PhD Susanne Knappe
email: knappe@psychologie.tu-dresden.de

PhD Katja Beesdo-Baum

PhD Hans-Ulrich Wittchen
email: wittchen@psychologie.tu-dresden.de

Familial risk factors in social anxiety disorder: Calling for a family-oriented approach for targeted prevention and early intervention

Keywords: social anxiety, family, parental psychopathology, intervention, prevention

Within the last decade, social anxiety disorder (SAD) has been identified as a highly prevalent und burdensome disorder. Both the characterization of its symptomatology and effective treatment options are widely documented. Studies particularly indicate that SAD aggregates in families and has its onset in early adolescence. Given the family as an important context for children"s cognitive, emotional and behavioural development, familial risk factors could be expected to significantly contribute to the reliable detection of populations at risk for SAD. Reviewing studies on familial risk factors for SAD argues for the importance of parental psychopathology and unfavourable family environment, but also denotes to several shortcomings such as cross-sectional designs, short follow-up periods, diverging methodologies and the focus on isolated factors. Using a prospective longitudinal study that covers the high risk period for SAD, including a broader spectrum of putative risk factors may help to overcome many of the methodological limitations. This review sets out to develop a more familyoriented approach for predicting the onset and maintenance of SAD that may be fruitful to derive targeted prevention and early intervention in SAD.

Facets of social anxiety: Shyness, social fears, social phobia and social anxiety disorder

The concept of social anxiety includes a variety of phenomena that may range from shyness to more or less isolated social fears, up to the clinically relevant diagnostic prototype of DSM-IV [START_REF]DSM-IV. Diagnostic and Statistical Manual of Mental Disorders[END_REF] defined social phobia or social anxiety disorder. To disentangle these terms is essential for an unbiased communication in clinical and research practice.

Shyness or timidy (sometimes also called diffidence or retentiveness) has often been associated with social anxiety [START_REF] Chavira | Scrutinizing the relationship between shyness and social phobia[END_REF][START_REF] Moore | Interactions between mothers and children: impacts of maternal and child anxiety[END_REF], and refers to a normal variation of behaviour in social situations. Shy individuals usually react inhibited in unfamiliar situations or in relation with unfamiliar people. Mostly, shyness is a transitory condition and people divest their shyness when getting used to the situation [START_REF] Cheek | Varieties of shyness in adolesence and adulthood[END_REF]. The majority of shy individuals do not experience extensive fear, dysfunctional cognitions, or panic-attack like bodily sensations, and thus do not suffer from severe functional impairment [START_REF] Heiser | Differentiating social phobia from shyness[END_REF]. Shyness constitutes a non-pathological, common personality trait or temperamental concept [START_REF] Kagan | Temperamental contributions to social behavior[END_REF]108]. Nevertheless, it may predispose a person to develop excessive social fears, especially when it is combined with substantial concerns about being shy and evidence that it has a detrimental effect of functioning [START_REF] Heiser | Differentiating social phobia from shyness[END_REF]88].

In contrast, social fears relate specifically to the consistent fear to act in a way that will be humiliating or embarrassing, or to be negatively evaluated by others. Social fears may manifest fairly isolated in only one or two situations, but may also be more pervasive in a wider range of situations.

They include performance fears such as fear of public speaking or speaking in front of others, writing in public, reading aloud, entering a room, or taking tests. Other social fears relate to social interactions such as initiating and/or maintaining a conversation, talking to or dealing with others, using public restrooms, eating and/or drinking in public, or going to (social) activities or participating in social events. Unreasonably strong fears were found in 22.3% of male and in 32.2% of female adolescents aged 14 to 24 years [115]. Similarly, at least one fifth of adults reported unreasonable strong fears [START_REF] Fehm | Social phobia above and below the diagnostic threshold: Prevalence, comorbidity and impairment in the general population[END_REF].

When social fears cause significant distress or interference with daily life, psychosocial functioning and role-fulfillments, the diagnosis of social phobia should be considered. According to the DSM-IV, social phobia is defined as the marked and persistent fear of social or performance situations in which the person is exposed to unfamiliar people or to possible scrutiny by others [START_REF]DSM-IV. Diagnostic and Statistical Manual of Mental Disorders[END_REF]. Since DSM-III-R [START_REF]DSM-III-R. Diagnostic and Statistical Manual of Mental Disorders[END_REF], a specifier was introduced to distinguish between a circumscribed, more discrete (nongeneralised) and a generalised form of social phobia. About one third of social phobia cases are classified as the generalised subtype. However, uncertainty about the (existence of a) threshold for the more severe form and its possible association with avoidant personality disorder [START_REF] Heimberg | The issue of subtypes in the diagnosis of social phobia[END_REF]106] are discussed.

The term "social anxiety disorder" was introduced in the DSM-IV-TR [START_REF]DSM-IV-TR. Diagnostic and Statistical Manual of Mental Disorders[END_REF], because "social phobia" could inadvertently suggest to represent a form of specific phobia, neglecting the severity associated especially with the more generalised subtype. Subsequently, we prefer the term social anxiety disorder (SAD), to better acknowledge the many facets and heterogeneity of social anxiety.

We first report on the epidemiology and natural course of SAD, and then emphasise the role of familial risk factors for its targeted prevention and early intervention, calling for a more family-oriented approach towards SAD.

Epidemiology of social anxiety disorder

SAD is very common, with lifetime prevalences ranging from 0.5% [110] in a Korean sample to 13.3% [START_REF] Kessler | Lifetime and 12-month prevalence of DSM-III-R psychiatric disorders in the United States: Results from the National Comorbidity Study[END_REF] in the US. Rates for 12-month prevalences or more narrow time frames similarly vary considerably from 0.4% [START_REF] Neufeld | Incidence of DIS/DSM-IV social phobia in adults[END_REF] to 7.9% [START_REF] Kessler | Lifetime and 12-month prevalence of DSM-III-R psychiatric disorders in the United States: Results from the National Comorbidity Study[END_REF], probably due to different sampling, study designs, assessment strategies and applied diagnostic criteria. Overall, the average lifetime prevalence is about 6.7% and about 2.0 to 3.0% [START_REF] Fehm | Size and burden of social phobia in Europe[END_REF] for the last 12 months. SAD is almost twice as often present in females than in males [START_REF] De Graaf | Predictors of first incidence of DSM III R psychiatric disorders in the general population: findings from the Netherlands Mental Health Survey and Incidence Study[END_REF][START_REF] Lépine | The epidemiology of anxiety disorders: Prevalence and societal costs[END_REF]115] and is associated with a decline in school or work performances, (school) refusal, and disinterest in age-appropriate activities [START_REF] Ollendick | The developmental psychopathology of social anxiety disorder[END_REF]. SAD often hampers affected individuals from pursuing their academic achievements, leading to lower educational attainment [START_REF] Katzelnick | Impact of generalized social anxiety disorder in managed care[END_REF][START_REF] Patel | The economic consequences of social phobia[END_REF], impaired work-performance, underemployment and lower household-income and subsequently high levels of financial dependence and lower socio-economic status [START_REF] Davidson | The epidemiology of social phobia: Findings from the Duke Epidemiological Catchment Area Study[END_REF][START_REF] Leon | The social costs of anxiety disorders[END_REF]. SAD places a substantial strain upon the health care system [START_REF] Alonso | Disability and quality of life impact of mental disorders in Europe: Results from the European Study of the Epidemiology of Mental Disorders (ESEMeD) project[END_REF], with only few costs related to SAD-specific treatment such as psychiatric and non-psychiatric care, hospitalization, and prescription drugs. Among the anxiety or mood disorders, SAD has the lowest treatment rates [START_REF] Issakidis | Service utilisation for anxiety in an Australian community sample[END_REF][START_REF] Patel | The economic consequences of social phobia[END_REF][START_REF] Runge | Wie häufig nehmen Jugendliche und junge Erwachsene mit Angststörungen eine psychotherapeutische Behandlung in Anspruch?[How frequent is psychotherapy utilisation by adolescents and young adults with anxiety disorders[END_REF], probably because the disorder is hardly detected by primary care physicians, affected individuals are afraid to disclose an illness condition, or because of financial strains and uncertainty over where to seek help [START_REF] Olfson | Barriers to the treatment of social anxiety[END_REF]. Less than 20% of affected individuals seek professional help [START_REF] Fehm | Size and burden of social phobia in Europe[END_REF][START_REF] Ormel | Disability and treatment of specific mental and physical disorders across the world[END_REF]. Most (indirect) costs are due to occupational disability like reduced productivity, absenteeism from work and suicide [START_REF] Katzelnick | Impact of generalized social anxiety disorder in managed care[END_REF][START_REF] Lépine | The epidemiology of anxiety disorders: Prevalence and societal costs[END_REF].

