MD, PhD Jordi Alonso
email: jalonso@imim.es

PhD Masha Petukhova
email: petukhova@hcp.med.harvard.edu

PhD Gemma Vilagut

MD Somnath Chatterji
email: chatterjis@who.int

PhD Steven Heeringa
email: sheering@isr.umich.edu

T Berdirhan Üstün

FICMS Ali Obaid Alhamzawi
email: ali_alhamzawi2000@yahoo.com

MD, PhD Laura Helena Andrade

MD Matthias Angermeyer
email: angermeyer@aon.at

PhD Evelyn Bromet
email: ebromet@notes.cc.sunysb.edu

PhD T Bedirhan Üstün

PhD Ronny Bruffaerts
email: ronny.bruffaerts@uz.kuleuven.ac.be

MD Giovanni De Girolamo
email: gdegirolamo@fatebenefratelli.it

MD, PhD Silvia Florescu
email: florescu.silvia@gmail.com

MD Oye Gureje
email: ogureje@comui.edu.ng

MD, MPH, PhD Josep Maria Haro
email: jmharo@sjd-ssm.com

MD Hristo Hinkov

MD, PhD Chi-Yi Hu
email: chiyihu@yahoo.com.cn

MD Elie G Karam
email: egkaram@idraac.org

MD, PhD Viviane Kovess
email: viviane.kovess@ehesp.fr

PhD Daphna Levinson
email: daphna.levinson@moh.health.gov.il

Maria Elena

PhD Medina Mora
email: medinam@imp.edu.mx

MD, MPH, FFPH Yosikazu Nakamura
email: nakamuyk@jichi.ac.jp

PhD Johan Ormel
email: j.ormel@med.umcg.nl

MD Jose Posada-Villa

MD Rajesh Sagar
email: rajeshsagar@rediffmail.com

PhD Kate M Scott
email: kate.scott@otago.ac.nz

Adley Tsang

MPH, PhD David R Williams
email: dwilliam@hsph.harvard.edu

PhD Ronald C Kessler
email: kessler@hcp.med.harvard.edu

Days Out of Role due to Common Physical and Mental Conditions: Results from the WHO World Mental Health Surveys

Keywords: Mental Disorders, Chronic Disease, Disability, Productivity loss, Prevalence, Burden of Disease

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

Introduction

A growing body of research aims to quantify the societal impacts of physical and mental disorders on functioning in order to influence social policy decisions about investing in healthcare. [START_REF] Acoem | Healthy workforce/healthy economy: the role of health, productivity, and disability management in addressing the nation's health care crisis: why an emphasis on the health of the workforce is vital to the health of the economy[END_REF][START_REF] Davis | Health and productivity among U.S. workers[END_REF][START_REF] Suhrcke | Economic costs of ill health in the European Region[END_REF][START_REF] Loeppke | Health and productivity as a business strategy: a multiemployer study[END_REF] One important component of this research examines effects of health problems on days out of role. For example, these studies have estimated that there are 3.6 billion annual health-related days out of role in the US [START_REF] Merikangas | The impact of comorbidity of mental and physical conditions on role disability in the US adult household population[END_REF] and that the annual human capital costs of health-related days out of role in Europe exceed 136 billion Euros. [START_REF] Andlin-Sobocki | Cost of disorders of the brain in Europe[END_REF] It is also important to quantify the relative importance of specific disorders in accounting for these effects and to evaluate the extent to which expanded outreach and best-practices treatment of the disorders associated with the largest losses reduce these effects. The first step in such a program of research should be to distinguish relative effects of specific disorders. This requires epidemiological data on a broad range of disorders so as to adjust for the high rates of comorbidity within and between physical and mental disorders. [START_REF] Korff | Global perspectives on mental-physical comorbidity[END_REF][START_REF] Stang | Mental and physical comorbid conditions and days in role among persons with arthritis[END_REF] While some studies of this sort have been carried out in the US, [START_REF] Merikangas | The impact of comorbidity of mental and physical conditions on role disability in the US adult household population[END_REF][START_REF] Stang | Mental and physical comorbid conditions and days in role among persons with arthritis[END_REF] we are not aware of any comparable studies in most other parts of the world.

The current report presents data of this sort from the World Health Organization (WHO) World Mental Health (WMH) Surveys (www.hcp.med.harvard.edu/WMH). The WMH initiative was launched by the WHO to carry out general population surveys in countries throughout the world to assess the prevalence and correlates of mental disorders. [START_REF] Kessler | The WHO World Mental Health Surveys: global perspectives on the epidemiology of mental disorders[END_REF] One section in the WMH interviews assesses the presence of commonly occurring chronic physical disorders in order to facilitate analysis of the comparative effects of mental and physical disorders on role functioning. WMH data from 24 countries are used in this report to examine these relative effects on days out of role in the month before interview. A novel method is used to estimate these effects in such a way as to adjust for comorbidity.

Methods

Samples

A total of 25 WMH surveys were carried out in 24 countries. (Table 1) Six of these countries are classified in 2007 by the World Bank 10 as low or lower-middle income countries (Colombia, India, Iraq, Nigeria, Peoples' Republic of China [PRC], Ukraine), six as upper-middle income countries (Brazil, Bulgaria, Lebanon, Mexico, Romania, South Africa), and twelve as higher income countries (Belgium, France, Germany, Israel, Italy, Japan, Netherlands, New Zealand, Northern Ireland, Portugal, Spain, and the United States). All surveys were based on probability samples of the adult household population of the participating countries. The samples were selected either to be nationally representative (in the majority of countries), representative of all urbanized areas in the country (Colombia, Mexico), or representative of particular regions of the country (Brazil, India, Japan, Nigeria, PRC). More details about sampling are provided elsewhere. 11 The weighted (by sample size) average response rate across surveys was 72.0%, ranging from 45.9% (France) to 95.2% (Iraq).

(Table 1 about here)

All WMH interviews were administered face-to-face by trained lay interviewers using training, and field quality control procedures described elsewhere. [START_REF] Kessler | The WHO World Mental Health Surveys: global perspectives on the epidemiology of mental disorders[END_REF][START_REF] Pennell | Implementation of the World Mental Health Surveys[END_REF][START_REF] Harkness | Translation Procedures and Translation Assessment in the World Mental Health Survey Initiative[END_REF] Informed consent was obtained before beginning interviews using procedures approved by the Institutional Review Board of the organization coordinating the survey in each

Measurement

Mental disorders: Mental disorders were assessed with Version 3.0 of the WHO Composite International Diagnostic Interview (CIDI), a fully structured lay-administered interview designed to generate research diagnoses of commonly mental disorders according to the definitions and criteria of both the DSM-IV and ICD-10 diagnostic systems. [START_REF] Kessler | The World Mental Health (WMH) Survey Initiative Version of the World Health Organization (WHO) Composite International Diagnostic Interview (CIDI)[END_REF] The nine mental disorders considered here include mood disorders (major depressive disorder, bipolar I-II disorder), anxiety disorders (panic disorder and/or agoraphobia, specific phobia, social phobia, generalized anxiety disorder, post-traumatic stress disorder) and substance disorders (alcohol abuse with and without dependence, drug abuse with and without dependence). Only disorders present in the 12 months before interview are considered. A clinical reappraisal study with blinded clinical follow-up interviews using the Structured Clinical Interview for DSM-IV (SCID) [START_REF] First | Structured Clinical Interview for DSM-IV Axis I Disorders, Research Version, Non-patient Edition (SCID-I/NP)[END_REF] in several WMH surveys (France, Italy, Spain, US) found generally good concordance between diagnoses based on the CIDI and those based on the SCID. [START_REF] Haro | Concordance of the Composite International Diagnostic Interview Version 3.0 (CIDI 3.0) with standardized clinical assessments in the WHO World Mental Health surveys[END_REF] Chronic physical disorders: Physical disorders were assessed with a standard chronic disorders checklist.

Checklists of this sort have been shown to yield more complete and accurate reports of disorder prevalence than estimates derived from responses to open-ended question. 17,[START_REF] Schoenborn | Summary health statistics for the U.S. population: National Health Interview Survey[END_REF] Reports based on such checklists have been shown in previous methodological studies to have moderate to good concordance with medical records. [START_REF] Knight | Estimating health needs: the impact of a checklist of conditions and quality of life measurement on health information derived from community surveys[END_REF][START_REF] Baker | What Do Self-reported, Objective Measures of Health Measure? National Bureau of Economic Research[END_REF] The ten conditions considered here are: arthritis, cancer, cardiovascular disorders (heart attack, heart disease, hypertension, stroke), chronic pain conditions (chronic back or neck pain, other chronic pain), diabetes, migraines or other frequent or severe headaches, insomnia, neurological disorders (multiple sclerosis, Parkinson's, epilepsy or seizures), digestive disorders (stomach or intestinal ulcers, irritable bowel disorder), and respiratory disorders (seasonal allergies, asthma, COPD, emphysema). The symptom-based disorders in this set (arthritis, pain disorders, heart attack, stroke) were assessed with respondents reports as to whether or not they ever experienced the disorder, while the remaining conditions were assessed with respondent reports of whether or not a doctor or other health professional ever told them they had the disorder. Questions about persistence were also asked about lifetime disorders that can remit. The focus in this report is on disorders present in the 12 months before interview.

Days out of role:

A modified version [START_REF] Korff | Modified WHODAS-II provides valid measure of global disability but filter items increased skewness[END_REF] of the WHO Disability Assessment Schedule (WHO-DAS) 22,[START_REF] Vazquez-Barquero | Versión en lengua española de un nuevo cuestionario de evaluación de discapacidades de la OMS (WHO-DAS-II): fase inicial de desarrollo y estudio piloto[END_REF] was used to ask respondents the number of days in the 30 days before interview (i.e., beginning yesterday and going back 30 days) they were totally unable to work or carry out your normal activities because of problems with either your physical health, your mental health, or your use of alcohol or drugs. Good concordance of these reports have been documented both with payroll records of employed people [START_REF] Kessler | Comorbid mental disorders account for the role impairment of commonly occurring chronic physical disorders: results from the National Comorbidity Survey[END_REF][START_REF] Revicki | The accuracy of self-reported disability days[END_REF] and with prospective daily diary reports. [START_REF] Kessler | Using the World Health Organization Health and Work Performance Questionnaire (HPQ) to evaluate the indirect workplace costs of illness[END_REF]

Statistical analysis

Multiple regression analysis was used to examine multivariate associations of the physical and mental disorders assessed in the survey with reported on days out of role in the past 30 days controlling for age, gender, employment status, and country. As substantial comorbidity was found among the disorders, [START_REF] Gureje | The pattern and nature of mental-physical comorbidity: specific or general?[END_REF] we included terms to capture the effects of comorbidity in the regression models. Given that the number of possible combinations of comorbid disorders in the data (2 [START_REF] Knight | Estimating health needs: the impact of a checklist of conditions and quality of life measurement on health information derived from community surveys[END_REF] -20 = 524,268) far exceeds the number of respondents, it was necessary to impose some structure on the terms used to capture the effects of comorbidity. This was done by including terms for total number of comorbid disorders and separate interaction terms for the extent to which the regression coefficient of each separate disorder changed as a function of number of comorbid disorders taking into account the size of the coefficients associated with the comorbid disorders. Nonlinear regression methods requiring iterative estimation procedures [START_REF] Gaf Seber | Nonlinear Regression[END_REF] were used for this purpose.

