Modeling Genetic and Environmental Factors to Increase Heritability and Ease the Identification of Candidate Genes for Birth Weight : A Twin Study Heritability estimates of birth weight have been inconsistent . Possible explanations are heritability changes during gestational age or the influence of covariates -LRB- e.g. chorionicity -RRB- . The aim of this study was to model birth weights of twins across gestational age and to quantify the genetic and environmental components . We intended to reduce the common environmental variance to increase heritability and thereby the chance of identifying candidate genes influencing the genetic variance of birth weight . Perinatal data were obtained from 4232 live-born twin pairs from the East Flanders Prospective Twin Survey , Belgium . Heritability of birth weights across gestational ages was estimated using a non-linear multivariate Gaussian regression with covariates in the means model and in covariance structure . Maternal , twin-specific , and placental factors were considered as covariates . Heritability of birth weight decreased during gestation from 25 to 42 weeks . However , adjusting for covariates increased the heritability over this time period , with the highest heritability for first-born twins of multipara with separate placentas , who were staying alive -LRB- from 52 % at 25 weeks to 30 % at 42 weeks -RRB- . Twin-specific factors revealed latent genetic components , whereas placental factors explained common and unique environmental factors . The number of placentas and site of the insertion of the umbilical cord masked the effect of chorionicity . Modeling genetic and environmental factors leads to a better estimate of their role in growth during gestation . For birth weight , mainly environmental factors were explained , resulting in an increase of the heritability and thereby the chance of finding genes influencing birth weight in linkage and association studies . Introduction Birth weight is a complex trait in which both environmental and genetic factors are involved . Low birth weight is not only associated with higher neonatal morbidity and mortality -LRB- Kramer et al. 2005 -RRB- , but also with post-natal psychopathology -LRB- Wichers et al. 2002 -RRB- . It is even stated that birth weight is a causal risk factor for child problem behavior and that the effects may well extend into adulthood -LRB- van Os et al. 2001 -RRB- . Heritability estimates for birth weight using twin pairs , based on structural equation modeling or study of the offspring of twins , have given inconsistent results ranging from 10 to 40 % -LRB- Baird et al. 2001 ; Clausson et al. 2000 ; Hur et al. 2005 ; Luciano et al. 2004 ; Magnus et al. 2001 ; van Baal and Boomsma 1998 ; van Dommelen et al. 2004 ; Vlietinck et al. 1989 ; Whitfield et al. 2001 -RRB- . The relatively low heritability estimates may be explained by unequal sharing of nutrients or unequal placental blood supply to the twin pair -LRB- Machin 1996 -RRB- . Vlietinck et.al . -LRB- Vlietinck et al. 1989 -RRB- revealed that chorionicity explained 12 % of the variance of birth weight with DC twins having a significantly higher similarity than MC twins . Random environment explained 23 % , additive genetic factors 22.5 % , and maternal age 0.7 % of the total variance . But , the largest source of variation on birth weight in this study was gestational age -LRB- 42 % -RRB- . Until now however , no study has focused on the change of variance of birth weight during gestation . We have shown that this variance increases during gestation -LRB- Gielen et al. 2007 -RRB- . As a consequent , heritability of birth weight may change over gestational age or different covariates may explain different parts of the variance-covariance structure . For example , it is more likely that at 37 weeks of gestation , nutrient factors -LRB- environmental factor -RRB- will explain a larger part of the covariance matrix as compared to 13 weeks of gestation . The advantage of twin studies , is that the total variance can be split up into genetic -LRB- A -RRB- , shared or common environmental -LRB- C -RRB- and unique environmental -LRB- residual , E -RRB- components , enabling an accurate estimation of heritability -LRB- = genetic variance/total variance -RRB- -LRB- Boomsma et al. 2002 -RRB- . The degree of heritability determines the power to localize and identify the genes involved -LRB- Snieder et al. 2003 -RRB- . For linkage and association studies a high degree of heritability is preferable . Since heritability is the proportion of phenotypic variation of a trait that can be attributed to genetic variation -LRB- Boomsma et al. 2002 -RRB- , reducing environmental and residual components will give an increase of the heritability . The latter can be achieved by adjusting for covariates that explain part of the environmental factors in the analysis . Additionally , a covariate may also explain part of the genetic variance and as a result this may provide an indication for finding genes for this covariate , but of course the drawback is that with this covariate in the model the success of finding the latent gene -LRB- s -RRB- will be reduced . Consequently , it is mandatory to determine how each covariate influences the genetic , common environmental and residual factors of the variance . Covariates that influence birth weight of twins are gestational age , maternal factors -LRB- parity and maternal age -RRB- , twin specific factors -LRB- zygosity , sex , birth order -RRB- , and placental factors -LRB- chorionicity , fusion of placentas and insertion of the umbilical cord -RRB- -LRB- Gielen et al. 2007 -RRB- . The aim of this twin study was to model birth weight across gestational ages and to quantify the genetic and environmental components in order to explain the effects of covariates on the variance of birth weight . We hypothesize that heritability changes during gestation and that covariates mainly explain the common environmental and residual factors of the variance . By entering these covariates in the means model and in the covariance matrix , heritability will increase and , as a consequence , the power to find genes by linkage and association studies will increase , although the effects of each covariate on the genetic and environmental component may be different . Materials and methods Subjects The study sample consisted of live born twin pairs selected from the East Flanders Prospective Twin Survey -LRB- EFPTS -RRB- , Belgium -LRB- Loos et al. 1998 -RRB- . Between July 1964 and the end of December 2002 , the EFPTS registered 6315 twin pairs who met the World Health Organization criteria for live born infants -LRB- birth weight ≥ 500 grams or gestational age ≥ 22 weeks , if birth weight unknown -RRB- . The vast majority consisted of twins of Caucasian origin . Methods of data collection have previously been described in detail -LRB- Gielen et al. 2006 ; Gielen et al. 2007 ; Loos et al. 2005 -RRB- . Briefly , a defined set of obstetric and perinatal data were recorded -LRB- including gestational age , mode of conception , maternal age , birth order , parity , sex and neonatal survival -RRB- and placentas were examined within 24 hours after delivery according to a standardized protocol . Based on the site of the cord insertion two groups were distinguished : -LRB- 1 -RRB- central insertion -LRB- central and eccentric insertions -RRB- , and -LRB- 2 -RRB- peripheral insertion . Gestational age was reported by the obstetrician , based on the last menstruation or a first trimester ultrasound investigation , and was calculated as the number of completed weeks of pregnancy . Zygosity was determined through sequential analysis based on sex , fetal membranes , umbilical cord blood groups -LRB- ABO , Rh , CcDEe , MNSs , Duffy , Kell -RRB- , placental alkaline phosphatase -LRB- Decorte et al. 1990 ; Vlietinck 1986 -RRB- , and , since 1982 , DNA fingerprints . Zygosity and chorionicity were determined with an accuracy of 99 % . Twin pairs of whom one or both children were stillborn -LRB- 205 pairs -RRB- or suffered from major congenital malformation -LRB- 120 pairs -RRB- were excluded -LRB- Loos et al. 2001 -RRB- . Two MC pairs were excluded because they were recorded as being MC but at birth had two separate placentas . Twin pairs with missing values in one or more covariates for one or both twins -LRB- birth weight , gestational age , parity , chorionicity , mode of conception , unknown whether staying alive postnatally , maternal age , placental type and weight , site of insertion of the umbilical cord -RRB- were excluded -LRB- 1756 pairs -RRB- -LRB- Gielen et al. 2007 -RRB- . Finally , 8464 twins -LRB- 4232 pairs -RRB- with complete datasets were analyzed . Statistical analysis The birth weights of pregnancies of different gestational ages were