Natural Course: Age of onset, duration, remission and patterns of course

Most adults with SAD fail to recall the first onset of SAD symptoms or to remember a time when SAD was not a problem. Age of onset from retrospective reports of clinical studies was located in late adolescence and adulthood [START_REF] Keller | Social anxiety disorder clinical course and outcome: Review of Harvard/Brown Anxiety Research Project (HARP) findings[END_REF]113]. Therein, age of onset may reflect the point in life at which symptoms have led to severe impairment requiring treatment, after the disorder has been present for a considerable proportion of years. In contrast, studies based on representative community samples that included adolescents, age of onset was corrected towards an earlier onset between ages 10 to 16.6 years [113]. The high risk period for SAD was found to increase steeply after the age of 9 and then gradually decreased [START_REF] Beesdo | Incidence of social anxiety disorder and the consistent risk for secondary depression in the first three decades of life[END_REF]. Onset of the disorder after 20 years of age is rare, and no further peaks for incidence are expected.

Mean duration for SAD symptoms ranges from 19 to 21 years in clinical studies, and from 19 to 25 years in community studies [START_REF] Fehm | Social phobia above and below the diagnostic threshold: Prevalence, comorbidity and impairment in the general population[END_REF]113]. SAD does therefore not appear to be a transitory condition in adolescence and young adulthood. In the absence of effective treatment, SAD follows a chronic, unremitting course, that may also increase the likelihood of comorbid or subsequent other mental disorders.

SAD symptoms may not be consistently present on the DSM-IV diagnostic threshold level, as affected individuals also experience periods of lower symptom load or even symptom-free-periods. For example, among those with a lifetime SAD diagnosis, often less than one third also report SAD within the last 12 months [START_REF] Becker | Incidence and prevalence rates of mental disorders in a community sample of young women: Results of the "Dresden Study[END_REF], and individuals rarely meet the threshold for SAD consistently at subsequent assessment waves [START_REF] Merikangas | Family and high-risk studies of social anxiety disorder[END_REF]. Accordingly, SAD may fluctuate in severity [START_REF] Becker | Incidence and prevalence rates of mental disorders in a community sample of young women: Results of the "Dresden Study[END_REF][START_REF] Last | A prospective study of childhood anxiety disorders[END_REF], but persists on a symptom level with frequent oscillations around the diagnostic threshold [114]. About half of SAD cases have syndromal shifts [START_REF] Keller | Social anxiety disorder clinical course and outcome: Review of Harvard/Brown Anxiety Research Project (HARP) findings[END_REF], which is similarly observed in other anxiety disorders. Adolescent SAD is associated with SAD in adulthood [START_REF] Pine | The risk for early-adulthood anxiety and depressive disorders in adolescents with anxiety and depressive disorders[END_REF], other anxiety [START_REF] Last | A prospective study of childhood anxiety disorders[END_REF], depressive [START_REF] Beesdo | Incidence of social anxiety disorder and the consistent risk for secondary depression in the first three decades of life[END_REF]100], and substance use disorders [START_REF] Kim-Cohen | Prior juvenile diagnosis in adults with mental disorders: developmental follow-back of a prospectivelongitudinal cohort[END_REF]117], suggesting both homotypic and heterotypic continuity of SAD.

Although effective treatment is available, the chance for complete and long-lasting remission from SAD is the lowest among all anxiety disorders, especially in the generalised subtype and in the presence of comorbidities like avoidant personality disorder [START_REF] Rapee | The etiology of social phobia: empirical evidence and an initial model[END_REF]. Remission is most likely in the first years after onset when complications of the disorder or comorbidities have not yet developed [118].

Remission rates in adults are often derived from the consecutive observational studies of the Harvard-Brown Anxiety Research Program (HARP). Depending on the lenght of follow-up periods, full remission rates vary between 35% and 75% [START_REF] Keller | Social anxiety disorder clinical course and outcome: Review of Harvard/Brown Anxiety Research Project (HARP) findings[END_REF]. Because remission criteria in the HARP studies refer to the absence of symptoms for only eight consecutive weeks, remission rates are however likely overestimated. The relapse rate, once recovery was achieved, was 34%, and increased to 75%, when depression is comorbid [START_REF] Bruce | Influence of psychiatric comorbidity on recovery and recurrence in generalized anxiety disorder, social phobia, and panic disorder: A 12-year prospective study[END_REF]. As time to second relapse was shorter than the time to first relapse, duration of the symptom-free interval appears to decrease over time. In children and adolescents, spontaneous remission rates are usually higher than in adults (up to 50% after 15 month [START_REF] Essau | Course and outcome of anxiety disorders in adolescents[END_REF]), but reduce when also subthreshold expressions of SAD and subsequent incidence of other anxiety disorders are considered [START_REF] Beesdo | Incidence of social anxiety disorder and the consistent risk for secondary depression in the first three decades of life[END_REF][START_REF] Last | A prospective study of childhood anxiety disorders[END_REF].

Probably due to the waxing and waning of symptoms, predictors for an unfavourable course of SAD remain unspecified. In the HARP-studies, female gender, early age of onset, longer duration of illness, lifetime history of various anxiety disorders, current comorbidity of anxiety or depressive disorders, low global functioning or measures of role functioning were not associated with lack of remission or stability of symptoms [START_REF] Reich | A prospective, follow-along study of the course of social phobia: II. Testing for basic predictors of course[END_REF]. In the same study, however, women were more likely to follow a chronic course than men, especially when the global functioning at study entry was low and they had a history of suicide attempts [118]. In adolescents, presence of SAD symptoms from baseline to 15 month follow-up was predicted by higher age, comorbid anxiety, somatoform and substance use disorders, but not by female gender, comorbid depressive disorders and attachment style [START_REF] Essau | Course and outcome of anxiety disorders in adolescents[END_REF]. ###Box 1 starts here### SAD is the most prevalent anxiety disorder, with first onset in late childhood and early adolescence. In the absence of effective treament, symptoms persist, often waxing and waning around the diagnostic threshold and increasing the likelihood for a range of comorbid disorders, and psychosocial maladjustment in the long run. Given the demonstrated size and burden of SAD, reliable factors are warranted to identify individuals at risk for SAD as early as possible, and to allocate them to prevention and once SAD has manifested, to targeted intervention.

###Box 1 ends here### (Familial) risk factors for social anxiety disorder

Traumatic events, peer relationships, social skills deficits, social cognition/information processing have been discussed as putative risk factors for SAD [START_REF] Rapee | The etiology of social phobia: empirical evidence and an initial model[END_REF]. Since the high risk phase for SAD onset is located in late childhood/ early adolescence, familial risk factors are likely to be particularly important for targeted prevention and intervention in SAD 1 .

Parental SAD and other mental disorders. Family and high risk studies in clinical and community samples demonstrated that SAD aggregates in families (Table 1). Socially anxious children are more likely to have parents with SAD.

-Please insert Table 1 about here -In family (genetic) studies that often excluded probands with comorbid anxiety disorders, relatives of socially phobics were at higher risk for social phobia exclusively [START_REF] Fyer | A direct interview family study of social phobia[END_REF][START_REF] Fyer | Specificity in familial aggregation of phobic disorders[END_REF][START_REF] Reich | Family history of psychiatric disorders in social phobia[END_REF]. Findings are defeasible by small or clinical samples, retrospective or indirect assessment of psychopathology in only one family member instead of direct interviews, and differences between parent and child reports.