As the outcome variable (a 0-30 measure of number of days out of role) was highly skewed, we investigated a number of different model specifications that included an ordinary least squares regression model and six generalized linear models (GLM) that considered the conjunction of two link functions (logarithmic or square root) and three error structures (constant, error variance proportional to the mean and error variance proportional to the mean squared). Standard diagnostic procedures to compare model fit [START_REF] Buntin | Too much ado about two-part models and transformation? Comparing methods of modeling Medicare expenditures[END_REF] showed that the OLS model and GLM with a log link function and constant variance were the two best-fitting models. (Detailed results of model comparison are available under request).

As the prediction equation includes interaction terms, the predictive effect of each disorder is distributed across a number of different coefficients. Simulation was used to produce a single term to summarize all these components effects. This was done by estimating the predicted value of the outcome for each respondent from the coefficients in the final model (the base estimate) and then repeating this exercise in modified form 19 different times, each time assuming that one of the 19 disorders no longer existed. The difference between the predicted mean of the outcome generated by the simulated estimate and the base estimate was divided by the number of respondents with the disorder in question to obtain the estimated individual-level effect of the disorder on the outcomes. The estimated societal-level effect of the disorder was then obtained by multiplying the individual-level estimate by the prevalence of the disorder. The same procedure was used to calculate total effects of any physical disorder, any mental disorder, and any disorder.

The fact that the WMH survey data are geographically clustered and weighted means that design-based methods were needed to obtain accurate estimates of standard errors and statistical significance. The Taylor series linearization method [START_REF] Wolter | Introduction to variance estimation[END_REF] implemented in SAS [START_REF]SAS/STAT® software, version 9.1 for Windows[END_REF] was used to do this for the basic model. The more computationally intensive method of Jack-knife Repeated Replications 30 implemented in a SAS macro that we wrote for this purpose was used to obtain standard errors of the simulated estimates of individual-level and societal-level disorder effects. Significance tests were consistently evaluated using .05-level, two-sided designbased tests.

More information about the methods used in this paper can be obtained from the authors on request.

Results

The distribution of days out of role

The mean number of health-related days out of role in the previous month was 1.2 in the total sample, with a range of 1.0-1.4 across countries in the three income levels. (Table 3) The overall mean can be decomposed into 12.8% of respondents who reported any days out of role (14.6% in lower, 10.4% in middle, and 13.1% in higher income countries) and a mean among those with any of 9.6 days (8.3 in lower, 9.3 in middle, and 10.3 in higher income countries). The distribution is skewed to the left, as indicated by the median among those with any days out of role, 4.2 in the total sample, (4.0 in lower, 4.3 in middle, and 4.3 in higher income countries) being lower than the mean.

(Table 3 about here)

Disorder prevalence estimates

More than half (58.0%) of respondents across countries reported one or more of the disorders considered here.

The proportion that reported at least one physical disorder (53.2%) is considerably higher than the proportion that reported any mental disorder (15.4%). Prevalence of having any physical disorder and any mental disorder both increase monotonically with country income level: from 45.6% in low/lower-middle, to 52.2% in uppermiddle, and 57.4% in high income countries for any physical disorder; and from 12.1% in low/lower-middle, to 15.4% for upper-middle, and 17.0% in high income countries for any mental disorder. This monotonic increase exists for 11 of the 19 individual disorders, with five others either having their lowest prevalence in low/lowermiddle income countries or having their highest prevalence in high income countries. The three exceptions are headaches and other chronic pain disorders, both of which have their highest prevalence in upper-middle income countries, and digestive disorders, with highest prevalence in low/lower-middle income countries.

(Table 4 about here)

The rank order of prevalence estimates across disorders is very similar in the three groups of countries, with rank-order correlations across groups in the range .87-.97. Chronic pain disorders are among the two most commonly reported disorders in all three groups of countries (21.6-23.7%), headaches among the top two in low/lower-middle and upper-middle income countries (14.5-19.4%), and cardiovascular disorders among the top two in upper-middle and high income countries (18.2-18.8%). Major depression is the most commonly reported mental disorder in all three groups of countries (4.9-6.2%) followed by specific phobia (4.2-6.0%). Other highly prevalent disorders in all groups of countries are arthritis (12.5-16.6%) and respiratory disorders (11.9-21.9%).

Respondents who reported any disorder had an average of 2.1 disorders. Comorbidity was the norm, with 55.9% of the respondents with a disorder reporting at least two. Odds-ratios (ORs) between pairs of disorders are largely positive (94.1% of all the 19x18/2 = 171 odds-ratios between pairs of disorders) and statistically significant (90.0% of all odds-ratios). The ORs are higher (median and inter-quartile range [IQR]) among pairs of physical (2.5, 1.9-3.0) and mental (7.2, 4.0-8.7) disorders than between physical-mental pairs (2.0, 1.4-2.6).

Similar patterns exist in each of the three groups of countries. (Detailed results of all ORs are available on request.) Mean days out of role per year vary by condition (Table 4). Individuals with panic (42.9), PTSD (42.7), and bipolar disorder (41.2) had the highest mean numbers of days out of role. Trends were similar across type of countries. Correlations between mean days out of role across conditions are high in each of the three income groups (Spearman rank-order correlations in the range .62 to .77). PTSD, panic, and GAD are among the top 6 conditions with highest mean days out of role in all 3 income groups. Individuals with any disorder had an average of 24.2 more days out of role in a year (31.1 days those with any mental, 24.5 those with any physical) than those with no conditions.

Table 5 shows the additional days totally out of role in a year among respondents with a disorder (individuallevel effect) adjusting by age, gender, marital status and employment, as well as the number and type of comorbid disorders. In lower income countries, bipolar disorder (36.5 additional days), neurological disorders (33.7), and panic (24.3) were the most disabling conditions. While in middle income countries were GAD (24.6 additional days), bipolar (23.4), neurological (18.6) and panic (17.6). And in higher income countries were pain (19.6 additional days), digestive (16.6), and PTSD (16.2). Rank correlations of individual effects of the conditions were low across country type (from .12 to .26). Interactions were found to be sub-additive for most disorders in all three income groups: the incremental increase in days out of role is smaller when a disorder occurs comorbidly compared to when the same disorder occurs in isolation (results are not shown but available on request). Also in table 5, the population attributable risk proportion (PARP) of days totally out of role for each condition is presented. Pain disorders contribute the highest proportion (21.5% on average), followed by headache/migraine (6.9%), cardiovascular (6.3%) and depression (5.1%). For each condition, PARPs tended to be fairly similar across country types, especially between lower and middle income countries (Spearman correlations 0.32).

(Table 5 about here)

Individual effects and PARPs for all countries combined are presented in figure 1. The figure clearly shows that the most impacting disorders according to their individual-level effect --most notably neurological, bipolar and pain disorders--(Figure 1A) are quite different from those that have the most impact at the societal level --pain, cardiovascular and depression (Figure 1B).

(Figure 1, about here)

Discussion

A number of limitations must be considered before interpreting our results. First, only a restricted set of the most common conditions was included in the analysis and some were pooled to form larger disorder groups. Some burdensome conditions, such as dementia and psychosis, were not included. While the conditions we did consider include are amongst those most commonly reported in previous population studies, [START_REF] Merikangas | The impact of comorbidity of mental and physical conditions on role disability in the US adult household population[END_REF] an expansion and disaggregation of these conditions is clearly needed in future studies. Secondly, diagnoses of chronic physical conditions were based on self reports. Prior research has demonstrated reasonable correspondence between selfreported chronic conditions such as diabetes, heart disease and asthma, and general practitioner records, [START_REF] Kriegsman | Self-reports and general practitioner information on the presence of chronic diseases in community dwelling elderly. A study on the accuracy of patients' self-reports and on determinants of inaccuracy[END_REF] but some bias might account for the generally higher prevalence estimates of these conditions in developed than developing countries. Likely, higher rates of medical treatment and detection in developed countries artificially inflate prevalence country differences. Thirdly, we only considered the days out of role that the respondents reported they were totally unable to do their work or usual activities. It is common that individuals perform their role activities less or worse than expected (e.g., presenteeism), [START_REF] Sanderson | Common mental disorders in the workforce: recent findings from descriptive and social epidemiology[END_REF] therefore information about days out of role underestimates total productivity loss. Finally, to increase validity of self-reporting, we assessed restriction of activities in the 30 days previous to the interview and then projected the numbers in this recall interval to the a whole year, improving the comparability with published literature [START_REF] Merikangas | The impact of comorbidity of mental and physical conditions on role disability in the US adult household population[END_REF] . But some mismatch between severity of the disease and its prevalence may exist: for more episodic conditions this recall period might have missed a severe exacerbation present in the previous year but not in the month before the interview. Due to the relative large number of events assessed, we expect this to cancel out with the opposite situation and overall not affect our estimates.

Within the context of these limitations, we identified a number of disorders that cause a great deal of disability.

For the overall sample (table 4), sufferers of bipolar disorder, PTSD and panic have the highest disability, followed closely by those with GAD and social phobia. These results show that mental disorders are among the most strongly associated with productivity loss. This finding is broadly consistent with previous studies. [START_REF] Polder | Cost of illness in the Netherlands[END_REF] Nevertheless, as comorbidity is so frequent, and especially so for mental disorders, the observed associations in previous studies, which failed to adjust adequately for comorbidity, could have been due not to the particular conditions themselves, but to their high comorbidity. This possibility was addressed in the analysis reported here by controlling for comorbidity. In Table 5 (individual effect) we observe that the rank ordering of disorders in terms of relative impacts on days out of role change when we adjust for comorbidity. It is consequently important to consider comorbidity in assessing the relative effects of particular disorderson days out of role.

Our results show that the 19 health conditions considered in our study account for almost two-thirds of all days of role in the general populations of the countries studied (PARP= 62.2%). Physical conditions accounted for 47.6% and mental disorders for 16.5%. It should be noted that the physical conditions include neurological disorders (epilepsy, multiple sclerosis and stroke), pain conditions (including severe headache/migraine) and insomnia. If insomnia was considered a mental disorder instead as a physical disorder, mental disorders would account for up to 20.3% of days out of role (data not shown, but available from the authors). Pain conditions are disabling and highly prevalent and they are, by far, the most important contributor to days totally out of role in the population. Also cardiovascular disorders, depression and migraine are major contributors to populationlevel days out of role. These conditions should be prioritized to improve the productivity of our societies.