analyzed using a non-linear multivariate Gaussian regression , MVN -LRB- -LRB- μ , Σ2 -RRB- where μ is the means model and Σ2 is the covariance matrix . Means model Growth was best modeled by a logistic growth curve -LRB- non-linear multivariate Gaussian regression -RRB- in which the time of conception -LRB- gestational age minus two weeks -RRB- was chosen as time point zero -LRB- where β1 t/m β3 are the coefficients of the logistic growth curve and Time is time since conception ; unadjusted model -RRB- -LRB- Neale and McArdle 2000 -RRB- . Furthermore , the two first weeks of pregnancy -LRB- the time between the last menstrual period and conception -RRB- were set to be zero . The logistic growth curve was modified to allow a change of growth rate -LRB- where β4 is the change in growth rate after the time point described by coefficient β5 -RRB- . The following covariates were considered : sex of the individual and of the co-twin , chorionicity , number of placentas , site of umbilical cord insertion , birth order , maternal age , mode of conception , parity , and neonatal death -LRB- Gielen et al. 2007 -RRB- . Because in 1964 all gestational ages were based on the last menstruation , while in 2002 almost all gestational ages were based on first trimester ultrasound and this might influence gestational age , we controlled for birth year . The covariates were consecutively added in the logistic growth curve , where X represents possible covariates and their interactions included in the model and β represents the corresponding coefficients . Finally , members of different groups were allowed to have different growth rates after a certain point in time as shown in the following equation where Z is a binary covariate -LRB- indicator for belonging to group Z -RRB- and β6 is the additional change in growth rate after the time point described by coefficient β7 for members of group Z. -LRB- adjusted model -RRB- . Examples of how to use the estimates can be found elsewhere -LRB- Gielen et al. 2007 -RRB- . Covariance matrix The twin model assumes that the total phenotypic variance can be decomposed into additive genetic effects -LRB- A -RRB- , dominance genetic effects -LRB- D -RRB- , effects of common environment -LRB- C -RRB- and effects of unique environment -LRB- E -RRB- -LRB- Neale and Maes 2002 -RRB- . The common environmental variance is assumed to be the same for both the MZ and DZ twin pairs and will account for the intra pair correlation between twin pair weights . MZ twins are assumed to share the same genetic variance -LRB- A and D -RRB- , whereas DZ twins share half of the additive genetic variance and a quarter of the dominance genetic variance . In the case of a covariance matrix containing an additive genetic effect , common environment , and unique environment -LRB- ACE -RRB- , the covariance matrix is respectively and for the MZ and DZ pairs . Because former analyses showed that a model in which the variance allowed to change linearly during gestational age fitted best -LRB- Gielen et al. 2007 -RRB- , we allowed the variance and covariance to change over time -LRB- for example , A = eα1 + eα2Time , C = eδ1 + eδ2Time , and ln -LRB- E -RRB- = σ1 + σ2Time , where Time is gestational age , not time since conception -RRB- . First , models containing the ACE and ADE components were fitted separately . Both models were fitted with only gestational age in the mean regression -LRB- unadjusted model -RRB- and with covariates in the mean regression -LRB- adjusted model -RRB- . The best fitting unadjusted and adjusted models were tested with the -LRB- co - -RRB- variance being allowed to change during gestation . Next , the covariates of the mean regression of the adjusted model were entered as moderators in the covariance matrix the same way the mean regression of the adjusted model was built . This model is called the full model -LRB- for example , ln -LRB- E -RRB- = σ1 + σ2 Time + σ3X where σ3 is the coefficient of covariate X -RRB- , which corresponds to the method described by Shaun Purcell -LRB- Purcell 2002 -RRB- . In this way , the covariance of the first-born twin was allowed to differ from the second born twin . The full model will show how heritability , A / -LRB- A + C + E -RRB- , depends on the representative state of the covariates in the model . For example , let us assume a model in which genetic , common and unique environmental components are defined as A = e7 .403 × Time , where the intercept α1 is set to zero C = e6 .688 × Time , where the intercept δ1 is set to zero In -LRB- E -RRB- = 5.4054 + 0.1435 × Time + 0.4324 × SB + 0.724 × ND − 0.163 × MP + 0.2441 × OPM , where SB is the second born , ND is neonatal death , MP is multiparity , and OPM is one placental mass . The heritability for a first-born twin with a separate placenta , staying alive after one week of birth , of a multiparous mother , in case of two separate placentas would be at 25 weeks and at 40 weeks of gestation Model selection Models were compared with the Akaike information criterion -LRB- AIC -RRB- -LRB- Akaike 1973 ; Lindsey and Jones 1998 -RRB- . Because the modeling process is exploratory , the inference criterion used for comparing the models under consideration is their ability to fit the observed data ; i.e. models are compared directly through their minimized minus log likelihood . When the numbers of parameters in models differ , they are penalized by adding the number of estimated parameters . Smaller values indicate more preferable models . This criterion allows direct comparison among models , which are not required to be nested . Effect of each separate covariate on the genetic and environmental component To explain the effect of each covariate on the -LRB- co - -RRB- variance of birth weight at different gestational ages , we removed a significant covariate completely from the full model -LRB- out of the mean regression and out of the covariance matrix -RRB- and compared the -LRB- co - -RRB- variances , so that the influence of that specific covariate became apparent . All analyses were conducted with R -LRB- Ihaka and Gentleman 1996 -RRB- version 2.0.1 using the twin library -LRB- Lindsey 2001 -RRB- . Results Heritability decreases during gestation For both the unadjusted model with only gestational age in the mean regression , and the adjusted model with covariates in the mean regression , the ACE model with additive genetic -LRB- A -RRB- , common environmental -LRB- C -RRB- , and unique environmental -LRB- E -RRB- variances fitted best . Allowing the covariance to change over time improved the fit -LRB- Table 1 -RRB- . Table 1Model fit statistics of ACE and ADE models for the unadjusted model with only gestational age in the mean regression , the adjusted model with covariates in the mean regression , and the full model with covariates in the mean regression and in the covariance matrix . The best fitting model -LRB- lowest AIC -RRB- is presented in boldUnadjusted model Adjusted model − Log likelihooddfAIC − Log likelihoodDfAICACE61696 .84845761703.8461350.49844161373.49 ADE61727 .45845761734.4561380.6844161403.6 CE61711 .88845861717.8861364.28844261386.28 AE61727 .45845861733.4561380.58844261402.58 E62189 .72845962194.7261826.93844361847.93 ACE ; A and C allowed to change over time61688 .44845761695.4461343.61844161366.61 Full modelACE ; A and C allowed to change over time and covariates in covariance matrix61297 .26843761324.26 A : Additive genetic effects , D : Dominance genetic effects , C : Effects of common environment , E : Effects of unique environment For both the unadjusted and adjusted ACE model , the common environmental variance was higher than the genetic variance . Although the variances of the adjusted model were lower , the proportions of the standardized variances of both models remained the same , and therefore heritability -LRB- standardized A-variance -RRB- . Heritability decreased from 38 % at 25 weeks to 15 % at 42 weeks -LRB- Fig. 1 -RRB- . Allowing the covariance to change during gestation increased the genetic and common environmental variances from 25 to 42 weeks . The common environmental variance was only a little bit higher than the genetic variance , thereby increasing heritability at the end of gestation -LRB- from 36 % at 25 weeks to 18 % at 42 weeks -RRB- . The model without an intercept for the genetic -LRB- α1 -RRB- and common environmental variance -LRB- δ1 -RRB- had a better fit , because it imposes these variance components to be zero at conception -LRB- Table 2 -RRB- . Fig. 1Genetic -LRB- A -RRB- , common environmental -LRB- C -RRB- and unique environmental -LRB- E -RRB- variance and standardized variance of birth weight of the unadjusted -LRB- unadj -RRB- model and the adjusted model -LRB- adj -RRB- . E-variance was allowed to change over time , but the A - and C-variance