Studies also sometimes fail to observe double-blind diagnostic evaluation. This susceptibility does not appear to be limited to parental SAD, as other parental anxiety, depressive or alcohol use disorders have also been shown to be associated with offspring SAD above [START_REF] Bögels | Familial correlates of social anxiety in children and adolescents[END_REF][START_REF] Fyer | A direct interview family study of social phobia[END_REF][START_REF] Knappe | The role of parental psychopathology and family eEnvironment for social phobia in the first three decades of life[END_REF][START_REF] Mcclure | Parental anxiety disorders, child anxiety disorders, and the perceived parent-child relationship in an Australian high Risk Sample[END_REF], and below the diagnostic threshold [START_REF] Knappe | Associations of familial risk factors with social fears and social phobia: Evidence for the continuum hypothesis in social anxiety disorder ?[END_REF]. The SAD-specific parent-to-offspring transmission though appears to be limited to DSM-IV threshold SAD [START_REF] Knappe | Associations of familial risk factors with social fears and social phobia: Evidence for the continuum hypothesis in social anxiety disorder ?[END_REF]. Thus, the familial transmission of SAD is probably characterised by low to modest specificity, and is likely to be determinated by cross-disorder risk factors, as well as family-based developmental conditions.

The familial aggregation is often reported to be stronger in the generalised subtype, indicating that this is the more familial form of the disorder. This conclusion is based on studies [START_REF] Fyer | A direct interview family study of social phobia[END_REF][START_REF] Mannuzza | Generalized social phobia: Reliability and validity[END_REF]100,101], which have also been subject to criticism. Findings were limited by the number and specific types of situations assessed, and there was no evidence that patients with the generalised subtype were more likely to transmit the generalised form [START_REF] Mannuzza | Generalized social phobia: Reliability and validity[END_REF]. Findings were not compared to relatives of nongeneralised social phobics [100], and have not yet been replicated in independent samples. In one of our own studies [START_REF] Knappe | The role of parental psychopathology and family eEnvironment for social phobia in the first three decades of life[END_REF], we used the number of social fears as a proxy for the generalised SAD subtype.

Therein, associations between the number of offsprings" social fears were most pronounced in the presence of parental SAD, other anxiety disorders, and depression, but not parental alcohol use disorders, suggesting a possible familial liability for internalising disorders.

Twin and adoption studies may help to further dissect the familial aggregation into that due to genetic and environmental factors. Up to date, no adoption study on SAD is available. Most of the twin studies have been conducted by Kendler and co-workers [START_REF] Kendler | Sex differences in genetic and environmental risk factors for irrational fears and phobias[END_REF][START_REF] Kendler | Fears and phobias: Reliability and heritability[END_REF][START_REF] Kendler | The genetic epidemiology of irrational fears and phobias in men[END_REF] using data of the Virginia Twin Registry (Table 2). Heritability of SAD ranges from 0.20 to 0.50, indicating that the phenotypic variation is moderately attributable to additive genetic factors. It is also suggested that men and women differ in the extent to which genetic and environmental factors contribute to SAD: Twin resemblance was best explained by (non-shared) factors of family-environment in females, and by (shared) genetic factors in males. Findings have been variously questionned. Particularly, those of the Kendler-studies were not always replicable (cf. [START_REF] Hettema | The structure of genetic and environmental risk factors for anxiety disorders in men and women[END_REF]; for findings in other twin samples also cf. [START_REF] Distel | Heritability of self-reported phobic fear[END_REF][START_REF] Middeldorp | Familial clustering of major depression and anxiety disorders in Australian and Dutch twins and siblings[END_REF]). For example, no effects of non-shared environment were found in the Missouri Adolescent Female Twin Study [START_REF] Nelson | Social phobia in a population-based female adolescent twin sample: co-morbidity and associated suicide-related symptoms[END_REF]. More generally, heritability estimates may be artificially inflated, as monozygotic twins may experience more similar environments than dizygotic twins. Also, dizygotic twins may differ from monozygotic twins in gestational periods in a way that could also lead to overestimation of heritability in monozygotic twins [START_REF] Elizabeth | Social anxiety disorder in children and youth: A research update on aetiological factors[END_REF]. So far, the overall heritability of SAD appears to be modest, and familial resemblance in both males and females with SAD is at least in part due to genetic factors [START_REF] Kendler | Sex differences in genetic and environmental risk factors for irrational fears and phobias[END_REF].

-Please insert Table 2 about here -Some studies have linked the familial aggregation of SAD with behavioural inhibition (BI; [START_REF] Biederman | Psychiatric correlates of behavioral inhibition in young children of parents with and without psychiatric disorders[END_REF][START_REF] Rosenbaum | Further evidence of an association between behavioral inhibition and anxiety disorders: results from a family study of children from a non-clinical sample[END_REF]. BI is defined as a temperamentally based disposition, characterised by fear, avoidance or restraint and withdrawal in both social and non-social unfamilial events [START_REF] Kagan | Temperamental contributions to social behavior[END_REF]. The association between BI and anxiety disorders was found to be more pronounced in children of parents with anxiety disorders [START_REF] Hirshfeld | Stable behavioral inhibition and its association with anxiety disorder[END_REF], and vice versa, increased rates of anxiety disorders were seen in parents of inhibited children. Hence, the association between BI and anxiety disorders is familial, perhaps genetic [START_REF] Elizabeth | Social anxiety disorder in children and youth: A research update on aetiological factors[END_REF],

and BI may serve as a genetic liability to develop SAD and other anxiety disorders. Accordingly, twin studies demonstrated a genetic component to BI, with heritability ranging between 0.4-0.7 at 14 to 24 month [START_REF] Dilalla | Genetic etiology of behavioral inhibition among 2year-old children[END_REF][START_REF] Robinson | The heritability of inhibited and uninhibited behavior: A twin study[END_REF]. Beyond BI, also the role of (heritable) anxiety related traits such as higher levels of neuroticism, lower levels of extraversion or high introversion [START_REF] Bienvenu | Low extraversion and high neuroticism as indices of genetic and environmental risk for social phobia, agoraphobia, and animal phobia[END_REF] and harm avoidance [START_REF] Lochner | Genetics and personality traits in patients with social anxiety disorder: A case-control study in South Africa[END_REF] have been found to be positively associated with lifetime SAD.

Both the familial aggregation and the associations with temperamental (risk) factors stimulated the search for genetic polymorphisms that contribute to the endophenotype of SAD [START_REF] Lochner | Genetics and personality traits in patients with social anxiety disorder: A case-control study in South Africa[END_REF]. Up to date, few studies examined potential candidate genes. Therein, the role of specific genes encoding components of serotonergic (5-HT) and dopaminergic pathways in patients with SAD appears crucial for SAD [START_REF] Domschke | Blushing propensity in social anxiety disorder: influence of serotonin transporter gene variation[END_REF][START_REF] Gelernter | Genome-wide linkage scan for loci predisposing to social phobia: Evidence for a chromosome 16 risk locus[END_REF]. Their true value however remains unclear until conditions for the expression of these candidate genes, and their association with other (anxiety) disorders have been clarified and validated.

Unfavourable family environment. Genetic factors appear to play only a modest (but nevertheless significant) role in the familial transmission of SAD. Some researchers suggest that family environment might be even more predictive for offsprings" mental health than parental diagnostic status [111].

Diverse family processes are suggested to provoke an enhancement of anxious and avoidant responses in children [START_REF] Barrett | Family enhancement of cognitive style in anxious and children[END_REF], such as insecure attachment [START_REF] Boer | Family and genetic influences: Is anxiety 'all in the family'?[END_REF]103], higher levels of expressed emotion

[104], excessive family cohesion [START_REF] Peleg-Popko | Marital quality, family patterns, and children's fears and social anxiety[END_REF]107], and disturbed family functioning [START_REF] Ballash | Family functioning, perceived control, and anxiety: A mediational model[END_REF]105]. Table 3 summarizes (however not exhaustively) studies on the associations between unfavourable parental rearing styles and offspring anxiety. Because few studies particularly focus on offsprings" SAD, studies on anxiety in general or on a broader symptomatic level were also taken into account. Parental rearing may be shared alike by all children in the family, but it is a unique factor, as the rearing style of parents is tailored to the specific child [START_REF] Boer | Family and genetic influences: Is anxiety 'all in the family'?[END_REF][START_REF] Hudson | Parental perceptions of overprotection: Specific to anxious children or shared between siblings?[END_REF].