Nonetheless, while PARPs indicate the theoretical proportion of outcome events that could be avoided if the exposure (the disorders in our study) was completely eliminated and are useful for identifying the burdensome targets for population intervention, it should be borne in mind that disability days avoided by removing one disorder might limit the opportunity of avoiding the same days by removing another condition. [START_REF] Steenland | An overview of methods for calculating the burden of disease due to specific risk factors[END_REF] With a few exceptions, results presented here are similar across the type of countries studied. We had anticipated that cultural, social and economic differences could modulate the association of disorders with days out of role. First, differences across type of countries in the average proportion of individuals reporting days totally unable to carry out normal activities were small. The mean number of yearly days among those with those with days out of role varied more across type of country. These differences might be due to a higher prevalence of disorders but also to a more developed welfare system in these countries. Some country differences nevertheless deserve further research.

Implications.

A first implication is the identification of the relative contribution of different common disorders to the population loss of productivity. Lowering the impact of common and disabling conditions such as pain, migraine, as well as cardiovascular and depression would have a major productivity returns. If we take into account that indirect costs are usually higher than direct medical and social services costs to care for disorders, [START_REF] Andlin-Sobocki | Cost of disorders of the brain in Europe[END_REF][START_REF] Andlin-Sobocki | Cost of disorders of the brain in Europe[END_REF][START_REF] Rice | Health economics and cost implications of anxiety and other mental disorders in the United States[END_REF][START_REF] Smit | Costs of nine common mental disorders: implications for curative and preventive psychiatry[END_REF] prevention and treatment of these disorders should be cost-effective.

Another implication is that interactions were found to be sub-additive in the best-fitting model. This does not mean that comorbidity is not highly disabling. On the contrary, there is clear evidence of its high burden. [START_REF] Turner | Effect of unrelated comorbid conditions on hypertension management[END_REF] But it does mean that disability increases at a decreasing rate when comorbid conditions exist. This finding has an important implication for the prevention of disability: addressing only one disorder (treatment or prevention) when it coexists with other disorders will render a less effective outcome than addressing all the coexisting conditions. 39 3 Most WMH surveys are based on stratified multistage clustered area probability household samples in which samples of areas equivalent to counties or municipalities in the US were selected in the first stage followed by one or more subsequent stages of geographic sampling (e.g., towns within counties, blocks within towns, households within blocks) to arrive at a sample of households, in each of which a listing of household members was created and one or two people were selected from this listing to be interviewed. No substitution was allowed when the originally sampled household resident could not be interviewed. These household samples were selected from Census area data in all countries other than France (where telephone directories were used to select households) and the Netherlands (where postal registries were used to select households). Several WMH surveys (Belgium, Germany, Italy) used municipal resident registries to select respondents without listing households. The Japanese sample is the only totally un-clustered sample, with households randomly selected in each of the four sample areas and one random respondent selected in each sample household. 18 of the 24 surveys are based on nationally representative (NR) household samples. [START_REF] Loeppke | Health and productivity as a business strategy: a multiemployer study[END_REF] Iraq, Israel, Romania, and South Africa did not have Part II sample. [START_REF] Merikangas | The impact of comorbidity of mental and physical conditions on role disability in the US adult household population[END_REF] The response rate is calculated as the ratio of the number of households in which an interview was completed to the number of households originally sampled, excluding from the denominator households known not to be eligible either because of being vacant at the time of initial contact or because the residents were unable to speak the designated languages of the survey. Mean response rate across all surveys is 72.0%. [START_REF] Andlin-Sobocki | Cost of disorders of the brain in Europe[END_REF] New Zealand interviewed respondents 16+ but for the purposes of cross-national comparisons we limit the sample to those 18+ [START_REF] Korff | Global perspectives on mental-physical comorbidity[END_REF] Only respondents from Part II who were asked questions about chronic conditions are included.

  country. Each interview had two parts. Part I, which was administered to all respondents, contained assessments of core mental disorders. All Part I respondents who met criteria for any core mental disorder plus a probability sub-sample of other Part I respondents were administered Part II, which assessed correlates and disorders of secondary interest to the study. The assessment of physical disorders was included in Part II. Part II is consequently the focus of the current report. The Part II data were weighted to adjust for the under-sampling of Part II non-cases and to adjust for residual discrepancies between sample and population distributions on a range of socio-demographic and geographic variables. The total number of Part II respondents across surveys was 62,971, with individual country sample sizes ranging from 602 (Lebanon) to 7,312 (New Zealand).

Table 1 . WMH Sample Characteristics by World Bank income categories 1 Survey 2 Sample Characteristics

 1 The World Bank. (2008). Data and Statistics. Accessed May 12, 2009 at: http://go.worldbank.org/D7SN0B8YU0 2 NSMH (The Colombian National Study of Mental Health); WMHI (World Mental Health India); IMHS (Iraq Mental Health Survey); NSMHW (The Nigerian Survey of Mental Health and Wellbeing); B-WMH (The Beijing World Mental Health Survey); S-WMH (The Shanghai World Mental Health Survey); CMDPSD (Comorbid Mental Disorders during Periods of Social Disruption); NSHS (Bulgaria National Survey of Health and Stress); LEBANON (Lebanese Evaluation of the Burden of Ailments and Needs of the Nation); M-NCS (The Mexico National Comorbidity Survey); RMHS (Romania Mental Health Survey); SASH (South Africa Health Survey); ESEMeD (The European Study

	3
	Field Dates	Age Range	Sample Size	Response Rate
			Part I	Part II	7

[START_REF] Acoem | Healthy workforce/healthy economy: the role of health, productivity, and disability management in addressing the nation's health care crisis: why an emphasis on the health of the workforce is vital to the health of the economy[END_REF]

Table 2 . Sample characteristics by country and country income. The WHO WMH Surveys

 2

						High School
			Age	Females	Not Married	or more	Not Working
	Country	N	Mean (SE)	% (SE)	% (SE)	% (SE)	% (SE)
	Lower income:
	Colombia	2381	36.6 (0.4)	54.5 (1.5)	43.4 (1.7)	46.4 (2.1)	46.4 (1.6)
	India
	(Pondicherry)

C:\temp\9476_1_art_file_70224_l73fn8.doc 27 Figure 1.

Yearly days totally out of role due to each of the 19 health conditions considered: A. among those suffering the condition ("individual effect"); and B. population attributable risk proportion (PARPs). WHO World Mental Health Surveys. A. Individual effects B. Population Attributable Risk Proportions (PARPs)

Wald F test (p-value);

Chi-square test of independence (p-value)

Test for difference between distributions of days out of role (among respondents with any) across income groups.

Acknowledgments

The analysis for this paper is carried out in conjunction with the World Health Organization World Mental Health (WMH) Survey Initiative. We thank the WMH staff for assistance with instrumentation, fieldwork, and data analysis. These activities were supported by the United States National Institute of Mental Health

Lebanese Ministry of Public Health, the WHO (Lebanon), Fogarty International, Act for Lebanon, anonymous private donations to IDRAAC, Lebanon, and unrestricted grants from Janssen Cilag, Eli Lilly, GlaxoSmithKline, Roche, and Novartis.The Mexican National Comorbidity Survey (MNCS) is supported by The National Institute of Psychiatry Ramon de la Fuente (INPRFMDIES 4280) and by the National Council on Science and Technology (CONACyT-G30544-H), with supplemental support from the PanAmerican Health Organization (PAHO). The Nigerian Survey of Mental Health and Wellbeing (NSMHW) is supported by the WHO (Geneva), the WHO (Nigeria), and the Federal Ministry of Health, Abuja, Nigeria. The Ukraine Comorbid Mental Disorders during Periods of Social Disruption (CMDPSD) study is funded by the US National Institute of Mental Health (RO1-MH61905). The US National Comorbidity Survey Replication (NCS-R) is supported by the National Institute of Mental Health (NIMH; U01-MH60220) with supplemental support from the National Institute of Drug Abuse (NIDA), the Substance Abuse and Mental Health Services Administration (SAMHSA), the Robert Wood Johnson Foundation (RWJF; Grant 044708), and the John W. Alden Trust.

Conflict of Interest

Dr. Kessler


Stella Marie Reamon-Buettner

Prof. Dr Juergen Borlak
email: borlak@item.fraunhofer.de

An Update on this Hypermutable Homeodomain Protein and its Role in Human Congenital Heart Disease (CHD)

Keywords: heart development, congenital heart disease, cardiac malformations, transcription factors, NKX2-5, mutations heart development, congenital heart disease, cardiac malformations, transcription factors, NKX2-5, mutations heart development, congenital heart disease, cardiac malformations, transcription factors, NKX2-5, mutations

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Table 1. Functional Consequences of Identified NKX2-5 Mutations in CHD: Functional Assays or Bioinformatic Analysis is contained in *doc.file NKX2-5 humu10-0256_Table 1.doc. Table 2. Detection Frequency of NKX2-5 Germline Mutations in Congenital Heart Disease is contained in *doc.file NKX2-5 humu10-0256_Table 2.doc.

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The single file NKX2-5 humu10-0256_Supp_ Mat. doc contains the following: Supp. Figure S1. Geographical distribution of germline NKX2-5 mutations in CHD along the coding sequence of the gene; Supp. Table S1. Studies on NKX2-5 Mutations Associated with Congenital Heart Disease (CHD); Supp. Table S2. Summary of Published Germline NKX2-5 Mutations in Patients with Congenital Heart Defects; Supp. Table S3. Functional Significance of Published Germline NKX2-5 Mutations in Patients with Congenital Heart Disease 3) Please check to see that the references follow the journal's standard format and are cited properly. All authors must be listed, for example. See our online Author Instructions http://www3.interscience.wiley.com/cgibin/jabout/38515/ForAuthors.html

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open access publication.More information on Wiley-Blackwell's policy is available at: http://www.interscience.wiley.com/openaccess 6) IMPORTANT INFORMATION REGARDING PREPRINTS a-Human Mutation is now publishing online preprints of accepted manuscripts prior to typesetting and page proof corrections. It is therefore crucial that you revise your manuscript carefully so that errors (typographical and grammatical) are corrected BEFORE the final accepted manuscript is posted online. The accepted preprint version will remain online until the corrected proofs are received and the typeset manuscript is finalized. At that time, the preprint version will be replaced with the final, typeset version online, in Early View.