notTable 2Regression coefficients of the covariance matrix of the unadjusted model , the adjusted model , and the full modelACLog -LRB- E -RRB- Log -LRB- estimate -RRB- SELog -LRB- estimate -RRB- SEestimateSEUnadjustedbIntercept10 .3700 0.173510.5800 0.11546.00550.3462 Gestational age -- -- 0.14050.0094 UnadjustedaIntercept0 .0000 -- 0.0000 -- 6.30610.3468 Gestational age6 .89300.16046.9420.12500.13170.0094 AdjustedbIntercept10 .28000.185710.48000.12226.14360.3495 Gestational age -- -- 0.13470.0094 AdjustedaIntercept0 .0000 -- 0.0000 -- 6.45520.3489 Gestational age6 .78300.17376.86000.12970.12560.0094 Full modelIntercept0 .0000 -- 0.0000 -- 5.40540.3963 Gestational age7 .04300.14216.68800.15710.14350.0103 Second born0 .43240.0642 Neonatal death0 .72400.1981 Multiparity − 0.16300.0491 One placental mass0 .24410.0636 Unadjusted : model with only gestational age in the mean regressionAdjusted : model with covariates in the mean regressionFull model : model with covariates in the mean regression and in the covariance matrixThe unadjusted and adjusted models are presented witha and withoutb A and C allowed changing during gestation and where Time is gestational age and X represent the covariates with the corresponding estimates β . Effect of covariates in the covariance matrix on heritability The full model with also covariates in the covariance matrix was the best fitting model -LRB- Table 1 ; for estimates of the means model see Table 3 -RRB- . The genetic variance became larger than the common environmental variance -LRB- Fig. 3 ; Table 2 -RRB- . Not all covariates of the mean regression explained part of the covariance matrix -LRB- Table 2 and Table 3 -RRB- . The genetic and environmental variance were only influenced by gestation , the unique environmental variance by parity , birth order , neonatal survival and number of placentas -LRB- estimates in Table 2 , path diagram in Fig. 2 -RRB- . Although these dichotomous covariates were only present in the unique environmental variance , heritability will nevertheless change , because heritability is the proportion of the total variance that can be explained by genetic variation.Table 3Regression coefficients of the full model with covariates in the mean regression and in the covariance matrixEstimateSEMeans modelCoefficients of the logistic growth curveb1 = saturation level at which growth stops3688 .098084.9100 b2 = growth rate5 .80610.1762 b3 = location parameter − 0.18420.0068 b4 = decrease in weight after inflection point0 .95630.3017 b5 = inflection pointa37 .59340.3216 b61 = decrease in weight for second bornb0 .10540.0021 b71 = inflection point for second borna30 .72210.8399 b62 = decrease in weight for neonatal deathb0 .57330.1846 b72 = inflection point for neonatal deatha29 .00001.098 b63 = decrease in weight for MZ MCb1 .14380.5059 b73 = inflection point for MZ MCa37 .78690.4227 CovariatesMultiparity0 .11650.0148 Male0 .14050.0118 Birth year -LRB- form 0 -LSB- 1964 -RSB- to 38 -LSB- 2002 -RSB- -RRB- 0.00180.0007 One placental mass − 0.05250.0127 Peripheral umbilical cord insertion − 0.05930.0227 Maternal age0 .00460.0014 Interaction termsBirth yeara Peripheral umbilical cord insertion − 0.00270.0009 Multiparitya Second born0 .03170.0131 aWeeks since conceptionbAfter inflection pointMZ MC = monozygotic monochorionic . Inflection point = weeks of gestational age since conception -LRB- Time -RRB- . Equation : b1 t/m b7 coefficients , Z = second born , Neonatal death or MZ MC , X = covariates , and β = coefficient of covariatesFig . 2Partial path diagram of the full model shown for one twin only . For the estimates and interactions , see table 1 the adjusted model . BWT1 = Birth weight twin 1 , Circle = latent variables , square = measured variables , triangle = definition variables in the means model , MZ MC = monozygotic monochorionic , A = additive genetic effects , C = effects of common environment , E = effects of unique environment First-born twins of multipara with two separate placentas , who stay alive neonatally , have the lowest unique environmental variance -LRB- Table 2 , Fig. 3 : Lowest -RRB- and therefore the highest heritability -LRB- from 52 % at 25 weeks to 30 % at 42 weeks -RRB- -LRB- Fig. 4 : Lowest -RRB- . Allowing a dichotomous variable to be fixed to another representative state showed that the unique environmental variance remained lower than in the adjusted model , except for twins who died neonatally -LRB- Fig. 3 -RRB- . However , heritability was always higher in the unadjusted model than in the adjusted model -LRB- Fig. 4 -RRB- . When the covariates were chosen to represent second born twins of primipara with one placental mass and who died neonatally , the unique environmental variance became extremely high -LRB- Fig. 3 : Highest -RRB- and heritability became lower than that of the adjusted model -LRB- Fig. 4 : Highest -RRB- . Fig. 3Genetic -LRB- A -RRB- , environmental -LRB- C -RRB- and residual -LRB- E -RRB- variance of birth weight of the adjusted -LRB- Adj -RRB- model and the full model -LRB- Full -RRB- . Legend : Lowest : E-variance for a first-born twin of a multipara with two separate placentas who is staying alive . Primipara : E-variance for a first-born twin of a primipara with two separate placentas who is staying alive . One placenta : E-variance for a first-born twin of a multipara with one placental mass who is staying alive . Second born : E-variance for a second born twin of a multipara with two separate placentas who is staying alive . Neonatal death -LRB- ND -RRB- : E-variance a first-born twin of a multipara with two separate placentas who died neonatally . Highest : E-variance for a second born twin of a primipara with one placental mass who died neonatallyFig . 4Heritability -LRB- standardized A-variance -RRB- of birth weight of the adjusted -LRB- Adj -RRB- and full model -LRB- Full -RRB- of Fig. 3 . Legend : Lowest : Standardized E-variance for a first-born twin of a multipara with two separate placentas who is staying alive . Primipara : Standardized E-variance for a first-born twin of a primipara with two separate placentas who is staying alive . One placenta : Standardized E-variance for a first-born twin of a multipara with one placental mass who is staying alive . Second born : Standardized E-variance for a second born twin of a multipara with two separate placentas who is staying alive . Neonatal death -LRB- ND -RRB- : Standardized E-variance a first-born twin of a multipara with two separate placentas who died neonatally . Highest : Standardized E-variance for a second born twin of a primipara with one placental mass who died neonatally Effect of each separate covariate on the genetic and environmental component Comparing the variances of the full model with and without a specific covariate showed that maternal factors -LRB- age and parity -RRB- had minor influence on the genetic variance , but no influence on heritability . Removing sex , birth order and neonatal survival gave an increase in genetic variance . However , only for sex , heritability increased if removed from the model . Removing the placental factors from the full means model resulted in a decrease of the genetic variance and a decrease of heritability . Insertion of the umbilical cord -LRB- unique for each twin -RRB- explained mainly the unique environmental variance , whereas number of placentas also explained common environment . Chorionicity or other placental factors Since chorionicity , number of placentas and insertion of the umbilical cord are related -LRB- MC twins always have one placenta and have more often a peripheral cord insertion -RRB- , as an additional step we built a model in which we replaced cord insertion and number of placentas with chorionicity -LRB- chorionicity model -RRB- . Although the AIC of the chorionicity model was higher , it roughly explained as much of the genetic and unique environmental variances but not of the common environmental variance -LRB- Fig. 5 -RRB- . Removing of either the two placental factors or chorionicity resulted in a decrease of genetic variance and decrease of heritability . The variances of the DC twins were comparable to the variances of two separate placentas , whereas variances of the MC twins were comparable to the variances of one placental mass -LRB- Fig. 5 -RRB- . Fig. 5Effect of either placental factors -LRB- site of insertion of umbilical cord and number of placentas -RRB- , or chorionicity on the variance and heritability of birth weight The curves represent the differences between the full model and the model without the specific covariate . The presented curves are the curves for the first born twin of a primipara , who is staying alive Discussion The aim of this study was to model birth weight at different gestational ages and quantify the genetic and environmental