-Please insert Table 3 about here -Retrospective studies in adolescents and young adults found lower levels of parental emotional warmth, higher levels of parental overprotection and rejection to be associated with offspring SAD [START_REF] Bögels | Familial correlates of social anxiety in children and adolescents[END_REF][START_REF] Knappe | The role of parental psychopathology and family eEnvironment for social phobia in the first three decades of life[END_REF][START_REF] Lieb | Parental psychopathology, parenting styles and the risk of social phobia in offspring: a prospectivelongitudinal community study[END_REF]. These findings have been questioned, as affected offsprings" reports may be subject to information processing [START_REF] Christensen | Social anxiety and interpersonal perception: a social relations model analysis[END_REF] or attributional bias due to their psychopathology [START_REF] Alfano | Cognitive correlates of social phobia among children and adolescents[END_REF]112].

Though this may apply to some findings, observational studies provide further evidence for the role of unfavourable family environment for offspring SAD [START_REF] Siqueland | Anxiety in children: perceived family environments and observed family interaction[END_REF]: For example, mothers of anxious children initiated more control, more involvement and negativity [START_REF] Hudson | Parent-child interactions and anxiety disorders: an observational study[END_REF] than mothers of unaffected offspring.

The complex steps of the transformation from parental rearing into particular behaviour or psychopathological disturbances are fairly unexplored. Findings are often interpreted in terms of social learning mechanisms [START_REF] Ollendick | Operant conditioning influences in childhood anxiety[END_REF], such as parental modelling of anxious or avoidance behaviour [START_REF] De Rosnay | Transmission of social anxiety from mother to infant: An experimental study using a social referencing paradigm[END_REF][START_REF] Muris | The role of parental fearfulness and modeling in children[END_REF][START_REF] Murray | The effects of maternal social phobia on mother-infant interactions and infant social responsiveness[END_REF],

or parental attitudes and actions [START_REF] Bögels | Family influences on dysfunctional thinking in anxious children[END_REF]111] by which parents actively manage the child"s behaviour [e.g., 77].

At least two types of family processes are probably involved in the familial transmission of (social) anxiety [START_REF] Boer | Family and genetic influences: Is anxiety 'all in the family'?[END_REF]: On the one hand, harbouring threat and/or providing insufficient protection from it, and promoting threat sensitivity and/or impeding the development of coping skills on the other hand may be contribute to offspring anxiety. Normatively, parents facilitate their children"s social interaction by arranging play dates, supporting participation in sport clubs or activities with peers. Parental overprotection or lack of emotional warmth may prevent offspring from acquiring social skills [START_REF] Rapee | The etiology of social phobia: empirical evidence and an initial model[END_REF], increase the offsprings" dependence upon their parents [116], prevent exposure and habituation to social interactions, thereby reinforcing the child"s anxiety and hampering the development of control, mastery and autonomy [89, 96 116]. Parents of anxious children may be more likely to become overinvolved with their child when attempting to reduce and prevent the child"s distress. This rearing behaviour would in turn reinforce the child"s anxiety by promoting negative beliefs about the world as a dangerous and uncontrollable place. Children who experience critical comments, rejection or feelings of shame by their parents may become preoccupied with evaluative components, leading to fear of negative evaluations, public self-consciousness and avoidance of social scrutiny [START_REF] Bruch | Familial and developmental antecendents of social phobia: Issues and findings[END_REF]. Overprotective parenting limits the offsprings" opportunities to access information that might disprove or correct inaccurate fear information, and preclude from unprejudiced and positive social interactions.

Otherwise, positive learning experiences from social interactions may be devaluated by parental critique.

Regardless of their plausibility, findings are challenged in two respects: First, McLeod, Wood, and Weisz [START_REF] Mcleod | Examining the association between parenting and childhood anxiety: A meta-analysis[END_REF] summarized that higher levels of parental "control" or "rejection accounted for only 6% and 4%, respectively, of the variance and therefore only moderately predict offspring anxiety. The association between parental rearing and offspring anxiety was largely affected by a range of methodological artefacts (conceptualizations of anxiety and parenting, assessment strategies, sample characteristics). Most importantly, rearing styles such as "control" or "rejection" are often conceptualized as bipolar dimensions with positive parenting at the one end and negative parenting at the other, probably ignoring underlying sub-dimensions of rearing behaviour. In fact, the amount of explained variance in offspring anxiety increased to 42% when autonomy-granting as one particular facet of parenting was considered. Second, findings do not sufficiently explain the underlying mechanisms of the relationship between unfavourable family environment and offspring SAD, and their specificity (i.e., relative to other anxiety disorders). Recent (experimental) studies suggest that it may not be parental rearing per se that leads to offspring SAD. Rather, some of the characteristic features of offspring SAD (e.g., interpretational bias, sensitivity to stressful or threatening events or avoidance behaviour) may be specifically transmitted through the cognitive style of parents [START_REF] Lester | Do anxious parents interpretive biases towards threat extend into their child's environment?[END_REF]:

Parents" verbal threat information may induce anxious cognitions, attentional bias to threat, and avoidance behaviour. In addition, non-verbal transmission processes such as observation of others reactions to social stimuli such as parental [START_REF] Muris | The role of parental fearfulness and modeling in children[END_REF] or maternal facial expressions [START_REF] Murray | Intergenerational transmission of social anxiety: The role of social referencing processes in infancy[END_REF], and maternal behaviour in social situations [START_REF] De Rosnay | Transmission of social anxiety from mother to infant: An experimental study using a social referencing paradigm[END_REF] may occur. There is however yet little evidence available on whether parent and offsprings" bias are correlated, and if a causal mechanism behind this association can be established. Interestingly, parents with higher trait anxiety may be more likely to disambiguate situations in a threatening way for their offspring, just like if parents were "training" their offspring towards a general tendency to interpret ambiguity in a threatening way [START_REF] Lester | Do anxious parents interpretive biases towards threat extend into their child's environment?[END_REF]. Also reciprocal influences remain understudied. ###Box 2 starts here### Parental psychopathology (e.g., genetics) appears to play only a modest, albeit non-specific role for the development of SAD. Family environment constitutes an important, and probably more specific familial risk factor for offspring SAD. The underlying mechanisms from parental psychopathology and/or parental rearing to offspring SAD are still under investigation, focussing on parents" harbouring of threat (or providing insufficient protection from it), and impeding the development of their offsprings" skills through parents" cognitive style. Understanding of the parent-to-offspring transmission is however hampared because studies often focus on either parental psychopathology or family environment.

###Box 2 ends here###

Interplay of parental psychopathology and family environment

Heritability estimates suggest that "genetic factors play a significant but by no means overwhelming role in the aetiology of phobia" [START_REF] Kendler | The genetic epidemiology of phobias in women: The interrelationship of agoraphobia, social phobia, situational phobia, and simple phobia[END_REF] (p. 279). Parental psychopathology may unspecifically prone an individual at risk to develop SAD (or any other mental disorder), but its interaction with individual specific factors (i.e., family environment) appears pivotal for the development and course of offspring SAD (in contrast to other offspring disorders): Affected parents have difficulties modelling appropriate coping strategies, by reacting towards their children"s fears negatively because they represent an aspect of themselves which they would rather deny, or becoming overly concerned about their children"s anxiety, resulting in overprotection and reduced opportunities for desensitization. In addition, affected parents may be limited in their ability to help their children to cope with SAD or to join social groups [START_REF] Ollendick | The developmental psychopathology of social anxiety disorder[END_REF].

