variome Project meeting[END_REF][START_REF] Kaput | Planning the Human Variome Project: The Spain report[END_REF].

These guidelines are largely an expansion in detail of the first generation guidelines proposed by Cotton andco-authors in 2005 (Cotton et al., 2005) which were rooted in the principles described by Knoppers and[START_REF] Knoppers | Ethical guideposts for allelic variation databases[END_REF][START_REF] Knoppers | Ethical guideposts for allelic variation databases[END_REF]. They were discussed and modified as a result of the international HVP planning meeting in Spain May 2008 attended by participants from a wide range of developed and emerging countries [START_REF] Kaput | Planning the Human Variome Project: The Spain report[END_REF]. Details of this can be found in the published meeting report and its supplementary information [START_REF] Kaput | Planning the Human Variome Project: The Spain report[END_REF]. The content and order of headings in the current version differs slightly from that of [START_REF] Cotton | Locus-specific databases: from ethical principles to practice[END_REF] shown in Box 1. Box 1:12 major points to consider pertaining to ethical issues arising in the curation of human Locus Specific variation Databases (LSDBs) 1. Clarify the main purpose of the particular database, 2. Define database policy with respect to sources of data.

3. Take Specific communities/cultures into account.

4. Take vulnerable persons into account.

5. Create an ethics oversight committee 6. Remove identifying information before submission to the database 7. Add further protection of confidentiality if needed.

8. Allow no further disclosure without consent. 9. Provide provision for removal of data from the database. 10. Be cautious in response to requests to an LSDB curator for a private opinion 11. Limit links to other LSDBs 12. Consider carefully the transfer of publicly available data from LSDBs to genome browsers

Background: Develop a common ethical framework

The goal of all such databases is the sharing of genomic and phenotypic information for the benefit of humanity. This requires the protection of privacy, which in this context is the right of the individual and members of their family to be protected against intrusion into their personal information and further intrusions ensuing from access to this, by publication of information. The balance between the public's interest in the value of the shared information and its interest in the strict protection of privacy has been widely discussed,(see footnote 1 which should read as follows:-(for example by the Academy of Medical Sciences UK in 2006 www.acmedsci.ac.uk/download.php?file=/images/project/Personal.pdf adverse comment in [START_REF] Matthews | Comment: Consent is crucial to medical research[END_REF] UK government report in 2009 on genomic medicine (http://www.publications.parliament.uk/pa/ld200809/ldselect/ldsctech/107/10702.htm), in a commentary from an Islamic perspective [START_REF] Aqeel | Islamic ethical framework for research into and prevention of genetic diseases[END_REF], from the USA [START_REF] Taylor | Personal genomes: when consent gets in the way[END_REF] This balance will be viewed differently in different cultures [START_REF] Aqeel | Islamic ethical framework for research into and prevention of genetic diseases[END_REF] and so international input into detailed guidelines is essential to ensure collective agreement which is requisite to effective collaboration. Harmonisation of standards will be a challenge. While the development of a common ethical framework must be nurtured by culture and countryspecific input, the converse also holds true: the guidelines will serve as reference for the developers of national laws and local ethics committees.

For many of the genes and for most of the issues dealt with below it seems likely that an independent group of well-informed individuals to oversee specific LSDBs not only at their initiation but on an ongoing basis will be essential. This general need is underlined by the 2008 revision of the Declaration of Helsinki http://www.wma.net/en/30publications/10policies/b3/17c.pdf which states that monitoring of ongoing studies must be put in place in addition to the initial approval by an ethics committee. Governance is thus necessary as new issues may appear in the course of a project or activity.