components . To our knowledge we are the first to show that heritability of birth weight seems to decrease during gestation , in our case from 38 % at 25 weeks to 15 % at 42 weeks . Entering the covariates of the mean regression as moderators in the covariance matrix , explained more of the common environmental variance than of the genetic variance . However , the unique environmental variance depended upon the representative state of the covariates and was lowest for first-born twins of multipara with two separate placentas , who stayed alive neonatally . Therefore these twins have the highest heritability : from 52 % at 25 weeks to 30 % at 42 weeks . Part of the explanation for a decrease in heritability during gestation could be the increasing demand for nutrients and oxygen in twin gestation , especially in the third trimester -LRB- Loos 2001 -RRB- . After about 29 weeks of gestation , intrauterine growth of twins deviates from growth of singletons , -LRB- Loos et al. 2005 ; Luke et al. 1993 ; Soucie et al. 2006 -RRB- , resulting in a lower birth weight and placental weight -LRB- per twin -RRB- -LRB- Gielen et al. 2006 ; Loos et al. 2005 ; Pinar et al. 1996 -RRB- . In addition , placental factors can be involved . In twin pregnancies , a lower placental weight , monochorionicity , one placental mass -LRB- one placenta or two fused -RRB- , and a peripheral cord insertion result in a lower birth weight -LRB- this study -RRB- -LRB- Gielen et al. 2007 ; Loos et al. 2001 -RRB- . Also , vascular anastomoses in MC twins result in higher discordancy rates -LRB- Machin et al. 1996 -RRB- . These placental factors can partly determine the -LRB- unequal -RRB- blood supply to the fetus and therefore the amount of nutrients and oxygen for each fetus , resulting in a higher degree of discordancy -LRB- Machin 1996 -RRB- and low heritability estimates in twins in late gestation . In this study , first-born twins of multipara with two separate placentas , who stay alive , were genetically the most homogeneous group . This group also had the highest birth weights and placental weights -LRB- Gielen et al. 2006 ; Gielen et al. 2007 -RRB- , suggesting that they may have minimal environmental restriction and therefore the highest heritability . Modeling common and unique environmental factors can increase heritability and therefore optimize finding genes responsible for the genetic variance in combined linkage and association studies . By partitioning the association effects into between twin pair and within twin pair components , true from false associations can be distinguished in combined linkage and association studies -LRB- Fulker et al. 1999 ; Posthuma et al. 2004 -RRB- . As a rule , the power in statistical analysis will increase as the sample size increases , if a quantitative trait locus -LRB- QTL -RRB- explains a larger part of the variance it is easier to find , and if multivariate models are used -LRB- Peeters 2005 -RRB- . For example , if a QTL effect explains 0.05 of the variance -LRB- α = 0.05 β = 0.80 -RRB- , with genetic factors explaining 20 % , and common and unique environmental factors each explaining 40 % of the variance , 6422 sib-pairs are needed . If genetic factors explain a larger part of the variance e.g. 40 % , and common and unique environmental factors each explaining 30 % of the variance , only 3751 sib-pairs are needed . If in the latter case the QTL effect is 0.10 even less sib-pairs -LRB- n = 1080 -RRB- are needed . Lack of statistical power may also be a reason for the inconsistent results of linkage studies to identify quantitative trait loci -LRB- QTLs -RRB- for birth weight . Only three genome wide linkage studies in different populations have identified QTLs on chromosome 6 -LRB- Arya et al. 2006 -RRB- , 7 -LRB- Fradin et al. 2006 -RRB- , and 11 -LRB- Lindsay et al. 2002 -RRB- -LRB- LOD > 3 -RRB- with relative small sample sizes -LRB- less then 413 sibships -LRB- Fradin et al. 2006 -RRB- , -LRB- Lindsay et al. 2002 -RRB- and 840 individuals from families -LRB- Arya et al. 2006 -RRB- -RRB- and only two covariates taken into account -LRB- sex and gestational age -RRB- . By building a means model and then entering the significant covariates in the covariance matrix , we controlled for environmental variables correlated with the genetic effects on the trait -LRB- gene environment correlation = rGE -RRB- -LRB- Purcell 2002 -RRB- . All the covariates with a unique environmental component , besides gestational age , must either explain differences between twins or describe gene environment interactions -LRB- Purcell 2002 ; van der Sluis et al. 2006 -RRB- . Parity is common for both twins , but because of the interaction with birth order in the mean regression , parity explains part of the unique environmental variance . Covariates also contain useful information in order to identify genes , which may be associated with the trait . In this study , only sex contained latent genetic information . For all other covariates used in this study , either heritability did not change , or environmental factors were explained with heritability depending on the representative state of the covariate . Since sex explained part of heritability , entering sex in the means model will mask genes that act differently according to sex and it is better to have these genes in the model instead of sex . In the past , Vlietinck et al -LRB- 1989 -RRB- showed an effect of chorionicity , although fusion of the placentas and site of insertion of the umbilical cord was not taken into account . Our analyses showed that only by replacing the placental factors cord insertion and number of placentas with chorionicity , the effect of chorionicity became visible , although the model fitted worse . Chorionicity certainly plays a role in growth -LRB- in mean regression -RRB- , but mainly after 40 weeks of gestation if fusion of the placentas and insertion of the umbilical cord are taken into account -LRB- Gielen et al. 2007 -RRB- . Consequently , in order to achieve a higher heritability by explaining environmental factors , number of placentas and site of the insertion of the umbilical cord are preferable above chorionicity . The strength of our study lies in the large sample size , accurate zygosity and chorionicity determination . This results in a decrease of the unique environmental variance and therefore a better estimation of heritability of birth weight . The large sample size further offers the opportunity to determine heritability at different gestational ages , although twins might not be representative for singletons due to differences in growth restriction . On the other hand , in studies without MZ and DZ twins , no difference can be made between genetic and common environmental factors , resulting in an overestimation of the genetic factors with , in case of birth weight , heritability estimates up to 80 % -LRB- Arya et al. 2006 ; Clausson et al. 2000 ; Magnus et al. 1984 ; Nance et al. 1983 -RRB- . Information on parental ethnicity was not always available . However , it has been shown that the genetic variance of birth weight does not differ between populations in which the majority -LRB- > 95 % -RRB- is Caucasian and Asian -LRB- Hur et al. 2005 -RRB- , suggesting that the genetic variance might be equal for populations . Since a vast majority of the inhabitants of East Flanders are Caucasians , we assume that our results are not biased by ethnicity . Finally , parental weight and height were not available . We assume however no biased results , since both traits are highly heritable -LRB- Carmichael and McGue 1995 ; Preece 1996 ; Silventoinen et al. 2001 -RRB- . Because the aim of linkage and association studies is to identify loci or alleles responsible for birth weight , adjusting for genetic factors will reduce the chance of finding them as stated above . But also , because we studied birth weight of twin pairs , rather than offspring of twins , the genes implicated in our study relate to fetal rather than maternal effects -LRB- Luciano et al. 2004 -RRB- . Therefore , maternal genes will be seen as common environment -LRB- Luciano et al. 2004 -RRB- . In conclusion , modeling genetic and environmental factors gives a better insight in factors influencing growth during gestation , and therefore birth weight . Heritability of birth weight changed during gestation with a decrease from 25 to 42 weeks with highest heritability for twins with the least environmental restriction -LRB- first-born twins of multipara with two separate placentas who stayed alive neonatally -RRB- . For this complex trait , modeling the covariance matrix with covariates , mainly environmental factors were explained , resulting in an increase of the heritability and subsequently the chance of finding genes by linkage and association studies .