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We proofread the revised manuscript carefully, and no substantial changes carried out except for some minor style editing (e.g. abstract), and updating of citations and references (e.g. [START_REF] Rauch | Comprehensive genotype-phenotype analysis in 230 patients with tetralogy of Fallot[END_REF][START_REF] De Luca | Familial transposition of great arteries caused by multiple mutations in laterality genes[END_REF].

b-It is essential that you submit a copyright transfer agreement (CTA) upon submission of your revised manuscript. This will avoid delay in publication of your article upon acceptance. If possible, the CTA must be signed by the corresponding author and should be signed by all contributing authors if practical. All authors must be made aware of the CTA and the rights it conveys to them. The CTA can be found here: www.wiley.com/go/ctaaus At the molecular and cellular levels, heart development entails precise orchestration of genetic events, as well as the interplay of master transcriptional regulators, signaling pathways and chromatin remodeling rendering the understanding of CHD a challenging task. But great efforts are underway towards the unravelling of mechanisms leading to CHD, as conveyed in recent reviews [START_REF] Bruneau | The developmental genetics of congenital heart disease[END_REF][START_REF] Nemer | Genetic insights into normal and abnormal heart development[END_REF][START_REF] Joziasse | Genes in congenital heart disease: atrioventricular valve formation[END_REF][START_REF] Bajolle | Genetics and embryological mechanisms of congenital heart disease[END_REF][START_REF] Leong | Fresh fields and pathways new: recent genetic insights into cardiac malformation[END_REF][START_REF] Combs | Heart valve development: regulatory networks in development and disease[END_REF]. Notably, the pivotal role of certain transcription factors in heart development has been demonstrated, and gene targeting of cardiac-specific transcription factor genes in animal models especially in mice has provided valuable insights into heart anomalies [START_REF] Nemer | Genetic insights into normal and abnormal heart development[END_REF].

Over the years, family-based studies identified mutations in several genes controlling heart development, especially transcription factor genes (see reviews, [START_REF] Clark | Transcription factors and congenital heart defects[END_REF][START_REF] Bruneau | The developmental genetics of congenital heart disease[END_REF][START_REF] Benson | Genetic origins of pediatric heart disease[END_REF]. Among these transcription factor genes with mutations associated with human CHD include NKX2-5 (MIM# 600584), GATA4 (MIM# 600576) and TBX5 (MIM# 601620) and these mutations are often observed in patients with atrial, ventricular and atrioventricular septation defects. Although these family studies have been useful in uncovering genes linked to CHD, population-based studies show so far, rare detection of mutations in unrelated patients with CHD (e.g. [START_REF] Rajagopal | Spectrum of heart disease associated with murine and human GATA4 mutation[END_REF][START_REF] Posch | Mutations in GATA4, NKX2.5, CRELD1, and BMP4 are infrequently found in patients with congenital cardiac septal defects[END_REF]; therefore, pointing to a multifactorial cause of disease. Furthermore, mutations exhibit variable expression, in which different cardiac malformations occur even from the same mutation, suggesting modifying factors and epigenetic effects contributing to disease F o r P e e r R e v i e w 4 manifestation. Indeed, a recent study which examined the effect of genetic background variation on the incidence of specific heart defects in heterozygous knock-out Nkx2-5 +/-mice and in crosses to two different strains revealed that such genetic modifiers to play an important role in buffering cardiac developmental pathways against a major perturbation, while directing the manifestation of disease in a few [START_REF] Winston | Heterogeneity of genetic modifiers ensures normal cardiac development[END_REF].

In this paper, we will review efforts in identifying disease-associated mutations in the transcription factor gene NKX2-5, and the clinical significance of such mutations in the pathogenesis of CHD. We summarize published germline NKX2-5 mutations and corresponding CHD phenotypes, their detection frequency, geographical location, as well as functional consequence as determined by assays or bioinformatic prediction. Finally, we explore different avenues in CHD pathogenesis, some of which challenge the well-accepted view of a single mutation as a direct cause of disease.

NKX2-5 and its Role in Cardiac Development and Disease

Nkx2-5 belongs to the NK-2 family of homeodomain-containing transcription factors, which are conserved in many organisms from flies to humans [START_REF] Bartlett | Examining the cardiac NK-2 genes in early heart development[END_REF]. Through the 60-amino-acid homeodomain (HD), Nkx2-5 interacts with DNA through a helix-turnhelix DNA-binding motif of three alpha helices, with helix 3 providing binding specificity.

Important for Nkx2-5 function are also other conserved regions namely, the transactivation (TN) domain, and the NK2-specific domain (NK2-SD). Nkx2-5 is a transcriptional regulator during early embryonic heart development, and its absence in mice models results in embryonic lethality and arrested heart development at the looping stage (see reviews, [START_REF] Harvey | Homeodomain factor Nkx2-5 in heart development and disease[END_REF][START_REF] Akazawa | Cardiac transcription factor Csx/Nkx2-5: Its role in cardiac development and diseases[END_REF][START_REF] Bartlett | Examining the cardiac NK-2 genes in early heart development[END_REF]. Nkx2-5 is also essential for the formation, maturation and maintenance of the conduction system [START_REF] Pashmforoush | Nkx2-5 pathways and congenital heart disease; loss of ventricular myocyte lineage specification leads to progressive cardiomyopathy and complete heart block[END_REF][START_REF] Jay | Nkx2-5 mutation causes anatomic hypoplasia of the cardiac conduction system[END_REF]. Nkx2-5 regulates the number of cells in the cardiac conduction system in a dose-dependent manner [START_REF] Jay | Nkx2-5 mutation causes anatomic hypoplasia of the cardiac conduction system[END_REF]. While null Nkx2-5 mice mutants appear to lack Anatomic hypoplasia of the conduction system may explain some of the defects in Nkx2-5 loss-of-function mutants. Notably, Nkx2-5 is expressed in both the first heart field (FHF) and the second heart field (SHF), the two distinct sources of cardiac progenitor cells contributing to different parts of the heart [START_REF] Buckingham | Building the mammalian heart from two sources of myocardial cells[END_REF]. This expression in progenitor cells indicates a higher role for Nkx2-5 in the hierarchy of transcriptional regulators of heart development.

Recent studies show further roles for Nkx2-5 in heart development and perinatal hearts. In mice, Nkx2-5 repression of Bmp2/Smad1 signaling regulates SHF proliferation and outflow tract (OFT) morphology [START_REF] Prall | An Nkx2-5/Bmp2/Smad1 negative feedback loop controls heart progenitor specification and proliferation[END_REF]. In the cardiac fields of Nkx2-5 mutants, genes controlling cardiac specification and maintenance of the progenitor state were upregulated, leading initially to progenitor overspecification, but subsequently to failed SHF proliferation and outflow tract (OFT) truncation. Nkx2-5-null embryos exhibited decrease in SHF proliferation, resulting in a smaller outflow tract and right ventricle. In vivo Cre-lox Pbased lineage tracing in mice showed that Nkx2-5-expressing cardiac progenitors contributed to the proepicardium [START_REF] Zhou | Nkx2-5-and Isl1-expressing cardiac progenitors contribute to proepicardium[END_REF]. Indeed, Nkx2-5 knockout resulted in abnormal proepicardial development, and decreased expression of the transcription factor Wilm's Tumor 1 (Wt1), which in the heart its expression is confined to the proepicardium and epicardium. Furthermore, inhibiting the function of Nkx2-5 homologs (nkx2.5 and nkx2.7) in zebrafish showed their involvement in the regulation of heart tube extension as well as ventricular and atrial cell number [START_REF] Targoff | Nkx genes regulate heart tube extension and exert differential effects on ventricular and atrial cell number[END_REF]. The nkx-deficient heart tube failed to elongate normally: its ventricular portion was atypically short and wide, and its atrial portion was disorganized and sprawling. This atrial phenotype was associated with a surplus of atrial cardiomyocytes; ventricular cell number decreased in nkx-deficient embryos later in development, when cardiac chambers are emerging. Lastly, it has also been demonstrated using tamoxifen-inducible Nkx2-5 knockout mice that Nkx2-5 function is critical not only during cardiac development, but also in perinatal hearts, by regulating expression of several important gene products involved in conduction and contraction [START_REF] Briggs | Perinatal loss of Nkx2-5 results in rapid conduction and contraction defects[END_REF]. The perinatal loss of Nkx2-5 resulted in rapid conduction and contraction defects within 4 days after the deletion of Nkx2-5 alleles.

Furthermore, using tamoxifen-inducible Cre transgene (Cre-ER), floxed-Nkx2-5 alleles were deleted beginning at 2 weeks of age [START_REF] Takeda | Slow progressive conduction and contraction defects in loss of Nkx2-5 mice after cardiomyocyte terminal differentiation[END_REF]. The loss of Nkx2-5 beginning at 2 weeks of age also resulted in conduction and contraction defects, but disease progression was slower than in perinatal loss of Nkx2-5.

An Update of NKX2-5 Germline Mutations

The human NKX2-5 gene maps to chromosome 5q34 and consists of two exons that encode a 324-amino-acid protein. Since the first report implicating NKX2-5 mutations in human CHD [START_REF] Schott | Congenital heart disease caused by mutations in the transcription factor NKX2-5[END_REF], the coding region of NKX2-5 has been consistently searched for additional disease-associated sequence alterations. There are at least 28 studies where germline NKX2-5 mutations have been analyzed or identified in CHD (Supp.Table S1).

NKX2-5 mutations were identified in different countries, but majority were found in studies conducted in the United States and affect different regions of the gene (Supp. Figure S1, Supp. Table S2). Some mutations may appear though to cluster geographically, such as the three deletions from Japan, and the two substitutions from Germany (Supp. Figure S1).

At least 41 different NKX2-5 germline mutations, most of which lead to amino acid change (nonsynonymous mutations) have been reported (Figure 1, Supp. Table S2). Of these 41 mutations, 33 are single nucleotide substitutions, 6 deletions and 2 insertions. The NKX2-5 mutations are spread along the gene, and except for one at a splice site, all are located in the coding region. Mutations affect conserved regions, where 2 are in the TN domain, 14 in the homeodomain (HD), and 2 are in the NK2-SD domain. Only 5 of 41 (12%) have been detected. However, p.R25C was also found in unaffected relatives and controls; hence, its pathogenicity remains unclear [START_REF] Goldmuntz | NKX2.5 mutations in patients with tetralogy of fallot[END_REF][START_REF] Akcaboy | The effect of p.Arg25Cys alteration in NKX2-5 on conotruncal heart anomalies: mutation or polymorphism?[END_REF][START_REF] Esposito | Molecular analysis of PRKAG2, LAMP2, and NKX2-5 genes in a cohort of 125 patients with accessory atrioventricular connection[END_REF][START_REF] Gioli-Pereira | NKX2.5 mutations in patients with non-syndromic congenital heart disease[END_REF]. Furthermore, NKX2-5 mutations have been identified in both familial and sporadic cases of CHD, in which 25 of 41 are familial, 14 are sporadic, and 2 are sporadic/familial cases (Figure 1, Supp. Table S2). Patients are heterozygous for the NKX2-5 mutations, and frequent CHD malformations in patients with NKX2-5 mutations are atrial septal defect (ASD) with or without atrioventricular block (AV) block. Specifically, 29 of 41 mutations were identified in patients with ASD II, 26 in AV block, and 25 in combined ASD II and AV block. Other CHD phenotypes were also found including Tetralogy of Fallot (TOF) in 7 mutations, as well as ventricular septal defect (VSD) in 9 mutations. A quick characterization of the 41 NKX2-5 mutations identified in patients with CHD is depicted in Figure 2.