Inevitably in these guidelines there is a strong emphasis on the validity and complexities of consent and the increasing difficulty of guaranteeing privacy in an era of electronic publishing and growing internet use. The authors would not wish to discourage the curation of LSDBs on this account. Current experience of curators indicates that the majority of patients and research participants are likely to be happy to share their data, although inevitably there will be exceptions, and participants' preference may change based on new understanding of clinical significance. So far curators have been as likely to receive complaints about the omission of a personal unique variant from the relevant database as its unexpected inclusion. The guidelines are presented in approximately the order in which the issues are encountered by the prospective curator:-1. Clarify the main purpose of the particular database, recognising that this may change over time. Who does the curator expect will use this LSDB and why?

This will allow evaluation of the exact information required or desirable and whether compliance with the remaining guidelines will be possible with a database open to the public.

It may be necessary to decide that at least part of the information should be restricted to identified persons. For discussion of robust methods for validation of identities of enquirers see the GEN2PHEN Knowledge Centre http://www.gen2phen.org/researcher-identification-primer .

Many LSDBs are used as a tool by diagnostic laboratories assessing the likelihood that the DNA change that they have found is the necessary and sufficient cause of a serious disease and can be used to inform treatment and/or prevention, including preimplantation and prenatal diagnosis and neonatal screening. However the data needed will vary in diffferent diseases so even for this use the ethical issues will be slightly different for each database. Some examples of questions which must be considered by curators at this stage are shown in Box 2 .The answers will allow the generation of a list of ethical requirements which any submitter must fulfil. They will inform decisions about any need for control of access and may also help in the determination of appropriate members of an ethical oversight committee as described later. Both those LSDBs which catalogue very rare or even unique changes relating to serious disease and those which deal with common variants of small individual effect should adhere to stringent rules of data standardization, validation, quantification, and transparency of sources, as described by participants at the HVP planning meeting [START_REF] Kaput | Planning the Human Variome Project: The Spain report[END_REF]. These aims should be clarified and explained in terms understandable by non specialists on the public part of the LSDB website.

Box 2 :Questions about aims and data required

How much detailed clinical data will be needed and will this be in the form of a link to another database? Is any family information needed, e.g. to support conclusions on pathogenicity?

Will an attempt be made to record every apparently unrelated case with the same mutation?

What ethnic and geographic origin data will be needed and for what purpose?

Will an attempt be made to record all known 'neutral' ('normal') variation?

Is the aim to evaluate the contribution of common variants to common diseases?

Is the goal to inform basic research into the mechanism of disease, e.g. modifier genes?

Is the aim to evaluate genetic variation in response to therapy in individual or populations?

Will the LSDB collect results of in vitro functional analyses?

Will the data include results from a cell or tissue culture of patient/participant material?

Will the database be used to assemble volunteers for new therapies such as mutation-specific strategies?

Is the interest mainly from an evolutionary perspective? 2. Define database policy with respect to sources of data. This will be dealt with in two main sections:-existing data and future data , and within each there is a section on data collected for research and that collected in a diagnostic setting. At the end of this section we .

EXISTING DATA

A. Published data

Usually, information which is already published and available electronically is assumed to have been collected by the appropriate standards that existed at the time of collection. By virtue of their public availability, these data are generally assumed to be usable in an LSDB.

Although it is possible that the person giving the consent for the research and publication may not have foreseen the full implications of web-based sharing of information, in most situations the likelihood of re-identification and/or misuse of data is considered low.

Whenever feasible, the LSDB curator is encouraged to inform the original data producer (defined as corresponding author for the publication) in order to explore whether any restrictions or modifications of data are appropriate . This recommendation is more for respect of free will and autonomy of patients than for fear of the risk of re-identification. The specifics of a particular rare disease may warrant more stringent monitoring of data; this should be addressed by the ethics oversight committee of the specific LSDB. The main obligation on the curator is to check the scientific accuracy as far as possible, including writing to authors when necessary. Curators should keep in mind that integration of published data may on occasion give rise to conclusions with serious implications for individuals or groups. Similar concerns have been discussed previously, for example, relating to accumulated data on CGH microarrays [START_REF] Tabor | Ethical implications of array comparative genomic hybridization in complex phenotypes: points to consider in research[END_REF]. Occasionally discussion with the oversight group might be needed before full public release of the integrated data.