Empirical evidence for an interaction of parental psychopathology and familial environment on

SAD is yet scarce. In one of our own studies [START_REF] Knappe | The role of parental psychopathology and family eEnvironment for social phobia in the first three decades of life[END_REF], we found the risk for offspring SAD to be particularly elevated in offspring whose parents had an anxiety, depressive or alcohol use disorder and who reported greater overprotection, lack of emotional warmth, dysfunctional problem solving, role behaviour or behaviour control, as compared to offspring of unaffected parents reporting a more favourable family environment. Notably, interactions with unfavourable parental rearing styles (overprotection, rejection, lack of emotional warmth) were most pronounced in offspring of parents with SAD. Similarly, parent-to-offspring transmission of cognitive bias was found to be more likely if parents have an anxiety disorder, and if parents" biases extend beyond their own environment into their offsprings" environment [START_REF] Lester | Do anxious parents interpretive biases towards threat extend into their child's environment?[END_REF], expected their offspring to make threatening interpretations of ambiguous situations [START_REF] Lester | Do anxious parents interpretive biases towards threat extend into their child's environment?[END_REF], demonstrated greater negative and less positive expectations on their children"s future performance, and responded to potentially threat provoking situations with catastrophizing [107] and avoidant solutions [START_REF] Cobham | Anxious children and their parents: What they do expect?[END_REF] than unaffected parents. The mediator/moderator role of parental psychopathology and unfavourable rearing styles has though yet to be examined.

Results of Turner et al.

[107] suggest a moderator role of parental psychopathology: Albeit they found that anxious parents did not restrict their children from playing or outgoing, anxious parents overtly expressed their concern and reported lower cohesion, expressiveness and higher conflict and achievement orientation than non-anxious parents. In addition, McClure et al. [START_REF] Mcclure | Parental anxiety disorders, child anxiety disorders, and the perceived parent-child relationship in an Australian high Risk Sample[END_REF] did not find a mediating role of parenting between maternal and child anxiety disorders, suggesting that affected children are more likely to perceive their parents negatively if their parents also have an anxiety disorder (in contrast: [START_REF] Knappe | The role of parental psychopathology and family eEnvironment for social phobia in the first three decades of life[END_REF]).

Also other factors such as offspring temperament likely exert their influence on the transactive parent-offspring-relationship, e.g., shy temperament may elicit overprotective parenting [START_REF] Manassis | The development of childhood anxiety disorders: toward an integrated model[END_REF][START_REF] Rubin | The transaction between parents' perceptions of their children's shyness and their parenting styles[END_REF].

Parents may react overinvolved to reduce the child"s distress, reinforcing the anxious temperament and keeping the child from learning or making the child rather focus on avoidance [START_REF] Rapee | The etiology of social phobia: empirical evidence and an initial model[END_REF]. Notably, parenting practices that promote child social anxiety are particularly likely to occur in anxious parents in the context of child inhibited, anxious or negative behaviour [START_REF] Murray | Intergenerational transmission of social anxiety: The role of social referencing processes in infancy[END_REF][START_REF] Rubin | The transaction between parents' perceptions of their children's shyness and their parenting styles[END_REF]. Anxious parents may overestimate their children"s vulnerability, anticipate catastrophic outcomes in potentially threatening situations, and respond by promoting avoidant solutions to challenges [START_REF] De Rosnay | Transmission of social anxiety from mother to infant: An experimental study using a social referencing paradigm[END_REF]116].

###Box 3 starts here###

In terms of a vulnerability-stress model, parental psychopathology may represent a more general (heritable) basis, that prones their offspring to develop SAD (and probably other disorders as well). In contrast, family environment factors such as dysfunctional parental rearing or disturbed family functioning are probably more specific risk factors for the onset and course of SAD-symptoms. In addition to their individual contributions, familial risk factors may also interact in terms of an accumulation of risk, as parents who pass on genes carrying psychological risks also tend to be the same parents who provide unfavourable rearing styles. Likewise, the expression of a specific genotype depends on the interplay of nature and nurture: On the one hand, the (genetic) susceptibility due to the familial aggregation of psychopathology may foster offspring"s" reaction to unfavourable family environment. On the other hand, unfavourable family environment may facilitate the outcome of the (genetic) transmission of parental psychopathology.

###Box 3 ends here###

Current limitations

Studies may provide insight into the possible developmental pathways for SAD. Nonetheless, conclusion are hampared due to methodological shortcomings in lieu of (diagnostic) assessment and study design.

On the one hand, SAD-diagnoses are based on non-standardised assessments including dimensional and categorical approaches (e.g., questionnaires, cut-off scores), but also on structured or standardised interviews (e.g, CIDI, SCID). Findings may be affected by information processing-and mood-congruent bias.

On the other hand, the majority of studies rather reflect correlational instead of causal relationships, thus evidence for a relationship between family environment factors and offspring anxiety is predominantly cross-sectional. Studies were unable to clearly discern whether the parental behaviours observed are responses to children"s psychopathology, or are antecedents and contributing risk factors. Therefore, whether the identified family factors actually cause childhood anxiety, still needs to be investigated. Further research that investigates mechanisms mediating the relationship between family factors and child anxiety is also called for.

Very few studies prospectively considered predictors for the natural course of the disorder, especially in young adolescent samples across the high risk period for SAD. Though it is likely that clinical characteristics of SAD, such as early age of onset, severity of symptoms, number of social fears and generalised subtype, avoidance and impairment, or comorbidities have an impact on the course of the disorder [START_REF] Noyes | Natural course of anxiety disorders[END_REF]; it remains basically unknown whether familial risk factors for the onset of SAD may also account for its further course. As mentioned above, the onset of SAD was predicted independently by parental psychopathology and unfavourable family environment, and also by their interaction. In contrast, the further course (e.g. persistence) of SAD was found to be rather predicted by characteristics of early SAD expressions [START_REF] Noyes | Natural course of anxiety disorders[END_REF] and unfavourable family environment [START_REF] Knappe | Do parental psychopathology and unfavorable family environment predict the persistence of social phobia?[END_REF]. Again, interactions suggest an accumulation of risk, namely that higher levels of parental overprotection were associated with higher persistence of offsprings" SAD when parental SAD was present. Accordingly, family environment (with parental rearing in particular) emerged as an important predictor for both the onset and course of the disorder. Findings argue for the prognostic value of early course characteristics for the future manifestation of SAD [START_REF] Noyes | Natural course of anxiety disorders[END_REF]. Course and outcome of SAD are however rarely characterised by simple linear relationships. Given the oscillating course of social phobia symptoms [START_REF] Merikangas | The spectrum of social phobia in the Zurich Cohort Study of Young Adults[END_REF]114], conclusions from the presence or absence of full diagnostic criteria are crude, neglecting the complexity and severity of the disorder, as also conditions below the diagnostic threshold lead to impairment in daily life and increase the risk for comorbid and subsequent psychopathology [START_REF] Fehm | Social phobia above and below the diagnostic threshold: Prevalence, comorbidity and impairment in the general population[END_REF].

Calling for a family-oriented approach in social anxiety disorder

During childhood and adolescence (i.e., the core SAD-incidence period), the family system probably represents one of the major developmental contexts, in which genetic predispositions cluster and interact with environmental factors. To consider the onset and course of SAD within a more family-oriented approach may help to expand our current knowledge about SAD-risk factors, and to provide a scientific basis for prevention and intervention: Assessment of familial risk factors may contribute to the reliable detection of high risk populations, and to delineate differential prevention strategies incorporating these familial contributions.

Increasing progress in studying gene-environment-interactions is underway, but still scarce for phobias [START_REF] Gregory | Finding gene-environment interactions for phobias[END_REF], and particularly for SAD [START_REF] Lau | Assessing gene-environment interactions on anxiety symptom subtypes across childhood and adolescence[END_REF]. To examine the independent and combined contributions of familial risk factors to SAD requires to longitudinally follow-up an unbiased, large representative sample across the high risk period for SAD onset and observe in its natural setting (instead of a small, cross-sectional clinically referred sample). Putative familial risk factors should be examined within one sample (instead of focusing on isolated factors), to allow for covariation among variables, but also to have sufficient sample size and statistical power to detect interaction between parental psychopathology and family environment on the risk for offspring SAD. The standardised assessment of a variety of psychopathology in offspring and their parents provides a reliable and valid basis for diagnoses. Finally, the frequent oscillations of SAD above and below the diagnostic threshold need to be considered when characterising the course of SAD.