O O O O O O O B O O O O O O O O B I O O O O B O O B I O O O O O O O B O O B I O O O O O O O O O O O O O O O O O O O O O B O B I O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B I O O O O O O O O O O O O O O O O B O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O B O O O O O O O O B I O O O O O O O B I O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B I O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O B I O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O B O B I O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O B O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O B I O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O B I O O O O O B I O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B I O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B I O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B I O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O B O B I O O O O O O O O O O O O O B O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O

birth weight
heritability
twins
gestational age


AVATAR : A database for genome-wide alternative splicing event detection using large scale ESTs and mRNAs In the past years , identification of alternative splicing -LRB- AS -RRB- variants has been gaining momentum . We developed AVATAR , a database for documenting AS using 5,469,433 human EST sequences and 26,159 human mRNA sequences . AVATAR contains 12000 alternative splicing sites identified by mapping ESTs and mRNAs with the whole human genome sequence . AVATAR also contains AS information for 6 eukaryotes . We mapped EST alignment information into a graph model where exons and introns are represented with vertices and edges , respectively . AVATAR can be queried using , -LRB- 1 -RRB- gene names , -LRB- 2 -RRB- number of identified AS events in a gene , -LRB- 3 -RRB- minimal number of ESTs supporting a splicing site , etc. as search parameters . The system provides visualized AS information for queried genes . Background Alternative splicing -LRB- AS -RRB- is an important mechanism for functional diversity in eukaryotic cells . AS allow processing of one pre-mRNA into different transcripts in a cell type . This results in protein diversity with each protein having distinct function . -LSB- 1 -- 2 -- 3 -RSB- To address this problem we used EST -LRB- short , single pass cDNA sequences generated from randomly selected library clones produced in a high throughput manner from different tissues , individuals and conditions -RRB- and mRNA sequences to detect AS variants . The detected variants -LRB- using 5,469,433 EST and 26,159 mRNA sequences -RRB- were stored in a database called AVATAR . Although , AS databases are available in the public domain , not many contain AS information for multiple eukaryotes -LRB- a comparison summarized in AVATAR web site -RRB- . Therefore , it is important to document AS information for multiple eukaryotes . Hence , we developed AVATAR containing AS information for six eukaryotes . Here , we describe AVATAR development , its content and utility . Methodology Dataset used The dbEST database -LRB- Jan 16 , 2004 -RRB- at NCBI contains nearly 5.4 million human EST sequences and this dataset is used in the current analysis . -LSB- 4 -RSB- The human genome sequences -LRB- CONTIG build 3.4 -RRB- in Genbank format is obtained from NCBI . -LSB- 5 -RSB- Gene information and mRNA sequence were downloaded from the NCBI RefSeq project . Identification of AS The identification of AS in AVATAR is performed in three steps -LRB- described below -RRB- as illustrated in Figure 1 . Step 1 : Alignment of EST and mRNA with the genome sequence EST sequences were aligned to the whole genome sequence using Mugup . -LSB- 6 -RSB- Mugup is a sequence alignment program developed in Windows platform . This procedure identified splice sites in the ESTs -LRB- Figure 1 panel A and B -RRB- . The matched regions and gaps correspond to exons and introns , respectively . EST and mRNA alignments with scores greater than 94 % were used for further analysis . Step 2 : Clustering EST and mRNA EST and mRNA were clustered according to their location in the genome -LRB- Figure 1 panel C -RRB- . EST and mRNA with overlapping regions were then assembled together . Step 3 : Detection of AS sites The mapping of EST alignment with genome sequence to intron positions helps to identify skipped exons and included exons . Searching AVATAR AVATAR can be queried using keywords . The keywords include accession number , gene name , gene isoform , gene location , cytogenetic locations , chromosome number and number of AS events . The database search produces AS visuals for queried gene . Utility to the Biological Community AVATAR is a collection of AS information for 6 eukaryotic organisms . The database can be queried simultaneously for 6 organisms . It can also be searched using gene names and desired number of AS events . EST sequences are error prone resulting in the detection of aberrant transcripts . Frequency of EST alignment at a specific site provides improved detection in AVATAR . Caveats AS information on paralogous genes in eukaryotic genomes are not included in AVATAR due to the difficulty in identifying their corresponding chromosomal locations using EST sequences . Future developments New EST sequences are generated in laboratories every day . Hence , it is a time consuming to keep AS databases updated due to the growth of genome and mRNA sequences . Hence , we are in the process of developing a computer agent which can update AVATAR automatically . We also plan to include tumor specific AS data .