B Data existing in diagnostic laboratories or as clinical reports

Available but unpublished diagnostic data are a major problem This is both because of limited clinical data but also because current practice does not usually ensure that those consenting to genetic testing have given permission for sharing (including scientific publication) of these data beyond the laboratory and clinical team undertaking the analysis.

LSDBs must be cautious in accepting unpublished data from any investigators or from accredited diagnostic laboratories, and consider issues that could limit the clinical accuracy of unpublished submissions, including standardization of clinical language, source of data, individual identification and consent.

It is suggested that where the quality of the data appears to be high and of significant clinical value but it is not feasible to obtain explicit consent, the decisions about which data should be uploaded and also which should be publicly displayed, protected by controlled access or displayed only in summary form should be made by the independent LSDB ethical oversight committee (see point 5). This committee must also be sensitive to different cultural views. In many cases it may be appropriate for these data to be anonymized, i.e. made 'not identifiable'

(see point 6 for explanation of ICH sample coding terminology ). Note that the current version of LSDB software LOVD [START_REF] Fokkema | LOVD: easy creation of a locus-specific sequence variation database using an "LSDB-in-a-box" approach[END_REF] has the option to store data that are not public but that can be queried. The result of a query hitting non-public data is a notification that there is such information in the database but that the curator needs to be contacted to get more information.

In some cases patients/families already report the data themselves (often with a copy of the lab result they obtained) and this can be encouraged with appropriate further information requested if needed. LSDBs should then have a consent form that should be signed by the self-submitter and by all relatives whose results the submitter forwards to the LSDB. This should be made clear to the person being asked for consent. It is essential that this is done without coercion in order to preserve the freedom of the consent. However the information provided to the patients and families should clearly explain the value of gathering such data and mention that in the long term, if data cannot be collected, interpretation of testing results may be less reliable or even impossible and development of future possibilities for treatment might be compromised. Although the curator should require a statement that the submitter has obtained appropriate consent in whatever way is acceptable in the country of origin of the data, the primary responsibility is that of the submitter. The curator should supply to the testing laboratory a clear explanation about the LSDB on an information sheet that the laboratory can provide to clinicians and patients.

A suggested form of wording as an addition to the consent for diagnostic testing (and which may also be appropriate for testing as part of a research project) is as follows:

i. I understand that the interpretation of DNA test results, including my own is based mainly on publicly available data from others who have been tested before me.

ii. I agree that the results of my DNA test and clinical examination may be added to these public data sets, in a manner which does not disclose my personal identity and which is in agreement with data protection law in my country.

iii. This information will then be available to help the diagnosis of others, and to further understanding about the disease. Improved understanding of the molecular mechanisms of disease may be important in developing new treatments and/or prevention.

iv. Any information which could identify me or members of my family may only be stored when a high standard of privacy and confidentiality (as defined and in accordance with national standards for health data) is maintained. However, unintentional third party cross examination of stored non-identified data, might indicate, but not prove, identity. Should this happen, third party users of the database will undertake not to explore this information further or to contact me v. I understand that I will not receive any payment for this. .

Take Specific communities/cultures into account. Identifiable groups such as Ashkenazi

Jews or Roma (Gypsies) may be particularly affected by a specific disease and thence become a major part of the relevant LSDB. Following consultation with the community, every effort must be made to take this into account and to provide privacy protection and respect cultural sensitivity ensuring that high ethical standards are maintained. A small specialised database gives the greatest chance of the unintended identifiability of one of the subjects. It may occasionally be necessary to store data only at summary level to preserve anonymity, as has been done in the Israeli and other National/Ethnic Mutation Databases (NEMDBs) [START_REF] Patrinos | National and ethnic mutation databases: recording populations' genography[END_REF][START_REF] Zlotogora | Documentation of inherited disorders and mutation frequencies in the different religious communities in Israel in the Israeli National Genetic Database[END_REF]. The cultural sensitivity of particular groups such as the Maori of New Zealand will need a step of local consultation before any sharing of DNA data, even for disorders not especially prevalent in that group.