Functional Significance of NKX2-5 Mutations

Of the 40 mutations identified in the coding region of NKX2-5, 25 are missense, 7 nonsense, 7 frameshift and 1 leading to an amino acid deletion. Most of these would affect protein function as predicted by bioinformatic methods or by functional assays (Table 1). So far, only 4 mutations have been predicted to have no deleterious effects on protein function.

Specifically, 17 mutations would lead to a truncated protein in which 11 affect both the homeodomain and NK2-SD. Notably, certain mutations that are not outright nonsense in the homeodomain would result in the breakage of the secondary structure (Figure 3) and /or affect highly conserved residues (Figure 4). Moreover, 16 mutations exhibited impaired function in all or any of the assays particularly DNA binding, transactivation activity, protein-protein interaction and nuclear localization (Table 1, Supp. Table S3). Essentially, NKX2-5 mutations activities, and protein-protein interactions particularly with GATA4 and TBX5 [START_REF] Kasahara | For the functional studies[END_REF][START_REF] Kasahara | Biochemical analyses of eight NKX2.5 homeodomain missense mutations causing atrioventricular block and cardiac anomalies[END_REF][START_REF] Zhu | Y259X 16 defective in functional assays* (DNA binding/transactivation activity/protein-protein interaction/ nuclear localization) c[END_REF][START_REF] Monzen | Dual effects of the homeobox transcription factor Csx/Nkx2-5 on cardiomyocytes[END_REF][START_REF] Inga | Functional dissection of sequence-specific NKX2-5 DNA binding domain mutations associated with human heart septation defects using a yeast-based system[END_REF][START_REF] Esposito | Molecular analysis of PRKAG2, LAMP2, and NKX2-5 genes in a cohort of 125 patients with accessory atrioventricular connection[END_REF].

Lack of Genotype-Phenotype Correlation

Despite NKX2-5 hypermutability and the pathogenic potentials of NKX2-5 germline mutations, there is lack of genotype-phenotype correlation of NKX2-5 mutations in CHD (see also review, [START_REF] Benson | Genetic origins of pediatric heart disease[END_REF]. Notably, the detection frequency of NKX2-5 mutations in sporadic cases of CHD is about 2% and in several studies none was found (Table 2). Most of the identified NKX2-5 mutations are unique and diverse cardiac malformations have been associated with NKX2-5 mutations (Figure 1). Furthermore, same CHD phenotypes have been exhibited by patients with different NKX2-5 mutations or same NKX2-5 mutation gave different CHD phenotypes in regards to severity even within families (see for instance, Figure 1 and Supp. Table S2). Indeed, more than half of identified NKX2-5 mutations are familial cases, and families have their private mutations.

Even as new studies on NKX2-5 mutations keep coming in, the picture remains basically unchanged. After genetic analysis of NKX2-5 mutations in 230 patients with TOF, a detection frequency of 0.9% was obtained [START_REF] Rauch | Comprehensive genotype-phenotype analysis in 230 patients with tetralogy of Fallot[END_REF]. Two patients with nonsyndromic TOF harbored an unknown variant each (i.e. p.C270Y and p.V315L), both located at the carboxy terminal and which did not show an effect on the transcriptional reporter assay used. The most frequent variant p.R25C was also detected in two patients. In another study, no NKX2-5 mutations were found in 96 patients with idiopathic atrial fibrillation, a condition of arrhythmia but no visible structural defects [START_REF] Boldt | Mutational analysis of the PITX2 and NKX2-5 genes in patients with idiopathic atrial fibrillation[END_REF]. After sequencing NKX2-5 in 34 patients with stroke and patent foramen ovale (PFO), no pathogenic mutations were obtained [START_REF] Belvis | Mutations in the NKX2-5 gene in patients with stroke and patent foramen ovale[END_REF]. All the while, NKX2-5 mutations are not exclusive to CHD. 9 Indeed, NKX2-5 mutations (p.A119S, p.R25C, p.R161P) were found in thyroid dysgenesis, in patients with or without cardiac anomalies [START_REF] Dentice | Missense mutation in the transcription factor NKX2-5: a novel molecular event in the pathogenesis of thyroid dysgenesis[END_REF], suggesting a broader role for NKX2-5 in organogenesis and pathophysiology.

There are several factors that may contribute to this lack of genotype-phenotype correlation. For one, genetic background can modify the phenotypic expression of single gene mutations in mice and humans [START_REF] Rajagopal | Spectrum of heart disease associated with murine and human GATA4 mutation[END_REF]. Indeed, modifier genes have been shown to affect NKX2-5 mutations in the pathogenesis of CHD [START_REF] Kaiser | Modifier genes in the pathogenesis of congenital heart disease[END_REF][START_REF] Winston | Heterogeneity of genetic modifiers ensures normal cardiac development[END_REF]. There is also the possible influence of synonymous (silent) mutations and dbSNPs affecting protein expression and function [START_REF] Sauna | Silent polymorphisms speak: how they affect pharmacogenomics and the treatment of cancer[END_REF]. Such silent variants and dbSNPs eventually serve as risk factors for CHD. Furthermore, buffering that can result from compensation by a normally functioning second allele or a duplicated gene or a pathway that maintains residual function may also explain reduced penetrance of mutations in sporadic cases of CHD (see review, [START_REF] Bentham | Genetic mechanisms controlling cardiovascular development[END_REF]. These authors proposed that the genetic architecture of sporadic CHD likely includes accumulation of rare nonsynonymous variants in cardiac developmental genes leading to mutational loading of cardiac developmental networks, copy number variation in cardiac developmental genes, and common variants that may not be obviously linked to cardiac development but may alter genetic buffering pathways. Moreover, as one review commented [START_REF] Weismann | The genetics of congenital heart disease: a review of recent developments[END_REF], germline mutations may not be compatible with survival of the developing embryo because many of the genes that control cardiac development also play critical roles in the development of other organ systems and in any case, would not likely cause CHD only.

Summing up, a single germline NKX2-5 mutation as the direct cause of disease or a simple Mendelian inheritance alone may not sufficiently explain disease pathogenesis. Needless to say, none of the patients studied so far are homozygous for NKX2-5 variant alleles.

Molecular Basis of CHD Pathogenesis

The landscape for CHD pathogenesis is complex. First of all, majority of CHD cases are sporadic in which patients have no family history of the disease. Indeed, the contribution of family history of any CHD among first-degree relatives to the overall prevalence of CHD is small, approximately 2-4% [START_REF] Oyen | Recurrence of congenital heart defects in families[END_REF]. There is clinical or phenotype heterogeneity within affected individuals, as well as within and between families. Disease association is difficult owing to lack of clear genotype-phenotype correlation of mutations. For instance, patients harboring the same NKX2-5 mutation have different malformations; or mutations in different genes give the same disease phenotype. Specifically, common cardiac malformations associated with NKX2-5 mutations are AV block, ASD, and VSD. But the same structural anomalies are also observed in mutations in other transcription factor genes (see reviews, [START_REF] Clark | Transcription factors and congenital heart defects[END_REF][START_REF] Nemer | Genetic insights into normal and abnormal heart development[END_REF]. ASD is associated with mutations involving GATA4, TBX5 and ZIC3; AV block in NKX2-5 and TBX5; or VSD in GATA4 and TBX5. Thus, CHD is considered as a multifactorial disease, likely resulting from gene-environment interactions which eventually determine disease penetrance and expressivity. Among the environmental risk factors for CHD are chemicals, drugs, infections, and maternal diseases (reviewed in [START_REF] Jenkins | Noninherited risk factors and congenital cardiovascular defects: current knowledge: a scientific statement from the American Heart Association Council on Cardiovascular Disease in the Young: endorsed by the American Academy of Pediatrics[END_REF].

At the molecular and cellular levels, heart development can practically go wrong in many directions leading to diverse cardiac malformations observed in CHD. Notably, heart development requires spatial and timely interactions of different transcription factors to regulate downstream targets, and breakdown of transcriptional interactions can lead to various CHD phenotypes (see reviews, [START_REF] Nemer | Genetic insights into normal and abnormal heart development[END_REF][START_REF] Bruneau | The developmental genetics of congenital heart disease[END_REF]. For example, Nkx2-5 through the homeodomain interacts physically and synergistically with Gata4 and Tbx5, such that abrogation of their interaction leads to CHD. Indeed, NKX2-5 mutations identified in patients affecting the homeodomain have been demonstrated to result in reduced interaction with Gata4 and Tbx5 [START_REF] Kasahara | Biochemical analyses of eight NKX2.5 homeodomain missense mutations causing atrioventricular block and cardiac anomalies[END_REF]. Recently, it has been shown that Nkx2-5 can Nkx2-5 and Mef2c are necessary for ventricle formation [START_REF] Vincentz | Cooperative interaction of Nkx2.5 and Mef2c transcription factors during heart development[END_REF]. While individual mutants for both Nkx2-5 and Mef2c had identifiable ventricles, double mutants displayed ventricular hypoplasia, a more severe cardiac phenotype. Besides transcription factors, Nkx2-5 can also interact with a histone methyltransferase to regulate target genes. A recent study linked Nkx2-5 to Wolf-Hirschhorn syndrome (MIM #194190) where cardiac septal defects are among the anomalies, through an association with Whsc1, an H3K36me3specific histone methyltransferase in mouse embryonic hearts [START_REF] Nimura | A histone H3 lysine 36 trimethyltransferase links Nkx2-5 to Wolf-Hirschhorn syndrome[END_REF]. The effects of Whsc1 haploinsufficiency increased in heterozygous Nkx2-5 mutant hearts, and reporter assays showed that Whsc1 negatively modulated the transcriptional activity of Nkx2-

5.