O O O B O O B I O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O B O O O O O B B O O O B B O O O O O B I O O O O O O O O O O B O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O B O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O B O B B O B O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

alternative splicing
est
sequence alignment
mrna
protein diversity
database
human
eukaryotes


A Highly Active and Tolerable Neoadjuvant Regimen Combining Paclitaxel , Carboplatin , 5-FU , and Radiation Therapy in Patients with Stage II and III Esophageal Cancer Background The present phase II study aimed to assess the feasibility and efficacy of a new paclitaxel-based neoadjuvant chemoradiation regimen followed by surgery in patients with stage II -- III esophageal cancer . The prognosis of patients with esophageal cancer who undergo surgery with a curative intention is poor , with a 5-year survival rate of 5 -- 20 % .1 In recent decades , several efforts have been made to improve outcome . Improved and standardized surgical techniques and supportive care as well as concentration of care have contributed to a better outcome ; however , further improvement of survival from a single modality approach seems unlikely .2,3 Neoadjuvant chemoradiation has become the focus of interest to improve long-term survival and reduce recurrence rates . Despite a large number of phase II and several phase III trials , the role of neoadjuvant chemoradiation remains unclear . Until now , only one phase III trial demonstrated a significant survival advantage .4 All neoadjuvant regimens used in these phase III trials were cisplatin-based , which leads to frequent toxicity-related dose reductions and delays and relatively low pathologic complete response rates -LRB- 10 -- 28 % -RRB- . New neoadjuvant chemoradiation regimens including drugs such as paclitaxel have shown encouraging complete response rates -LRB- 29 -- 39 % -RRB- and R0 resection rates .5 -- 7 Since pathologic complete response and R0 resection have been shown to be associated with improved survival ,8,9 it can be expected that these new regimens lead to better survival rates . Therefore , the optimal neoadjuvant treatment regimen with high pCR rates and minimal toxicity has to be determined . Paclitaxel interferes with mitotic spindle function by enhancing the rate and yield of microtubule assembly and preventing microtubule depolymerization .10 Paclitaxel is a potent radiosensitizer because it synchronizes cells at G2/M , the phase of the cell cycle that is particularly sensitive to radiotherapy .11,12 Furthermore , paclitaxel radiosensitization persists well after the period of G2/M synchronization , suggesting that other factors may also relate to its ability to enhance radiotherapy .13 The p53 gene is required for initiation of apoptosis in response to most chemotherapeutic agents and radiotherapy . In vitro , paclitaxel-mediated blockade at the G2/M has shown to activate cell-cycle control pathways that induce apoptosis independent of p53 .14 Therefore , paclitaxel-based neoadjuvant chemoradiation seems a rational treatment approach for esophageal cancer . A recent study by Meluch et al. 6 has reported an encouraging complete response rate of 38 % with a paclitaxel-based regimen . However , toxicity was severe as only 54 % of the patients received a full dose of the neoadjuvant regimen , because of complicating esophagitis and leucopenia . The goal of this study was to assess the feasibility and efficacy of a new paclitaxel-based neoadjuvant chemoradiation protocol in which we applied a reduced chemotherapy dose compared with the protocol of Meluch et al. 6 PATIENTS AND METHODS Patients Referral to our institute was accomplished as a result of agreements between hospitals affiliated to the Comprehensive Cancer Centre South in the Netherlands to concentrate surgical care of patients with esophageal cancer . The protocol of this prospective clinical phase II trial was approved by the Medical Ethics Committee , and from all patients written informed consent was obtained . All consecutive patients with a potentially resectable stage II and III esophageal cancer who were referred to our Centre between January 2002 and November 2004 were found eligible . Further eligibility criteria included : patient age between 18 and 75 years , WHO performance status ≤ 2 , written informed consent , central venous catheter , and adequate hematological , renal , and hepatic functions defined as white blood cell count > 3.5 × 109/L , platelet count > 100 × 109/L , creatinine level < 120 μmol/L and/or creatinine clearance > 60 mL/min , and normal hepatic enzyme levels . Patients with distant metastatic disease or tumor positive supraclavicular lymph nodes were excluded from entry into the protocol . However , M1a disease in the regional lymph nodes was not . Pretreatment staging evaluation included clinical examination , upper gastrointestinal endoscopy with histological biopsies of the tumor , computed tomography -LRB- CT -RRB- scan of chest and upper abdomen , and ultrasound of the neck . Endoscopic ultrasound -LRB- EUS -RRB- was performed when a T1 tumor could not be excluded by conventional techniques . More recently , PET imaging was introduced to exclude distant metastases . Neoadjuvant Treatment The neoadjuvant regimen consisted of paclitaxel 175 mg/m2 intravenously and carboplatin AUC 5 intravenously -LRB- IV -RRB- on day 1 and 22 , and 5-FU 200 mg/m2 continuous infusion on day 1 -- 42 . All patients were treated prophylactic with dexamethasone 20 mg and a 5HT3 antagonist IV followed by ranitidine 50 mg IV and clemastine 2 mg IV . Radiotherapy was performed with a linear accelerator with a minimal photon energy of 6 MV . The total dose of 45 Gy was given in 25 fractions of 1.8 Gy once daily , starting on day 1 . The radiation fields encompassed the primary tumor and enlarged lymph nodes , if any , defined by endoscopy , CT scan , and EUS , surrounded by 5-cm proximal and distal margins and a 2-cm radial margin . Neoadjuvant treatment was given on a fully outpatient base . Dose modifications were made for a toxic reaction as defined according to the criteria of the World Health Organization -LRB- WHO -RRB- .15 Surgery Surgery was planned 6 -- 8 weeks after completion of neoadjuvant treatment . For tumors in the distal third of the esophagus , a transhiatal approach was used whenever possible . A thransthoracic Ivor-Lewis technique was used for tumors in the proximal and middle third of the esophagus . Esophagogastric continuity was reestablished using the stomach with a cervical anastomosis in all patients . A feeding jejunostomy was placed , and enteral feeding was started 24 hours after surgery . Data Collection The following data were prospectively collected : age , sex , WHO performance status , location of the tumor , histology of the tumor , comorbidity , toxicity of neoadjuvant treatment , type of resection , and postoperative course and complications . Histological Examination All pathology reports and specimens were centrally reviewed by one pathologist -LRB- GL -RRB- . Pathologic assessment was performed after standard hematoxylin-eosin -LRB- H&E -RRB- staining . The assessment included a determination of the histological type of the tumor , the depth of invasion of the tumor , whether there was nodal involvement , and the status of circumferential , proximal , and distal resection margins . The tumor stage was defined according to the classification of the American Joint Committee on Cancer .16 Patients with no residual viable tumor cells in the surgical specimen were classified as having a pathologic complete response -LRB- pCR -RRB- . In addition , response after chemoradiation of the primary tumor was classified according to the Tumor Regression Grade -LRB- TRG -RRB- as described by Mandard et al. 17 Categories according to the TRG ranged from TRG1 , complete regression , to TRG 5 with no regressive changes . TRG 1 -- 3 scores were defined as a major response to chemoradiation . Statistical Analysis The analyses were performed on the basis of intention-to-treat . Survival time was calculated as the duration from the day of start of chemoradiation therapy until death or last day of follow-up , and recurrence free interval was calculated from the day of surgery until the day of diagnosis of recurrence . Overall survival and disease-free survival were estimated using the Kaplan-Meier method . Median survival time was obtained