Take vulnerable persons into account.

Persons who do not have the capacity to consent either because of disability or young age are especially vulnerable. In some disorders, this will apply to many of the patients/ participants and regular external review of procedures for obtaining consent from appropriate relatives/ representatives or other suitable authority should be in place. Usually this will be part of the remit of the LSDB oversight committee. A database which only accepts publicly available data would still benefit from some independent ethical review and this is strongly recommended This could be provided either by a specific oversight committee as described above, or perhaps by an international HGVS or HVP group who could advise a number of databases 6. Remove identifying information before submission to database. Every effort must be made to ensure that the individuals whose DNA variation is displayed in an LSDB are not individually identifiable. With increasing availability of total genomic sequence and the enormous amount of personal information retrievable from websites, absolute certainty of non identifiability is no longer guaranteed [START_REF] Barash | Threats to privacy protection[END_REF][START_REF] Homer | Resolving individuals contributing trace amounts of DNA to highly complex mixtures using high-density SNP genotyping microarrays[END_REF][START_REF] Lowrance | Ethics. Identifiability in genomic research[END_REF][START_REF] Walter | A little privacy, please. Computer scientist latanya sweeney helps to save confidentiality with "anonymizing" programs, "deidentifiers" and other clever algorithms. Whether they are enough, however, is another question[END_REF]. However, with care, the risk of identification from an LSDB will be very low in almost every case, particularly if data from genome-wide analyses such as SNP genotyping data are not associated with the mutation. (see point 7 for possible These definitions will be used here and are explained below:-Decide whether data should be 'coded' (also called 're-identifiable') or 'anonymized' (also called 'de-identified'). For the purposes of these guidelines, 'coded' is taken to mean removal of all existing identifiers as far as is compatible with usefulness of the data and substitution by proxy identifiers which are used in the database. The link between the existing identifier and the proxy identifier could be maintained either by the submitter acting as the 'honest broker' between the hospital records and the LSDB or within a securely non-public area of the LSDB. It would be desirable that each proxy identifier be unique and generated by a standard coding mechanism, perhaps by some national or international body. This would avoid inadvertent duplication of identifiers that might arise if the process was left to individual LSDB curators. For published data, and especially for recent publications, many cases are already coded and then classified as 're-identifiable' specifically for publication.

These codes might be acceptable if not recorded in hospital notes. However, new coding for the database would be safer. Unpublished data, unless anonymized as explained below, should always be re-coded to make them re-identifiable and not directly identifiable. In the case of unpublished data on a rare disorder it is advisable that no link between a particular entry and the submitter should be displayed, and that ethnic origin and geographic data on the donor are not visible. This is recommended even if the clinical data displayed are minimal.

Anonymized (or 'de-identified') here means that identifiers and any information which might be used as clues to identity through other links are permanently removed and the link to the ID used by the submitter is destroyed. This would limit the usefulness of the data, particularly with regard to long term phenotype follow-up data and any late correction of wrong information but also possibly in ways that cannot currently be predicted. It also makes withdrawal of consent impossible and is not the approach of choice.

If the decision is to proceed with coded or in other words 're-identifiable' data, although the original identifier is replaced by a code, many other pieces of information give possible clues to identity and will need to be removed to avoid unauthorised re-identification and, in many countries, to obey privacy laws. In most published cases, all data given in the publication would be acceptable to include in the database, including geographical location and ethnic group of individuals studied (both for cases and for population controls) and clinical details of patients. Limited family details present in the original report which help in the interpretation of the mutation may also be included. A link to the original publication is acceptable and useful. Note that data on frequency and population/ethnicity can be very helpful in the design of cost-effective targeted diagnostics and/or treatment protocols.For unpublished data, the specific nature of the LSDB will be considered by the ethical oversight committee in recommending which data can be collected and displayed. Special consideration should be given to rare mutations (see point 7).