A complex network of signaling molecules and their signal transduction pathways participates in the precise orchestration of the many morphogenetic events in heart development especially in the specification, proliferation and differentiation of cardiac progenitor cells (reviewed in Wagner and Siddiqui, 2007a;Wagner and Siddiqui, 2007b;[START_REF] Niessen | Notch signaling in cardiac development[END_REF][START_REF] Nemir | Functional role of Notch signaling in the developing and postnatal heart[END_REF][START_REF] Rochais | Signaling pathways controlling second heart field development[END_REF]. Shown to regulate cardiogenesis include bone morphogenetic proteins (BMP), fibroblast growth factors (FGF), Wnt, Hedgehog, Nodal, retinoic acid, neuregulin, vascular endothelial growth factor (VEGF) and Notch signaling pathways. Errors in these signaling pathways can impair heart development. For instance, mutations in the Notch signaling pathway [START_REF] Mcbride | NOTCH1 mutations in individuals with left ventricular outflow tract malformations reduce ligand-induced signaling[END_REF] or in the Nodal signaling pathway [START_REF] Roessler | Reduced NODAL signaling strength via mutation of several pathway members including FOXH1 is linked to human heart defects and holoprosencephaly[END_REF] [START_REF] Lickert | Baf60c is essential for function of BAF chromatin remodelling complexes in heart development[END_REF]. Furthermore, Baf180, a specific subunit of the PBAF form of the BAF complex is required for cardiac and placental development [START_REF] Wang | Polybromo protein BAF180 functions in mammalian cardiac chamber maturation[END_REF]. Ablation of Baf180 in mouse embryos resulted in severe hypoplastic ventricle, ventricular septal defect and trophoblast placental defects. Recently, it has been shown that myocardial trabeculation requires Brg1, a chromatin remodeling protein, (Stankunas et al., 2008a). Trabeculation defects occurred in the absence of endocardial Brg1 which resulted from the derepression of a secreted matrix metalloproteinase, ADAMTS1, which normally only increases in expression later in development to prevent excessive trabeculation.

Thus, there is justification for proposing the concept of one heart disease-several mechanisms-several genes to explain the heterogeneity of certain cardiac malformations, which is not by chance but by the heterogeneity of mechanisms [START_REF] Bajolle | Genetics and embryological mechanisms of congenital heart disease[END_REF].

According to this concept, a certain malformation may originate from different embryology mechanisms, which in turn controlled by multiple genes. Given as example is common arterial trunk (CAT), which may result from a participation defect of progenitors from the second cardiac field and/or a migration defect of the neural crest cells and/or a rotation defect of the myocardium and/or a formation abnormality of the endocardial cushions. Further example is atrioventricular valve formation which is regulated by different genetic pathways [START_REF] Joziasse | Genes in congenital heart disease: atrioventricular valve formation[END_REF]. Disturbances in each subset of a particular pathway, i.e. ligand receptor, transcription factor or extracellular matric proteins can all result in similar cardiac defects.

Recently, a study showed that interactions between alleles of three Pbx genes, which encode homeodomain transcription factors, are sufficient to determine the phenotypic presentation of CHD in mice (Stankunas et al., 2008b). A major role is served by Pbx1, whose

Learning from the Leipzig Collection of Malformed Hearts

Model organisms especially mice provide insights into the molecular mechanisms leading to CHD. But results can be species-specific and only a minority of mutations in these genes have been related to CHD in humans (see [START_REF] Bruneau | The developmental genetics of congenital heart disease[END_REF]. Furthermore, genetic studies on CHD are usually based on blood samples and such analysis may not reveal complete genetic alterations leading to the disease. To gain insights into the molecular causes of CHD, we conducted in the past genetic analysis of NKX2-5 and other cardiac-specific transcription factor genes, most recently on HAND1 (MIM# 602406), in a rare collection of morphologically, well-characterized explanted, malformed hearts stored in formalin for many years (Reamon Buettner and Borlak, 2004;Reamon-Buettner et al., 2004;2008;[START_REF] Esposito | numbering reflects cDNA numbering with +1 corresponding to the A of the ATG translation initiation codon in the reference sequence NM_0004387.3, according to journal guidelines[END_REF]. This research identified somatic mutations of cardiac-specific transcription factor genes as a possible disease-causing mechanism as discussed below.

Direct sequencing of different cardiac-specific transcription factor genes in 'affected' tissues of malformed hearts revealed frequent disease-associated mutations which were mainly absent in 'unaffected' tissues in the same patient's heart. This finding indicates somatic mosaicism and somatic origin of mutations. Many patients displayed multiple deleterious mutations not only in one but several transcription factor genes. While for some investigators in the field, it would be easiest to explain the findings from the Leipzig heart collection as artifacts on the basis of degraded DNA owing to many years of storage in formalin solution [START_REF] Draus | Investigation of somatic NKX2-5 mutations in congenital heart disease[END_REF], the study on such a rare collection of malformed hearts may lead to important insights on heart development and CHD. Here are some of the pros and cons of the scientific discussion.

Somatic mutations as a molecular rationale for CHD

Somatic mutations may result from environmental insults and epigenetic instability to provide a molecular rationale for CHD. Although somatic mutations are well known in cancer, they can also occur in non-cancer diseases. For example, somatic mutations in the gene TEK encoding the endothelial cell tyrosine kinase receptor TIE2 were identified in lesions from patients with sporadic venous malformations [START_REF] Limaye | Somatic mutations in angiopoietin receptor gene TEK cause solitary and multiple sporadic venous malformations[END_REF]. The somatic mutations included a frequent p.L914F substitution and several double mutations in cis, all of which resulted in ligand-independent TIE2 hyperphosphorylation in vitro. Somatic mutations were also identified in GJA5 gene encoding cardiac gap-junction protein connexin 40 in cardiac tissues of patients with atrial fibrillation [START_REF] Gollob | Somatic mutations in the connexin 40 gene (GJA5) in atrial fibrillation[END_REF]. Atrial fibrillation is the most common type of cardiac arrhythmia and a leading cause of cardiovascular morbidity, particularly stroke.

Multiple mutations in complex cardiac malformations

The presence of multiple mutations in patients with complex heart malformations may be a reflection of disease severity. Indeed, there are now studies to show that the presence of multiple disease-causing mutations in the same gene or in different genes can be an indication of disease severity [START_REF] Kelly | Multiple mutations in genetic cardiovascular disease: a marker of disease severity?[END_REF]. In familial hypertrophic cardiomyopathy (FHC) which is characterized by genetic and clinical heterogeneity, patients with two FHCcausing mutations typically had more severe phenotype (i.e. earlier onset of disease, greater left ventricular hypertrophy and a higher incidence of sudden cardiac death events) compared (Tsoutsman et al., 2008a;Tsoutsman et al., 2008b). Furthermore, in familial transposition of great arteries (TGA) some patients harbored more than one mutation in laterality genes which presumably influenced cardiac phenotype [START_REF] De Luca | Familial transposition of great arteries caused by multiple mutations in laterality genes[END_REF].

A structural malformation may result from combined effects of deleterious mutations in several transcription factor genes

Certain malformations may have resulted from combined effects of deleterious mutations in several transcription factor genes. In the hypoplastic hearts group, a frameshift mutation p.A126fs (HAND1) was frequently detected in hypoplastic ventricles [START_REF] Reamon-Buettner | A loss-of-function mutation in the binding domain of HAND1 predicts hypoplasia of the human hearts[END_REF]. But further analysis led to the detection of additional loss-of-function mutations affecting the 2nd zinc finger of GATA4 and homeodomain of NKX2-5 (unpublished results). For example, in the hypoplastic left ventricle of patient A82, we detected in HAND1 p.A126fs; in GATA4 p.R266X; and in NKX2-5 homozygous for p.L116P, p.T178M, p.K192T.

Indications of transient hypermutability/ genetic instability in cardiac tissues of CHD patients

Hypermutability varied among the different transcription factor genes, but mutations were non-random and closely-spaced. The multiple mutations observed in the affected tissues were possibly the result of various haplotypes. Specifically, depending on the stretch of DNA being analyzed, the closely-spaced sequence alterations could involve those in the coding or non-coding regions, and when in the coding region, mutations could be synonymous or nonsynonymous. For example, in two patients affected by VSD, we found 11 non-random, closely-spaced mutations in NKX2-5 within 362-bp spanning intronic and coding sequences of exon 2. In this window, the separation of an NKX2-5 mutation to the next ranged from 3 bp to 121 bp, but majority was less than 30 bp. Moreover, after cloning of a PCR fragment 'heterozygous' for the multiple mutations in an analyzed transcription factor gene, more than the two expected haplotypes or alleles were found. In some cases, all the variants occurred in cis. The occurrence of closely-spaced multiple mutations in human genes has been suggested to be possible signatures of transient hypermutability, a mutational mechanism with the potential to generate multiple synchronous mutations, resulting from a perturbation of one of the many transactions that maintain genetic stability [START_REF] Chen | Closely-spaced multiple mutations as potential signatures of transient hypermutability in human genes[END_REF]. Specifically, transient hypermutability may result from (1) the deregulated expression of, or conformational change in, either a replicative DNA polymerase or another protein involved in the maintenance of replication fidelity; (2) the disruption of the balance of the nucleotide pool; or (3) the recruitment of error-prone DNA polymerases in DNA replication or repair (reviewed in [START_REF] Chen | Closely-spaced multiple mutations as potential signatures of transient hypermutability in human genes[END_REF].

Future Directions

Understanding the molecular process leading to CHD should provide important insights into the etiology and treatment of patients. This is a difficult task as CHD is complex entailing defects in multiple genes and different pathways. A good grasp of CHD would thus require a whole-genome functional approach, in which for a specific structural defect a combination of different genes and associated pathways involving transcriptional machinery, signal transduction, and chromatin remodeling will be simultaneously interrogated. But with high-throughput technologies in different 'omics' available, as well as accumulating evidence from animal models, this may be no longer a monumental undertaking. Eventually, there is the task ahead to translate discoveries to human CHD, although in future genetic studies corresponding molecular events may have been identified by then to explain genetic heterogeneity, reduced penetrance and variable expressivity owing to complex inheritance of the disease. Nevertheless, there is also a need to better understand environmental factors that possibly cause CHD, such as intrauterine stress shortly after conception owing to environmental contaminants. Indeed, there is evidence that in utero DNA damage from environmental pollution is associated with somatic gene mutation in newborns [START_REF] Perera | In utero DNA damage from environmental pollution is associated with somatic gene mutation in newborns[END_REF], and that there are indications for increased risk of CHD in polluted areas [START_REF] Brook | Air pollution and cardiovascular disease: a statement for healthcare professionals from the Expert Panel on Population and Prevention Science of the American Heart Association[END_REF]. Additional research areas should be addressed: 1. To gain deeper knowledge of how epigenetic regulation influence molecular mechanisms of heart development and its disruptions in CHD. Epigenetics describes heritable changes in gene expression that are not coded in the DNA sequence, but an interplay of DNA methylation, histone modifications and expression of non-coding RNAs, in the regulation of gene expression patterns. Epigenetic mechanisms are essential for development and differentiation, but they can become misdirected leading to birth malformations and various human diseases. Indeed, efforts are now underway in this direction, where for instance, deletion of miRNAs in mice during cardiogenesis has been shown to result in double-outlet right ventricle with a concurrent ventricular septal defect [START_REF] Saxena | miRNA-processing enzyme Dicer is necessary for cardiac outflow tract alignment and chamber septation[END_REF]. 2. To decipher mechanisms of chromatin remodeling and DNA repair in heart development and CHD. Essentially, DNA is packaged, through histone and non-histone proteins, into a condensed structure termed chromatin. The nucleosome is the basic repeating unit of chromatin, consisting of about 147-bp of DNA wrapped approximately two times around a histone core comprising two copies of each of the histone proteins H2A, H2B, H3 and H4.