from the time corresponding to 50 % survival based on the Kaplan-Meier survival curve . The log-rank test was used to assess survival differences . Significance was defined at a confidence level of P < .05 . Statistical analyses were performed with SPSS version 15 . RESULTS Patient Characteristics Fifty-two patients with stage II -- III esophageal carcinoma were treated in the period between January 2002 and November 2004 . Two patients were found ineligible . One patient had a history of Hodgkin 's disease and squamous cell carcinoma of the tongue . Previous treatment of this patient included external beam radiotherapy on the chest . One patient had intellectual disability -LRB- mental retardation -RRB- and was extremely anxious . Therefore , our study population consisted of 50 patients . All 50 patients signed the written informed consent and were included . Patient characteristics are detailed in Table 1 . TABLE 1 . Patient characteristicsNo . of patients n = 50Age -LRB- years -RRB- Median60 Range34 -- 75Sex Male44 Female6WHO performance status Median1 Range0 -- 2ASA classification I24 II24 III2 IV -- Tumor location Upper third2 Middle third1 Lower third47Histology Adenocarcinoma42 Squamous cell carcinoma8 Toxicity Related to Neoadjuvant Treatment Forty-two patients -LRB- 84 % -RRB- completed full neoadjuvant treatment . Three patients discontinued chemotherapy prematurely due to grade II hand-foot syndrome , four patients due to esophagitis , and one due to grade IV leucopenia . Forty-seven patients -LRB- 94 % -RRB- received at least 5 weeks chemotherapy ; the remaining three patients discontinued chemotherapy in week 4 . Full dose of radiotherapy was received by 49 patients -LRB- 98 % -RRB- . Grade III leucopenia occurred in 23 patients -LRB- 46 % -RRB- , without febrile episodes . No other hematologic toxicity was recorded . All 50 patients experienced esophagitis , usually mild -LRB- ≤ grade 2 -RRB- . A total of 13 patients -LRB- 26 % -RRB- needed nasogastric enteral feeding . Infection of the indwelling central venous catheter occurred in two patients . There were no neoadjuvant treatment related deaths . However , one patient died of myocardial infarction 3 weeks after completing neoadjuvant treatment . Postoperative Complications Surgery was performed after a median of 8.1 weeks -LRB- range 6.0 -- 17.9 weeks -RRB- . No significant difference was observed between responders -LRB- TRG 1 -- 3 -RRB- and nonresponders -LRB- TRG 4 -- 5 -RRB- with respect to the time between the end of chemoradiation and surgery . Two patients showed metastatic disease at the time of surgery ; hence , 47 patients underwent surgery with a curative intention . Transhiatal esophageal resection was performed in 44 patients ; a transthoracic Ivor-Lewis resection was performed in three patients . The postoperative course was uneventful in 23 patients -LRB- 49 % -RRB- . There were four postoperative deaths -LRB- 8.5 % -RRB- . Two patients died of necrosis of the gastric tube . One patient developed a fistula between the gastric conduit and the trachea and died of pulmonary sepsis . One patient died of a progressive chylothorax . Other postoperative complications included major anastomotic leakage -LRB- n = 5 -RRB- , pulmonary complications -LRB- n = 15 -RRB- , recurrent nerve palsy -LRB- temporary n = 4 , permanent n = 1 -RRB- , and cardiac arrhythmias -LRB- n = 3 -RRB- . An overview is outlined in Table 2 . TABLE 2 . Postoperative complicationsType of complicationNo . of patients -LRB- % -RRB- Major anastomotic leakage5 -LRB- 11 -RRB- Pulmonary15 -LRB- 32 -RRB- Pneumonia7 Emphysema4 ARDS1 Mediastinal infection1 Trachea-esoph fistula1 Chylothorax1Recurrent nerve palsy Permanent1 -LRB- 2 -RRB- Temporary4 -LRB- 9 -RRB- Cardiac arrhythmias3 -LRB- 6 -RRB- Wound infection2 -LRB- 4 -RRB- Diaphragmatic hernia1 -LRB- 2 -RRB- Hospital stay -LRB- days -RRB- Median15 Range9 -- 83ICU/MCU stay -LRB- days -RRB- Median2 Range2 -- 18Ventilation time -LRB- days -RRB- Median1Range0 -- 9 Pathologic Response to Neoadjuvant Treatment Pathologic complete regression in the primary tumor was achieved in 18 of 47 operated patients -LRB- 38.3 % -RRB- . However , in two patients the primary tumor showed complete response , but microscopic residual tumor was detected in locoregional lymph nodes . The ypTNM stages of the remaining patients were : pT1N0M0 in six patients -LRB- 12.8 % -RRB- , pT2-3N0M0 in 12 patients -LRB- 25.5 % -RRB- , pT1-2N1M0 in four patients -LRB- 8.5 % -RRB- , and pT3N1M0/pT4N0 -1 M0 in seven patients -LRB- 14.9 % -RRB- . Subsequent postoperative tumor stages are outlined in Table 3 . Postoperative tumor regression grades -LRB- TRG -RRB- scored by an independent pathologist are outlined in Table 4 . Eighteen patients -LRB- 38.3 % -RRB- showed a complete tumor regression -LRB- TRG1 -RRB- , and 38 patients -LRB- 80.9 % -RRB- displayed a major response -LRB- TRG1-3 -RRB- after neoadjuvant treatment . Two patients had positive margin involvement of the resected specimen ; therefore , a R0 resection was achieved in 45 of 47 patients -LRB- 95.7 % -RRB- . TABLE 3 . Pathological stage of patients treated with neoadjuvant chemoradiation and surgeryStage , ypTNMNo . of patients -LRB- % -RRB- 018 -LRB- 38.3 -RRB- I6 -LRB- 12.8 -RRB- II a12 -LRB- 25.5 -RRB- II b4 -LRB- 8.5 -RRB- III7 -LRB- 14.9 -RRB- TABLE 4 . Tumor regression grade -LRB- TRG -RRB- of patients treated with neoadjuvant chemoradiation and surgeryTRGaNo . of patients -LRB- % -RRB- 1 -- Absence of histologically identifiable residual cancer and fibrosis extending through the different layers of the esophageal wall18 -LRB- 38.3 -RRB- 2 -- Presence of rare residual cancer cells scattered through the fibrosis11 -LRB- 23.4 -RRB- 3 -- Increase in the number of residual cancer cells , but fibrosis still predominated9 -LRB- 19.1 -RRB- 4 -- Residual cancer outgrowing fibrosis7 -LRB- 14.9 -RRB- 5 -- Absence of regressive changes2 -LRB- 4.3 -RRB- a TRG according to Mandard et al. 17 Survival and Pattern of Failure All 50 patients were included in the survival analysis . The median follow-up for surviving patients was 41.5 months -LRB- range 21 -- 59 months -RRB- . The median overall survival and disease-free survival were 44 and 50 months , respectively . The estimated 3 - and 5-year overall survival rates were 55.6 and 47.8 % , respectively , whereas the estimated 3 - and 5-year disease-free survival rates were 52.2 and 41.8 % , respectively . The Kaplan-Meier curves for overall and disease-free survival are shown in Fig. 1 and 2 . FIG. 1.Kaplan-Meier curve of overall survival of all patients -LRB- n = 50 -RRB- . FIG. 2.Kaplan-Meier curve of disease-free survival -LRB- n = 50 -RRB- . Patients with TRG 1 -- 3 showed a significant improved overall and disease-free survival compared with patients with TRG 4 -- 5 . Median survival time in TRG 1 -- 3 patients was not yet reached and 23 months in patients with TRG 4 -- 5 . The estimated 3 - and 5-year overall survival rates were 61.0 and 55.9 % for the patients with a TRG 1 -- 3 and 33.3 and 16.7 % for the patients with a TRG 4 -- 5 . Median disease-free survival in TRG 1 -- 3 patients was not yet reached and 11 months in patients with TRG 4 -- 5 . The estimated 3-year disease-free survival rates for patients with TRG 1 -- 3 was 58.6 vs 25.0 % for patients with TRG 4 -- 5 . Comparing the TRG 1 -- 3 patients with TRG 4 -- 5 patients , the log-rank test of equality of the survival curves generated a P value of .03 for overall survival and .02 for disease-free survival time in favor of patients with TRG1 -- 3 . The Kaplan-Meier curve for disease-free survival according to tumor regression grade is shown in Fig. 3 . FIG. 3.Kaplan-Meier curve of disease-free survival by tumor regression grade -LRB- TRG -RRB- . TRG1 -- 3 -LRB- n = 38 -RRB- : major response , TRG 4 -- 5 -LRB- n = 9 -RRB- : minor or no response -LRB- P = .0183 -RRB- . Recurrent disease was found in 20 patients surviving postoperatively -LRB- 20 of 43 , 47 % -RRB- Five of them were still alive at the time of the analysis . Recurrence was only locoregional in three patients . Distant metastases occurred in 16 patients , and in one patient recurrence presented with locoregional and pulmonary metastases . To date 22 patients have died . Of these 22 patients , three died from nondisease-related causes and were presumed disease free . Of the 19 patients who had disease-related deaths , four patients died of postoperative complications , and three patients died of complications