Add further protection of confidentiality if needed.

This may be necessary in the case of rare or unique mutations in rare diseases, unique combinations of clinical features or where higher protection is required for some other reason. The oversight committee will be valuable photographs or detailed pedigrees) access to these data may have to be restricted by appropriate registration and approval for access. A possible solution here which could also be applied to any unpublished data for which explicit consent is not certain would be to display the mutation in the database with no other data at all. Someone with a genuine reason for wanting to know if this mutation causes disease could then click on a tool which would send an email to the curator to explore the possibility of finding more information (see section 8). There are several variations to this approach and the display of every variant recorded including those for which no other information can be displayed publicly makes accurate enquiries easier for the user than requiring the enquirer to specify in correct format a variant which is not displayed As mentioned in point 2, one of us (JdD) has already provided a similar facility for LOVD databases [START_REF] Fokkema | LOVD: easy creation of a locus-specific sequence variation database using an "LSDB-in-a-box" approach[END_REF].

Allow no further disclosure without consent. Information about a particular entry

beyond what is publicly viewable in the database should not be supplied by a curator to an enquirer unless consent for this has been explicitly provided. The request should be referred to the submitter who will use using professional judgement in their response.This will usually require seeking further explicit consent from the patient. If there is any doubt the independent oversight committee should be consulted . ensure that he/she is aware of his/her LSDB entry and has the right to withdraw it.This should also be the case if an adult previously judged incompetent becomes competent. However, it should be made clear that whilst it will be possible to eradicate information which was originally displayed from the database, it may not be possible to eradicate it from other sources which have used this information for example in an overview publication.

Provide provision

10. Be cautious in response to requests to an LSDB curator for a private opinion on whether a particular variant is pathogenic, especially if any of the information used is not published. Add a disclaimer about responsibility for the clinical use of the opinion and be cautious of a 'virtual medical advisor relationship'. From the medico-legal point of view, it is safest to obtain clinical interpretation from published data. If unpublished information is used, a careful record should be kept. Recommendations of the IARC Working Group on Unclassified Genetic Variants encourage that classification of pathogenicity be carried out not by individuals but by teams of experts that can carefully evaluate all lines of evidence [START_REF] Greenblatt | Locus-specific databases and recommendations to strengthen their contribution to the classification of variants in cancer susceptibility genes[END_REF][START_REF] Tavtigian | for the IARC Unclassified Genetic Variants Working Group, Assessing pathogenicity: overview of results from the IARC Unclassified Genetic Variants Working Group[END_REF].

11. Limit links to other LSDBs. If mutations in more than one gene are relevant to a particular disease, it may be useful to record the variation of both genes in the same individual and link the entries so that the fact that it is one person is recorded. This facility is already available on at least one LSDB platform (LOVD) and can be of great value in the interpretation of results. However, logically it may eventually extend to enough genes to allow identification of the individual. At this point, the considerations of the ethics of large scale resequencing will be relevant (see [START_REF] Lowrance | Ethics. Identifiability in genomic research[END_REF]. For example, a recent investigation into DNA variants causing X-linked mental retardation included substantial amounts of sequence information on the coding regions of X-linked genes (Tarpey et al.,

  consent form which we propose might be appropriate in future, particularly for results obtained in the course of clinical testing.