The merits of such a family-oriented approach appear promising: The standardised assessment of offspring and parental psychopathology allows considering the associations of parental SAD and other disorders including their comorbidity with offspring SAD. Also, the specificity of the familial transmission of SAD can be explored in greater detail. Recent findings indicate at least some specificity: For example, Murray et al. [START_REF] Murray | The effects of maternal social phobia on mother-infant interactions and infant social responsiveness[END_REF] found that socially phobic mothers appeared more anxious, engaged less with the stranger, and were less encouraging to infant than mothers with generalised anxiety disorder or healthy controls. In addition, conditions of SAD above and below the DSM-IV diagnostic threshold can also be modelled, broadening the concept of SAD to social fears, and thereby investigating whether there is a continuum of social fears, social phobia and its subtypes, or even a graded relationship between familial risk with offspring SAD status in terms of a doseresponse-relationship [START_REF] Knappe | Associations of familial risk factors with social fears and social phobia: Evidence for the continuum hypothesis in social anxiety disorder ?[END_REF].

Summary and conclusion

There is an antagonism between the high prevalence of SAD in the community, associated individual and societal costs, and the minority of SAD cases that receive treatment. Up to date, knowledge about reliable risk factors, their interplay and predictors for the onset and course of SAD is too limited to derive precise strategies for prevention and intervention. Given the early onset of SAD in adolescence and young adulthood, the substantial familial aggregation and the family as an important context for cognitive, emotional and behavioural development, parental psychopathology and unfavourable family environment appear as promising targets for research. Taking into account clinical features of SAD (i.e., different types of social fears, interaction-vs. performance-related social fears, age at first onset, anxiety-related cognitions), and other putative risk factors (i.e., somatic conditions, natal complications) may further help to illustrate the development and phenomenolgy of this earlyonset anxiety disorder. There is a lack of longitudinal studies, prospectively covering the high risk period and assessing a wider range of putative risk factors to collect detailed and methodological sound data on SAD. Though such studies are extremely time-consuming, and expensive in regard to costs and personnel required, they allow us to examine the associations and interactions of familial risk factors with the onset and course of SAD, to thereby constitute a valuable basis for targeted prevention and early intervention in adolescents and young adults at risk.

Table 1 : The familial aggregation of social anxiety disorder

 1 Note: *calculated from publication; when both direct and indirect information on relative's diagnostic status was available, best-estimate diagnoses were derived Abbreviations in alphabetical order: CIDI Composite International Diagnostic Interview; DSM Diagnostic and Statistical Manual of Mental Disorders; EDSP Early Developmental Stages of Psychopathology-Study; FH-RDC Family History Research Diagnostic Criteria; FH-PD Family History for DSM-III anxiety and Personality Disorders; NIHM-DIS National Institute of Mental Health-Diagnostic Interview Schedule; SADS Schedule for Affective Disorders and Schizophrenia; SADS-LA Schedule for Affective Disorders and Schizophrenia, adopted for Lifetime Anxiety Disorders; SCID Structured Clinical Interview for DSM-Disorders; SPIKE Structured Psychopathological Interview and Rating of the Social Consequences for Epidemiology

	proband characteristics	1st degree relative characteristics	lifetime prevalence

Table 2 : Twin and genetic studies on social fears and social anxiety disorder

 2

				heritability
	study	traits / study outcome	population	age in years

Table 3 : Associations between unfavourable parental rearing styles and offspring anxiety / social anxiety disorder

 3

	parental rearing	parental characteristics	offspring characteristics		results	reference
	assessed with	N	assessment	N	age in	assessment
					years
	behavioral observation	95 mothers	ADIS-IV-P	43 anxious	7-15	ADIS-IV-C,	Mothers of anxious children	Hudson &	[41]
	during cognitive tasks			offspring, 20		CBCL,	showed more involvement and	Rapee, 2001
				oppositional		RCMAS	more negativity than mothers of
				deviant			controls, especially when chidlren
				children, 32			were younger than 10 years of
				controls			age.
	behavioral observation	68 mothers	ADIS-IV	68	7-15	K-SADS	Mothers of anxious children were	Moore et al.,	[74]
	of interactions						less warm, irrespective of their	2004
							own diagnostic status.
	Do you feel that you	45 families	ADIS-IV-P	45 anxious,	7-16	ADIS-IV-C	Parents (especially mothers)	Hudson &	[43]
	relate more to either	with two		33 non-			perceive themselves as more	Rapee, 2005
	child a or b? Do you	children, of		anxious			protective of the anxious child
	feel you are more	whom one		children			than of the non-anxious sibling.
	protective of (need to	had an
	look after) either child	anxiety
	a or b? In comparison	disorder,
	to other parents, do	and 33 non-
	you feel your are more	referred
	protective of both of	families
	the children?
	PBI	-		224	7-18	SADS	SADs reported lower levels of	Merikangas et	[72]
							maternal emotional warmth. For	al., 2003
							overprotection, no differences
							between affected and unaffected
							individuals were found.
	EMBU-C	190	-	64 offspring	8-18	SAS	Offspring with SAD reported lower	Bögels et al.,
				with SAD,			family sociability and emotional	2001
				126 controls			warmth, more parental rejection;
							parents of SAD-offspring reported
							higher dependence upon the
							opinion of others, no differences
							found for overprotection or
							parental encouragement.

Table 3 : Associations between unfavourable parental rearing styles and offspring anxiety / social anxiety disorder (continued)

 3 Abbreviations in alphabetical order: ADIS-IV-P/C Anxiety Disorders Interview Schedule for Children, Parents Version; CBCL Child Behavior Checklist; CIDI Composite International Diagnostic Interview; CRPBI Childs Report of Parental Behavior Inventory; CRPR Childs Report of Parental Behavior Inventory; EMBU-C Egna Minnen Betraffande Uppfostran (e.g. Questionnaire of Recalled Parental Rearing Practices); FEE Fragebogen zum Erinnerten Elterlichen Erziehungsverhalten (Questionnaire of Recalled Parental Rearing Behavior); FEQ Family Expressiveness Questionnaire; K-SADS Kiddie-Schedule for Affective Disorders and Schizophrenia for School-Age Children; PBI Parental Bonding Instrument; PSP Preschool Socioaffective Profile; RCMAS Revised Children's Manifest Anxiety Scale; SAD Social Anxiety Disorder; SAS, SAS-A Social Anxiety Scale (for Adolescents); SASC-R Social Anxiety Scale for Children-Revised; SCID Structured Clinical Interview for DSM-Disorders; SIAS Social Interaction Anxiety Scale; SPAI-C Social Phobia and Anxiety Inventory for Children, SPS Social Phobia Scale

	behavioral observation	17 mothers,	-	17 anxiety-	9-12	ADIS-P if	Anxiety-disordered children rated	Siqueland et	[96]
	of family decision	17 fathers		disordered		age <12	their mothers (but not their	al., 1996
	making task, CRBI			offspring		ADIS-C if	fathers) as more restrictive, less
						age >=12	accepting.
						years
	FEQ	-	-	3649	9-17	SASC-R /	SADs perceived greater parental	Johnson et al.,	[45]
						SAS-A	shame overconcern, restriction of	2005
							family sociability.
	FEQ	404 parents	-	2708	12-18	RCMAS,	SADs rated their parents as more	Caster,	[18]
						SAS-A,	socially isolating, more concerned	Inderbitzen &
						CDI	about others opinions, more	Hope, 1999
							ashamed of their shyess and poor
							performance, less socially active.
	FEE	-	-	1395	14-17	CIDI	SADs reported more parental	Lieb et al.,	[64]
							overprotection, rejection, and	2000
							lower levels of emotional warmth.
	CRBI	816 mothers	SCID, K-	816	mean	K-SADS-E	Maternal control predicted	McClure et al.,	[69]
		with	SADS-E		15.0		offspring anxiety.	2001
		depressive
		symptoms
	EMBU	-	-	16 SAD, 43	18+		SADs rated their parents as more	Arrindel et al.,	[6]
				with			rejective, overprotective, and less	1989
				agoraphobia			emotionally warm than children
				100 controls			with agoraphobia and healthy
							controls.
	PBI	-	-	8232	18+	CIDI	Lack of maternal and paternal	Heider et al.,	[37]
							care, maternal overprotection, but	2008
							not maternal or paternal
							authoritianism were associated
							with SAD.