from local recurrence , whereas 12 patients died due to systemic failure that included metastases to lung , liver , brain , and bone . DISCUSSION This study shows the results of a phase II efficacy study , with a modified chemoradiation regimen according to Meluch et al. 6 A reduction of the dose of chemotherapy components of this regimen resulted in a decrease of toxicity with preservation of its high efficacy rate . The pathologic complete tumor regression rate of 38.3 % and R0 resection rate of 95.7 % are among the highest described in the literature . Toxicity and morbidity of this regimen compared with the original scheme were mild and acceptable . Numerous phase II trials using neoadjuvant chemoradiation showed marked downstaging and suggested survival benefits . In most trials , chemotherapy regimens included cisplatin and 5-FU . However , randomized controlled trials -LRB- RCT -RRB- comparing chemoradiation followed by surgery with surgery alone have yielded inconclusive results .4,18 -- 22 One of these trials showed significant improvement in survival in the patients who received combined-modality therapy .4 The relevance of this study was disputed because the 3-year survival rate of 6 % in the control arm was exceptionally low . Two additional trials showed a possible benefit of neoadjuvant chemoradiation without reaching a statistical significance .19,22 Furthermore , the power of these trials to detect modest but clinically relevant differences was limited by small patient numbers . In a meta-analysis report by Fiorica et al. 23 six RCTs were pooled comparing neoadjuvant chemoradiation and surgery versus surgery alone . A significant difference in 3-year mortality favoring neoadjuvant chemoradiation with surgery versus surgery alone was detected -LRB- OR 0.53 ; 95 % CI 0.31 -- 0.93 ; P = .03 NNT = 10 -RRB- . It was concluded that neoadjuvant chemoradiation and surgery significantly improved 3-year survival compared with surgery alone in patients with resectable esophageal cancer . However , a careful interpretation of these results is warranted , because this meta-analysis was based on summarized patient data . Neoadjuvant chemoradiation induces acute treatment-related toxicity . These effects include myelotoxicity , esophagitis , nausea , and vomiting . The regimen used in this phase II trial produced moderate toxicity and is comparable with reported literature on neoadjuvant chemoradiation regimens including paclitaxel .5 -- 7,24,25 The highly effective regimen used by Meluch et al. 6 was modified to reduce toxicity . The modification consisted of dose reduction of 5-FU 200 mg/m2 instead of 225 mg/m2 , paclitaxel 175 mg/m2 instead of 200 mg/m2 and carboplatin AUC 5.0 instead of AUC 6.0 . This modification resulted in a reduction of toxicity . Forty-two patients -LRB- 84 % -RRB- received full doses of the planned 6 weeks chemotherapy , whereas in the group of Meluch et al. only 54 % of patients received full dose chemotherapy . A relative large proportion of the patients -LRB- 26 % -RRB- received preoperative nasogastric enteral feeding . This was partly due to our liberal use of nasogastric enteral feeding , since in our experience optimizing preoperative nutritional status in patients treated with a neoadjuvant chemoradiaton protocol is critical . The neoadjuvant treatment was delivered on a fully outpatient basis . Further improvement of our regimen may be obtained by replacing 5-FU for novel oral fluoropyrimidines . In colorectal cancer , the novel fluoropyrimidines such as capecitabine and UFT show equal if not superior efficacy and improved patient convenience . The reported postoperative mortality in the literature for neoadjuvant chemoradiation followed by surgery ranges from 0 to 29 % -LRB- median 9 % -RRB- . A nationwide study in the United States demonstrated in-hospital mortality rates of high-volume versus low-volume centers of 8.4 vs 20.3 % .2 A comparative Dutch study found in-hospital mortality rates of 4.9 vs 12.1 % .26 Therefore , the in-hospital mortality rate of 8.5 % in our trial is within these boundaries . Several studies reported an increase in postoperative mortality after neoadjuvant chemoradiation . The aforementioned recent meta-analysis of six randomized trials confirmed a significant effect of chemoradiation on postoperative mortality -LRB- OR 2.10 95 % CI 1.18 -- 3.73 , P < .01 , NNT = 25 -RRB- .23 A relatively short interval between completion of neoadjuvant therapy and surgery of 2 -- 4 weeks is noted in these trials and might be of influence in the relative high mortality rates described in these trials . We decided to perform surgery after at least 6 weeks to achieve an adequate downstaging and permit maximal recovery ; in addition , surgery was postponed in some patients due to a slow recovery from the chemoradiation . We believe that the timing of surgery is important . Performing an esophagectomy too soon after neoadjuvant chemoradiation may lead to increased morbidity due to prolonged myelosuppression and hence an increased risk for developing postoperative complications . Until recently , neoadjuvant chemoradiation studies have been performed using cisplatin/5-FU based regimens with a variation in dose and a variation in radiotherapy schemes . Moderate complete response rates of 8 -- 26 % and R0 resection rates of 55 -- 84 % have been described in these studies .4,19,20,22,27 -- 29 With the implementation of regimens including paclitaxel , 5-FU , and cisplatin , complete response rates of 32 -- 38 % and R0 resection rates of 78 -- 95 % have been documented .6,7,25,30 Our study reports a complete pathologic response of 38.3 % , which is high . Furthermore , a R0 resection rate of 95.7 % is among the highest described in the literature . The dismal prognosis of esophageal cancer has discouraged many clinicians . For many patients , the prognosis was supposed to be dependent on the presence of indolent distant metastasis despite the type of operative treatment given . In a recent phase III trial , however , extended radical transthoracic esophagectomy showed a trend to a better survival when compared with a less radical transhiatal esophagectomy , indicating that an adequate locoregional treatment may have a beneficial effect on survival .3 However , in both groups a R0 resection rate of only 72 % was noted . This observation is an important rationale for neoadjuvant combined multimodality treatment . One of the goals of neoadjuvant chemoradiation therapy is increasing curative R0 resection rates by downsizing the tumor and theoretically eradicating micrometastasis . Achieving R0 resection in locally advanced -LRB- stage II -- III -RRB- esophageal cancer has been identified as a prognostic factor .8,9 Furthermore , complete pathologic response has been associated with an improved survival and local control .30,31 In the present study a major response , tumor regression grade 1 -- 3 , resulted in a significant increase in overall and disease-free survival . Since the time between surgery and the end of the chemoradiation did not significantly differ between the responders and nonresponders , a difference in response could not be explained by a lag-time difference . Thus , a regimen with high complete pathologic response and R0-resection rates will be very likely to improve survival . However , with our relatively short follow-up we can not report definitive long-term survival data yet . In conclusion , although the discussion regarding the value of neoadjuvant therapy is ongoing , the neoadjuvant treatment regimens used for the last 20 years produced only modest benefit , at best . Improving chemoradiation regimens as in our study , by incorporating modern chemotherapeutics such as paclitaxel in combination with modern 3-D conformal radiotherapy seems to be promising . Further improvement of the systemic therapy seems to be critical as survival is mainly determined by recurrences at metastatic sites . Data of ongoing phase III trials including novel chemoradiation protocols have to be awaited in the near future .

O O O O O O O O B O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O B I O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O B I O O O O O B O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O 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O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

esophageal cancer
neoadjuvant chemoradiation
paclitaxel
pathologic complete response
tumor regression grade