  In future research projects, consent forms should specifically indicate what data will be included in a publicly available database and describe their possible intended uses. The clinical significance of published data will contiue to change as new findings emerge and the ethical repercussions will depend on many different variables.It may be appropriate to include agreement in the original consent about the need for re-contact or for delegation of decision-making to an ethical committee for future unforeseen uses and implicationsB Diagnostic DataWith regard to consent, we would strongly recommend that informing donors of the possibility of transmission of data to an LSDB should in the future be part of the consent form for all genetic testing, together with an explanation of why this is useful, and how their privacy will be secured. Refusal to allow inclusion of data in an LSDB should not affect genetic testing since this would contravene the traditional commitment of medicine as exemplified in the UK General Medical Council guidelines 2006'Make the care of your patient your first concern' http://www.gmc-uk.org/Good_Medical_Practice_0510.pdf_32611016.pdf

  This wording will need modification in certain circumstances and if any part of the interpretation of the result to be shown on the database depends on family history or testing of other members of the family, this should be considered and consent sought if appropriate.

  an ethics oversight committee. A variation database which accepts genotype and phenotype data not already in the public domain (or that are in the public domain but whose combination and integration are foreseen to change the degree of identifiability of persons) and makes them widely available, should have an independent and well-informed oversight group. This should be drawn from several disciplines and from relevant society stakeholders, including patient groups, in order to review the particular ethical issues arising in relation to that LSDB. They should recommend constraints needed in uploading and displaying data and decide on any requirement for control of access or for anonymization. The delegation of such decisions to a committee with a long term remit will balance the difficulty of having truly informed consent in such a fast moving field. The decisions of this committee may require formal ethical approval either from their own institution or a national body.

  unique variants). There is now a set of definitions including sample coding terminology agreed internationally and recognised by all constituents of the International Conference on Harmonisation (ICH) that has become official in 2008 http://www.fda.gov/downloads/RegulatoryInformation/Guidances/ucm129296.pdf

  here. The database will have limited usefulness if important genotype phenotype data cannot be released because this information alone might allow identification of the individual. In diseases where very detailed clinical data have been collected (especially clinical

  data were regarded as too sensitive for the full set of variants for each patient to be entered onto the LSDBs in the most informative way. See http://www.LOVD.nl/MR for the summary data submitted.12. Consider carefully the transfer of publicly available data from LSDBs to genome browsers. Some of the genetic variations collected and displayed by curators of LSDBs are now visible also in one or several of the main tools used by scientists worldwide analysing the human genome, for example at the National Center for Bioinformatic Information (NCBI) at Bethesda and the UCSC Genome Browser at Santa Cruz. This does not raise entirely new ethical issues except in the need for adequate recognition of the work of the LSDB curator. It makes the misuse of data for re-identification slightly more likely and may also increase the chance of a mistake being widely disseminated in a short time. Further discussion of the sharing of data with genome browsers can be found elsewhere[START_REF] Den Dunnen | Sharing data between LSDBs and central repositories[END_REF].

  and in 2003 from the French National Bioethics advisory Committee http://www.ccne-ethique.fr/docs/en/avis076.pdf ) end of footnote

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for removal of data from database. The

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	parents or guardians who
	have given consent for a child's or incompetent adult's information to be included in the
	LSDB should be made aware of their right to withdraw this information at any time (unless
	data are truly not identifiable). The LSDB should make available information in order to
	facilitate this task. If a child reaches the age of consent (16 in many but not all countries)
	and is capable of making a decision, those who previously authorised data sharing should

Acknowledgements

We would like to thank Professor Ruth Chadwick, chair of the HUGO Ethics Committee and other members of that committee in particular Dr Thomas H. Murray, President of The Hastings Center in New York for reading the manuscript and providing valuable criticism.

We also thank an anonymous referee for constructive comments. Before this stage many people contributed helpful suggestions for the improvement on the manuscript. Particular thanks are due to Professor Bartha Knoppers, PhD, Director of the Centre of Genomics and Policy, Faculty of Medicine, Department of Human Genetics, McGill and former Chair of the International Ethics committee of the Human Genome Organization (HUGO).Thanks also to Professor Finlay Macrae, at the Royal Melbourne Hospital, for constructive comments on the manuscript and to members of the working group on ethics at the HVP planning meeting

These guidelines have been reviewed by the Ethics Committee of the Human