Selection of the studies was based on literature research in PubMed, Web of Science and PsycInfo, using the keywords "social anxiety", "anxiety", "social phobia", "family", "parents", including studies from 1989 to present. With regard to studies on family environment, we focus on studies in offspring ≤ 18 years of age.


MD Nadia A Sutedja

Yvonne Van Der Schouw

Kathelijn Fischer

PhD Eefje M Sizoo

Mark Huisman

Jan Veldink

MD, PhD Leonard H Van Den Berg

MD ; Y T Van Der Schouw

PhD ; K Fischer

MD ; M H B Huisman

MD J H Veldink

Huisman

Julius Veldink

Beneficial vascular risk profile is associated with amyotrophic lateral sclerosis

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

INTRODUCTION

Studies on angiogenic factors have suggested a role for altered vascular homeostasis in amyotrophic lateral sclerosis (ALS) pathogenesis. In several human populations, VEGF haplotypes associated with low vascular endothelial growth factor (VEGF) levels are more prevalent among ALS patients, and mice expressing reduced VEGF levels develop motor neuron degeneration. [START_REF] Lambrechts | VEGF is a modifier of amyotrophic lateral sclerosis in mice and humans and protects motoneurons against ischemic death[END_REF][START_REF] Van Vught | Lack of association between VEGF polymorphisms and ALS in a Dutch population[END_REF][START_REF] Oosthuyse | Deletion of the hypoxia-response element in the vascular endothelial growth factor promoter causes motor neuron degeneration[END_REF] Moreover, the functionally similar angiogenin has been associated with ALS. [START_REF] Greenway | ANG mutations segregate with familial and 'sporadic' amyotrophic lateral sclerosis[END_REF][START_REF] Cronin | Elevated serum angiogenin levels in ALS[END_REF][START_REF] Wu | Angiogenin loss-of-function mutations in amyotrophic lateral sclerosis[END_REF] Vasculature damage may be an early pathological event leading to motor neuron degeneration in the transgenic mutated SOD1 mouse model of ALS. [START_REF] Zhong | ALS-causing SOD1 mutants generate vascular changes prior to motor neuron degeneration[END_REF] Findings have suggested vascular risk factors to contribute to neurodegeneration in Alzheimer's disease. [START_REF] Regan | Relationship of vascular risk to the progression of Alzheimer disease[END_REF][START_REF] Luchsinger | Aggregation of vascular risk factors and risk of incident Alzheimer disease[END_REF][START_REF] Honig | Atherosclerosis and AD: analysis of data from the US National Alzheimer's Coordinating Center[END_REF][START_REF] Helzner | Contribution of vascular risk factors to the progression in Alzheimer disease[END_REF] Only one study examined the presence of multiple vascular risk factors in ALS patients, [START_REF] Armon | Antecedent medical diseases in patients with amyotrophic lateral sclerosis. A population-based case-controlled study in Rochester, Minn, 1925 through 1987[END_REF] but remained inconclusive due to a small study size. Other studies have focused on blood levels of specific vascular parameters: reports of higher plasma homocysteine [START_REF] Zoccolella | Elevated plasma homocysteine levels in patients with amyotrophic lateral sclerosis[END_REF] and higher lipid [START_REF] Dupuis | Dyslipidemia is a protective factor in amyotrophic lateral sclerosis[END_REF] levels seem to suggest atherogenic risk factors in ALS; however, the lack of association between lipid levels and ALS in a more recent study was not able to reinforce this hypothesis. [START_REF] Chio | Lower serum lipid levels are related to respiratory impairment in patients with ALS[END_REF] Moreover, the implication of the protective effect of higher lipid levels on disease progression found in one study, [START_REF] Dupuis | Dyslipidemia is a protective factor in amyotrophic lateral sclerosis[END_REF] but not in another study, [START_REF] Chio | Lower serum lipid levels are related to respiratory impairment in patients with ALS[END_REF] requires further elucidation. These different results have been suggested to be partly explained by an association of lower lipid status with lower respiratory function. [START_REF] Chio | Lower serum lipid levels are related to respiratory impairment in patients with ALS[END_REF] The aim of the present study was to assess the hypothesis that a higher risk profile for vascular disease, measured by clinical and biochemical indicators, was associated with susceptibility for developing ALS and to assess the association with survival in patients, after adjusting for confounders.

METHODS

Patients

Between July 1, 2004 and July 1, 2009, patients diagnosed with sporadic ALS at the University Medical Centre Utrecht, a tertiary referral clinic in The Netherlands, were recruited. Diagnosis was made according to the El Escorial Criteria after exclusion of other conditions. Patients diagnosed with possible, probable or definite ALS according to the El Escorial criteria were included. Age and site of onset of disease were recorded. Onset of disease was defined as the time of initial weakness, dysarthria or dysphagia. The study protocol was approved by the institutional ethical committee of the University Medical Centre Utrecht.

Questionnaire study

In the questionnaire study, 334 patients and 538 controls were included. Controls were derived from two sources. Each case was asked to approach an individual meeting the following criteria: 1) not a spouse, partner, or blood relative, 2) age difference of 5 years or less, 3) same sex. Also, the general practitioner of the patient was asked to select a control randomly from his clinic meeting the same criteria. Demographic characteristics (age, sex, level of education), and occurrence of traditional vascular risk factors and disease were ascertained by a questionnaire. Hypertension, hypercholesterolemia, and diabetes were classified as present when use of disease-specific medication was reported. Data on cigarette-smoking habits, height and weight during adulthood were collected. Body mass index (BMI) was calculated and overweight was defined as BMI ≥ 25 kg/m 2 . In patients with ALS, only data referring to the period before onset of first symptoms were analyzed. Occurrence of vascular disease was ascertained by questions relating to ever having had a myocardial infarction (MI), angina pectoris (AP), stroke and transient ischemic attacks (TIA), or peripheral bypass or angioplasty (peripheral vascular disease (PVD)).

Blood sample study

In the blood sample study, 303 patients and 2100 controls were included. There is overlap between the two patient cohorts from the questionnaire and blood sample studies. Both cohorts are derived from the same source patient population (tertiary referral centre). In both cohorts patients were sampled for inclusion. Controls were recruited from participants enrolled in two prospective studies in The Netherlands described elsewhere. [START_REF] Muller | Cardiovascular disease and cognitive performance in middle-aged and elderly men[END_REF][START_REF] Aleman | Vascular risk factors and cognitive function in a sample of independently living men[END_REF][START_REF] Boker | Prospect-EPIC Utrecht: study design and characteristics of the cohort population. European Prospective Investigation into Cancer and Nutrition[END_REF] The HAMLET study was a single-centre, population-based cohort study in 400 men, aged 40 to 80 years, who lived independently. It was designed to explore vascular risk factors. [START_REF] Muller | Cardiovascular disease and cognitive performance in middle-aged and elderly men[END_REF][START_REF] Aleman | Vascular risk factors and cognitive function in a sample of independently living men[END_REF] Volunteers were recruited by means of invitation letters to a random sample of the municipal register of Utrecht and a database of potential volunteers nominated by volunteers of previous studies. The PROSPECT-EPIC study was a population-based cohort study of 17357 healthy women aged 50-70 recruited from breast cancer screening participants designed to assess the relation between nutrition and cancer. [START_REF] Boker | Prospect-EPIC Utrecht: study design and characteristics of the cohort population. European Prospective Investigation into Cancer and Nutrition[END_REF] From this EPIC-cohort, 1700 women were randomly sampled for blood analysis.