Through chromatin remodeling, histone-DNA is disrupted resulting in a 'relaxed' chromatin, which then allows transcriptional machinery contact DNA to turn genes 'on' or 'off'. But more important, chromatin remodeling also facilitates DNA-damage-response proteins access DNA lesions and eventually initiate repair to maintain genome integrity (for reviews, [START_REF] Osley | Altering nucleosomes during DNA double-strand break repair in yeast[END_REF][START_REF] Osley | ATP-dependent chromatin remodeling factors and DNA damage repair[END_REF]. Chromatin remodeling factors may involve those modifying enzymes that catalyze post-translational modifications of residues on both histone core and tails; or those enzymes that use ATP hydrolysis to disrupt histone-DNA leading to structurally showing an essential role of chromatin remodeling with respect to transcriptional regulation in heart development and CHD [START_REF] Lickert | Baf60c is essential for function of BAF chromatin remodelling complexes in heart development[END_REF][START_REF] Wang | Polybromo protein BAF180 functions in mammalian cardiac chamber maturation[END_REF], none has been found so far on DNA repair. The role of DNA repair would be of utmost importance during early heart development, notably in embryonic cells and committed precursors of the mesoderm.

3. To identify molecular causes of hypermutability of genes and subsequent contribution to CHD, and explore whether activation-induced cytidine deaminase (AID) causes somatic hypermutability of cardiogenic genes in CHD. AID is a master regulator of diversification of antibody genes by initiating the processes of somatic hypermutation (SHM), gene conversion (IGC), and class-switch recombination (CSR) in B-lymphocytes (see reviews, [START_REF] Teng | Immunoglobulin somatic hypermutation[END_REF][START_REF] Liu | Balancing AID and DNA repair during somatic hypermutation[END_REF]. Essentially, AID induces somatic hypermutability of immunoglobulin (Ig) genes by cytosine deamination resulting in the generation of a uracil and thus converting a C:G basepair into a U:G mismatch, which is then processed into a mutation by different mechanisms. For instance, DNA replication over the uracil leads to C to T or G to A transition mutations. Alternatively, removal of the uracil by uracil-DNA glycosylase (UNG) generates an abasic site in the DNA, which when replicated across yields transition and transversion mutations at the C:G basepair. The U:G mismatch can also be recognized by the mismatched repair factors allowing the generation of either mutations at A:T basepairs by error prone patch repair or a nicked DNA recombination substrate.

However, there are now evidences for AID to deaminate not only Ig genes, but many more genes including numerous oncogenes implicated in B-cell tumorigenesis [START_REF] Liu | Balancing AID and DNA repair during somatic hypermutation[END_REF]. Indeed, AID can be potentially functional in a variety mammalian of non-B cells triggering genome-wide hypermutation and consequently to genetic instability and neoplasm [START_REF] Perez-Duran | Oncogenic events triggered by AID, the adverse effect of antibody diversification[END_REF]. [START_REF] Kasahara | Loss of function and inhibitory effects of human CSX/NKX2.5 homeoprotein mutations associated with congenital heart disease[END_REF] no DNA binding, no transactivation, inhibited transcriptional activity of NKX2-5 WT (WT + Q170X less transactivation than in WT alone) [START_REF] Zhu | Y259X 16 defective in functional assays* (DNA binding/transactivation activity/protein-protein interaction/ nuclear localization) c[END_REF] loss of transactivation, WT + Q170X less transactivation than in WT alone, no DNA binding, diffused localization in nucleus and cytoplasm, inhibited synergistic NKX2-5-GATA4 interaction [START_REF] Monzen | Dual effects of the homeobox transcription factor Csx/Nkx2-5 on cardiomyocytes[END_REF] reduced expression and transactivation of cardiac-specific genes, inhibited cardiomyocyte differentiation, promoted stress-induced cell death of cardiomyocytes functional assay/ in vitro cardiomyocyte differentiation system 20. c.512T>C p.L171P [START_REF] Kasahara | Biochemical analyses of eight NKX2.5 homeodomain missense mutations causing atrioventricular block and cardiac anomalies[END_REF] HD [START_REF] Kasahara | Biochemical analyses of eight NKX2.5 homeodomain missense mutations causing atrioventricular block and cardiac anomalies[END_REF] anomalous nuclear localization, reduced DNA-protein binding reduced transactivation, less efficient interaction with GATA4 and TBX5 functional assay 21. c.533C>T p.T178M [START_REF] Schott | Congenital heart disease caused by mutations in the transcription factor NKX2-5[END_REF][START_REF] Hirayama-Yamada | Phenotypes with GATA4 or NKX2.5 mutations in familial atrial septal defect[END_REF]) [START_REF] Elliott | Cardiac homeobox gene NKX2-5 mutations and congenital heart disease: associations with atrial septal defect and hypoplastic left heart syndrome[END_REF] HD [START_REF] Kasahara | Loss of function and inhibitory effects of human CSX/NKX2.5 homeoprotein mutations associated with congenital heart disease[END_REF], markedly reduced DNA binding and transactivation [START_REF] Zhu | Y259X 16 defective in functional assays* (DNA binding/transactivation activity/protein-protein interaction/ nuclear localization) c[END_REF] reduced transactivation, inhibited transcriptional activity of NKX2-5 WT (WT + T178M less transactivation than in WT alone, reduced DNA binding), inhibited NKX2-5-GATA4 synergistic interaction functional assay/ in vitro cardiomyocyte differentiation system

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Human Mutation

F o r P e e r R e v i e w [START_REF] Monzen | Dual effects of the homeobox transcription factor Csx/Nkx2-5 on cardiomyocytes[END_REF] reduced expression and transactivation of cardiac-specific genes, inhibited cardiomyocyte differentiation, promoted stress-induced cell death of cardiomyocytes [START_REF] Kasahara | Biochemical analyses of eight NKX2.5 homeodomain missense mutations causing atrioventricular block and cardiac anomalies[END_REF], anomalous nuclear localization, less repressor activity than GAL4-NKX2-5_WT (repressor) [START_REF] Inga | Functional dissection of sequence-specific NKX2-5 DNA binding domain mutations associated with human heart septation defects using a yeast-based system[END_REF] HD [START_REF] Kasahara | Biochemical analyses of eight NKX2.5 homeodomain missense mutations causing atrioventricular block and cardiac anomalies[END_REF] reduced DNA binding, reduced transactivation, reduced interaction with TBX5 functional assay 24. c.564C>A p.N188K [START_REF] Benson | Mutations in the cardiac transcription factor NKX2.5 affect diverse cardiac developmental pathways[END_REF] HD [START_REF] Kasahara | Loss of function and inhibitory effects of human CSX/NKX2.5 homeoprotein mutations associated with congenital heart disease[END_REF] markedly reduced DNA binding and transactivation [START_REF] Kasahara | Biochemical analyses of eight NKX2.5 homeodomain missense mutations causing atrioventricular block and cardiac anomalies[END_REF] less repressor activity than GAL4-NKX2-5_WT (repressor) functional assay 25. c.565C>G p.R189G [START_REF] Benson | Mutations in the cardiac transcription factor NKX2.5 affect diverse cardiac developmental pathways[END_REF] HD [START_REF] Kasahara | Loss of function and inhibitory effects of human CSX/NKX2.5 homeoprotein mutations associated with congenital heart disease[END_REF] markedly reduced DNA binding and transactivation, inhibited transcriptional activity of NKX2-5 WT (WT + R189G much less transactivation than in WT alone) [START_REF] Kasahara | Biochemical analyses of eight NKX2.5 homeodomain missense mutations causing atrioventricular block and cardiac anomalies[END_REF] 28. c.572A>G p.Y191C [START_REF] Benson | Mutations in the cardiac transcription factor NKX2.5 affect diverse cardiac developmental pathways[END_REF] HD [START_REF] Kasahara | Loss of function and inhibitory effects of human CSX/NKX2.5 homeoprotein mutations associated with congenital heart disease[END_REF], [START_REF] Kasahara | Biochemical analyses of eight NKX2.5 homeodomain missense mutations causing atrioventricular block and cardiac anomalies[END_REF] markedly reduced DNA binding and transactivation, inhibited transcriptional activity of NKX2-5 WT (WT + Y191C less transactivation than in WT alone) functional assay 29. c.592C>T p.Q198X [START_REF] Schott | Congenital heart disease caused by mutations in the transcription factor NKX2-5[END_REF]) [START_REF] Hosoda | Familial atrial septal defect and atrioventricular conduction disturbance associated with a point mutation in the cardiac homeobox gene CSX/NKX2-5 in a Japanese patient[END_REF] 3' coding region [START_REF] Kasahara | Loss of function and inhibitory effects of human CSX/NKX2.5 homeoprotein mutations associated with congenital heart disease[END_REF] reduced DNA binding affinity as dimer [START_REF] Zhu | Y259X 16 defective in functional assays* (DNA binding/transactivation activity/protein-protein interaction/ nuclear localization) c[END_REF] reduced transactivation, WT + Q198X much increased transactivation than in WT alone, enhanced NKX2-5-GATA4 synergistic interaction, overexpression in cultured cardiomyocytes induced apoptosis [START_REF] Monzen | Dual effects of the homeobox transcription factor Csx/Nkx2-5 on cardiomyocytes[END_REF] lower cardiomyocyte differentiation efficiency, promoted stressinduced cell death of cardiomyocytes functional assay / in vitro cardiomyocyte differentiation system 30. c.605_606delTG p.L202fs [START_REF] Sarkozy | Spectrum of atrial septal defects associated with mutations of NKX2.5 and GATA4 transcription factors[END_REF]

  Figures and Tables should be prepared in a single MS Word *.doc file labeled 'Supp_Mat', with Figures preceding Tables. Each table/figure should be accompanied with its legend.

  in Human Congenital Heart Disease (CHD) Stella Marie Reamon-Buettner and Juergen Borlak* Molecular Medicine and Medical Biotechnology, Fraunhofer Institute of Toxicology and Experimental Medicine, Nikolai-Fuchs-Strasse 1, D-30625 Hannover, Germany. *Correspondence to Prof. Dr. Juergen Borlak, Molecular Medicine and Medical Biotechnology, Fraunhofer Institute of Toxicology and Experimental Medicine, Nikolai-Fuchs-Strasse 1, 30625 Hannover, Germany. Tel: +49-511-5350-559; Fax +49-511-5350-573; E-mail: borlak@item.fraunhofer.de Contract grant sponsors: Ministry of Science and Culture, Lower Saxony, Germany; Contract grant number: 25A.5-76251-99-3/00 Introduction Cardiac malformations in congenital heart disease (CHD) result from errors during heart development, a process which is very complex and completed in a relatively short period of time. Essentially within eight weeks of embryogenesis, the human heart undergoes several stages of development involving cardiac crescent formation, linear heart tube formation, cardiac looping, chamber formation and maturation, septation and valve formation.

  the atrioventricular node, heterozygotes have half the number of cells.

  once in unrelated individuals, with c.73C>T (p.R25C) being frequently

  resulted in reduced or loss of DNA binding, transactivation

  Mef2c, and the physical, functional, and genetic interactions between

  have been identified in patients with various cardiac anomalies. Furthermore, there are now studies showing that defects in chromatin remodeling can lead to CHD. Chromatin remodeling allows transcriptional machinery access to gene regulatory elements and is essential in heart development. Silencing of Smarcd3, which encodes Baf60c, a subunit of the Brg1-associated-factor (BAF) chromatin remodeling in ES-cell-derived mouse embryos severe cardiac malformations including hypoplastic atrium, hypoplastic right ventricle, single ventricle, abnormal outflow tract and severely impaired trabecular formation

  persistent truncus arteriosus (PTA). Reduction or absence of Pbx2 or Pbx3 leads to Pbx1 haploinsufficiency and specific malformations that resemble Tetralogy of Fallot, overriding aorta with ventricular septal defect, and bicuspid aortic valves. Disruption of Meis1, which encodes a Pbx DNA-binding partner, results in cardiac anomalies that resemble those caused by Pbx mutations. Altogether, these findings among others challenge the well-accepted paradigm in autosomal dominant monogenic disease that 1 mutation in a single gene as the direct cause of disease, and have major implication in clinical evaluation, diagnosis, and management of families with a genetic cardiovascular disease (see for instance,[START_REF] Kelly | Multiple mutations in genetic cardiovascular disease: a marker of disease severity?[END_REF].

  or to sliding or eviction of nucleosomes. Although there are now studies

Figure 1 .

 1 Figure 1. Summary of germline NKX2-5 mutations in CHD, detection frequency in sporadic

Figure 2 .

 2 Figure 2. Facts about germline NKX2-5 mutations.

Figure 3 .

 3 Figure 3. Predictions of the effect of NKX2-5 mutations on the secondary structure of the

Figure 4 .

 4 Figure 4. NKX2-5 mutations affect conserved regions of the homeodomain. Protein modelling

  Summary of germline NKX2-5 mutations in CHD, detection frequency in sporadic or familial cases, and associated cardiac malformations. 215x189mm (300 x 300 DPI) Predictions of the effect of NKX2-5 mutations on the secondary structure of the protein. NKX2-5 interacts with DNA through a helix-turn-helix DNA-binding motif of three alpha helices within the homeodomain (HD). The amino acids in the alpha helices are underlined and those affected by NKX2-5 mutations are indicated by asterisks. Some NKX2-5 mutations can lead to breakage of the structure (arrows), and thus a truncated protein. For predictions, amino acids in the Swiss Model for NKX2-5 (P52952) (http://swissmodel.expasy.org) were mutated using tools contained in Swiss-PdbViewer (http://spdbv.vital-it.ch/), and the resulting structure viewed through Protein Explorer (http://proteinexplorer.org). 227x188mm (200 x 200 DPI) Predictions of the effect of NKX2-5 mutations on the secondary structure of the protein. NKX2-5 interacts with DNA through a helix-turn-helix DNA-binding motif of three alpha helices within the homeodomain (HD). The amino acids in the alpha helices are underlined and those affected by NKX2-5 mutations are indicated by asterisks. Some NKX2-5 mutations can lead to breakage of the structure (arrows), and thus a truncated protein. For predictions, amino acids in the Swiss Model for NKX2-5 (P52952) (http://swissmodel.expasy.org) were mutated using tools contained in Swiss-PdbViewer (http://spdbv.vital-it.ch/), and the resulting structure viewed through Protein Explorer (http://proteinexplorer.org). 227x188mm (200 x 200 DPI) affect conserved regions of the homeodomain. Protein modelling and identification of conserved regions were undertaken using Consurf (http://consurf.tau.ac.il/) and Chimera (http://www.cgl.ucsf.edu/chimera/). 187x155mm (300 x 300 DPI) affect conserved regions of the homeodomain. Protein modelling and identification of conserved regions were undertaken using Consurf (http://consurf.tau.ac.il/) and Chimera (http://www.cgl.ucsf.edu/chimera/). 187x155mm (300 x 300 DPI) Geographical distribution of germline NKX2-5 mutations in CHD along the coding sequence of the gene.

Table 1 .

 1 Functional Consequences of Identified NKX2-5 Mutations in CHD: Functional Assays or Bioinformatic Analysis

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

Table 2 .

 2 Detection Frequency of NKX2-5 Germline Mutations in Congenital Heart Disease

	Mutation-
	Positive	Patients	Frequency	Study	Country
	Patients	Analyzed	(%)		Study/Patients
	1	109	0.9	(Ikeda et al., 2002)	Japan
	18	608	3.0	(McElhinney et al., 2003)	USA
	2	146	1.4	(Elliott et al., 2003)	Australia, USA
	0	227	0	(Hobbs et al., 2005)	USA
	3	16	18.8	(Hirayama-Yamada et al., 2005) Japan
	0	205	0	(Posch et al., 2008)	Germany
	1	72	1.4	(Akcaboy et al., 2008)	Turkey
	2	159	1.3	(Gioli-Pereira et al., 2010)	Brazil
	0	230	0	(Zhang et al., 2009a)	China
	0	62	0	(Zhang et al., 2009b)	China
	6	125	4.8	(Esposito et al., 2009)	Italy
	0	104	0	(Hamanoue et al., 2009)	Indonesia
	33	2063	2.0	Total

Table S1 .

 S1 Studies on NKX2-5 Mutations Associated with Congenital Heart Disease (CHD)

			2009)				7 with ASD, 1 VSD)
	17. (c.479A>C)**		(Rifai et al., 2006)	HD	damaging			SIFT/Polyphen
	21. c.533C>T p.Q160P		p.T178M	HD	AV block,	familial	n = 12	(Schott et al.,	USA
					ASD II		(2 families; 12 AV block,	1998)
	18. c.498_499 insC	(Sarkozy et al., 2005) HD	11 ASD II) truncated protein affecting HD, NK-SD
	c.533C>T p.E167fs				AV block,	familial	n = 2	(Hirayama-	Japan
					ASD II, atrial		1 AV block+ASD II, 1	Yamada et al.,
	Ref. No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 c.533C>T 22. c.554G>T 23. c.561G>C 24. c.564C>A p.Q170X 19. c.508C>T	Authors/Year Schott et al., 1998 Benson et al., 1999 Hosoda et al., 1999 Goldmuntz et al., 2001 Watanabe et al., 2002 Ikeda et al., 2002 Gutierrez-Roelens et al., 2002 McElhinney et al., 2003 Elliott et al., 2003 Kasahara and Benson, 2004 Sarkozy et al., 2005 Hobbs et al., 2005 Hirayama-Yamada et al., 2005 Gutierrez-Roelens et al., 2006 Rifai et al., 2006 Konig et al., 2006 F o r P e e r R e v i e w NKX2-5 Germline Mutation present absent x x x x x x x x x x x x x function x reduced LV LV function ) x tricuspid valve, abnormal TV, 1 reduced x fibrillation (AF) ASD II+AF) ASD II, HLHS familial n = 3 (1 phenotypically normal family member also positive; 1 ASD II +HLHS, 2 ASD II) p.W185L HD AV block, ASD II, VSD familial n = 3 (1 AV block +ASD II, 2 ASD II+ muscular VSD (swiss-cheese VSD) p.Q187H HD AV block, ASD II, anomalous systemic venous return familial n = 6 (2 positive members had anomalous venous return) p.N188K HD AV block, ASD II, abnormal familial n = 5 ( 5 AV block, 5 ASD II, 3 F o r P e e r R e v i e w (Schott et al., 1998) HD	2005) (Elliott et al., 2003) (Sarkozy et al., 2005) (Gutierrez-Roelens et al., 2002) (Benson et al., 1999)	Australia Italy Belgium USA
	17 18 19 25. c.565C>G	Majumdar et al., 2006 Pabst et al., 2008 Akcaboy et al., 2008 p.R189G	HD	tricuspid valve, x II abnormal x AV block, ASD	x familial	reduced LV function ASD, 1 abnormal TV, 3 n = 5 (5 AV block ), 4	1999) (Benson et al.,	USA
	20	Posch et al., 2008		reduced LV	x
	21	Khetyar et al., 2008		function	x
	22	Bjornstad and Leren, 2009		x
	23	Esposito et al., 2009		x
	24	Draus, Jr. et al., 2009		x
	25	Hamanoue et al., 2009			x
	26	Zhang et al., 2009a			x
	27	Zhang et al., 2009b			x
	28	Gioli-Pereira et al., 2010		x

John Wiley & Sons, Inc.Human Mutation

ACKNOWLEDGEMENTS

We thank the Institute of Anatomy, University of Leipzig for providing the heart collection; to Dr. Mike Resnick and Dr. Alberto Inga for the fruitful discussions; and A. Roskowetz and A.

Hiemisch, for the excellent technical support. The financial support of the Lower Saxony Ministry of Science and Culture, Germany to JB is greatly appreciated.

Contract grant sponsors: Ministry of Science and Culture, Lower Saxony, Germany; Contract grant number: 25A.

http://www.hgvs.org/dblist/glsdb.html

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

Supp. Table S2 [START_REF] Benson | Mutations in the cardiac transcription factor NKX2.5 affect diverse cardiac developmental pathways[END_REF] intronic, splice donor site [START_REF] Kasahara | Loss of function and inhibitory effects of human CSX/NKX2.5 homeoprotein mutations associated with congenital heart disease[END_REF] abolished intron splicing, poor accumulation of mutant protein in the cell, no DNA binding,and transactivation functional assay **supplied based on description in the paper or written in accordance with recommended mutation nomenclature. Nucleotide numbering reflects cDNA numbering with +1 corresponding to the A of the ATG translation initiation codon in the reference sequence NM_0004387.3, according to journal guidelines (www.hgvs.org/mutomen). The initiation codon is codon 1.