Total cholesterol (TC), low-density lipoprotein (LDL), high-density lipoprotein (HDL), and its derived total LDL/HDL ratio, high sensitive (hs)-CRP, and homocysteine, have been shown to be independently associated with other vascular risk factors and disease. Nonfasting blood samples from patients with ALS at time of diagnosis, non-fasting blood samples from PROSPECT-controls and fasting blood samples from HAMLET-controls were collected. TC, LDL, HDL, glucose, hs-CRP were measured in all participants of PROSPECT (n = 1700) and HAMLET (n = 400). Homocysteine was measured in all (n = 400) HAMLET and in a subset of the PROSPECT subjects (n = 950). TC, LDL, HDL, and homocysteine in Sutedja 7 plasma were measured using an enzymatic assay. [START_REF] Van Der | Non-transferrin-bound iron and risk of coronary heart disease in postmenopausal women[END_REF] Hs-CRP was measured in serum in patients and HAMLET control subjects. We adjusted for the plasma measurements of hs-CRP in the PROSPECT study by multiplying all values by 1.13. Details of the measurements are provided elsewhere. [START_REF] Aleman | Vascular risk factors and cognitive function in a sample of independently living men[END_REF][START_REF] Van Der | Non-transferrin-bound iron and risk of coronary heart disease in postmenopausal women[END_REF][START_REF] Dalmeijer | Prospective study on dietary intakes of folate, betaine, and choline and cardiovascular disease risk in women[END_REF][START_REF] Hr | C-reactive protein and aortic stiffness and wave reflection in middle-aged and elderly men from the community[END_REF] Functional vital capacity percent of predicted (FVC) was measured in all patients at time of blood sampling.

Statistical analysis

We compared the prevalence of measures of cardiovascular risk in the ALS group to that of the control group. To determine whether ALS was associated with self-reported cardiovascular risk factors and events, logistic regression analysis was performed. In order to determine the increase in ALS risk with each unit of increment in serum values, logistic regression analysis adjusting for age was performed for women and men separately, because age and sex distribution differed among patients and controls.

FVC is known to be associated with survival. To assess this possible confounder in the survival analysis, the association between FVC and lipid levels was tested by linear regression. To determine whether survival was associated with self-reported cardiovascular risk factors and events, and blood levels of vascular risk factors, a univariate Cox regression and a multivariate Cox regression were performed, adjusting for age, site at onset and FVC at time of blood sampling, confounders significantly associated with survival. To determine whether serum values were influenced by duration of disease in patients with ALS, linear regression was used to study the association between blood levels and onset-diagnosis interval. A two-sided p-value < 0.05 was considered significant. The study protocol was approved by the institutional ethical committee of the University Medical Centre Utrecht. All patients gave informed consent prior to the study. Sutedja 8

RESULTS

Participants

Table 1 shows the characteristics of 334 patients and 538 controls in the questionnaire study (table 1a) and 303 patients (131 women and 172 men) and 2100 controls (1700 women and 400 men) in the blood sample study (table 1b). In ALS patients, age and site at onset were similar to those reported in previous population-based studies. [START_REF] Beghi | The epidemiology of ALS and the role of population-based registries[END_REF] The response rate of the participants in the questionnaire study was 80%. Characteristics of patients with ALS who completed questionnaires did not differ from those of patients with ALS for whom blood samples were available. Patient characteristics of participants and non-participants were similar. In the questionnaire study, sex and age were similar among patients and controls. In the blood sample study, patients were significantly older and there were more men than in the control population; therefore, we adjusted for these confounders.

Vascular risk factors and ALS

Table 2 shows the frequency of self-reported vascular risk factors in patients and controls.

Compared to controls, fewer patients used cholesterol-lowering agents (OR=0.6, 95% CI 0.4-0.9, p=0.008) or were overweight (OR=0.7, 95% CI 0.5-1.0, p=0.02); moreover, patients had a lower BMI (OR=0.9, 95% CI 0.9-1.0, p=0.001).

Table 3 shows the blood levels of biochemical indicators of vascular risk in patients and controls. All lipid values were consistent with lower vascular risk in patients: TC and LDL were significantly lower, and HDL was significantly higher. LDL/HDL ratio was significantly lower in patients with ALS (in women, OR=0.4, 95% CI 0.3-0.6, p<0.001; in men, OR=0.5, 95% CI 0.4-0.6, p<0.001). The lipid profile in patients who completed the questionnaire did not significantly differ from those who did not complete questionnaires.

Sutedja 9

Significantly lower homocysteine levels were found in patients with ALS (in women: OR=0.9, 95% CI 0.9-1.0, p=0.02; in men: OR=0.9, 95% CI 0.8-0.9, p<0.001). No significant associations between hs-CRP levels and ALS-status were observed. Stratification for disease duration did not show differences in measured blood levels (data not shown).

Cholesterol levels and survival in ALS

FVC and site at onset are known to be associated with survival. In patients with ALS, mean FVC was 87%. Table 4 shows the lipid status according to respiratory function at the time of blood sampling. Mean LDL-and TC levels were significantly lower among patients with a lower FVC. Endpoints (i.e. death) were reached in 207 out of 331 patients with ALS in the questionnaire study. Median survival was 3.5 years. In the blood sample study, endpoints were reached in 101 of 131 female patients with ALS and 124 of 172 male patients. Median survival was 2.6 years (in women as well as in men). Univariate Cox regression showed a significantly increased survival for patients with a high LDL/HDL ratio in the male (HR=0.8, p=0.01) and total group (HR=0.9, p=0.04) (table 5). This association disappeared in the multivariate Cox regression, after adjusting for age, site at onset and FVC (table 5).

Sutedja 10

DISCUSSION

The results of our clinical and biochemical studies were all consistent with a more favourable vascular risk profile in patients with ALS than in controls; besides having lower homocysteine levels, ALS patients used cholesterol-lowering agents less frequently, had a lower premorbid BMI and obesity rate, and had a more favourable lipid profile. The finding of shorter survival for ALS patients with low lipid levels is in agreement with one previous study [START_REF] Dupuis | Dyslipidemia is a protective factor in amyotrophic lateral sclerosis[END_REF] , and suggests that a more favourable lipid profile is a risk factor as well as a disease modifying factor in ALS. However, we also observed a significant association between low FVC and lower lipid levels, suggesting an increased energetic demand or inflammatory status due to the increased respiratory effort. [START_REF] Chio | Lower serum lipid levels are related to respiratory impairment in patients with ALS[END_REF] The significantly shorter survival of patients with a favourable lipid profile was no longer observed after adjusting for FVC indicating the effect of lipid levels on survival may be explained by this confounder. This may represent a state of debilitation in which poor FVC correlates with advanced disease and therefore poor nutrition. However, a causal effect of lipid levels on survival mediated by FVC cannot be excluded.

Only one previous study including 45 patients with ALS and 90 controls examined the presence of multiple vascular risk factors in ALS patients but was underpowered to detect an association between ALS and risk factors identified by chart review. [START_REF] Armon | Antecedent medical diseases in patients with amyotrophic lateral sclerosis. A population-based case-controlled study in Rochester, Minn, 1925 through 1987[END_REF] The favourable lipid profile in patients with ALS in our study is in contrast to the reported higher [START_REF] Dupuis | Dyslipidemia is a protective factor in amyotrophic lateral sclerosis[END_REF] or similar [START_REF] Chio | Lower serum lipid levels are related to respiratory impairment in patients with ALS[END_REF] blood cholesterol levels observed in patients with ALS in previous studies. These apparent discrepancies may have been caused by differences in the control population. In one study, [START_REF] Dupuis | Dyslipidemia is a protective factor in amyotrophic lateral sclerosis[END_REF] lipid levels in controls were lower than in our study, which could be explained by an underrepresentation of persons with high cholesterol levels in the control population due to selection of health-conscious individuals as they used as controls visitors for routine cholesterol work-up in the hospital after excluding those with disorders associated with vascular disease. In a more recent study, [START_REF] Chio | Lower serum lipid levels are related to respiratory impairment in patients with ALS[END_REF] the lipid levels in the controls were lower than in Sutedja 11 our study but controls taking lipid lowering drugs and those with diabetes mellitus were excluded which may have resulted in an underrepresentation of persons with high vascular risk and high cholesterol levels. [START_REF] Chio | Lower serum lipid levels are related to respiratory impairment in patients with ALS[END_REF] Consistent with an earlier report, [START_REF] Scarmeas | Premorbid weight, body mass, and varsity athletics in ALS[END_REF] our study showed an increased incidence of ALS among premorbid, leaner individuals, which has been linked to an increased metabolic rate in patients with ALS and in mouse models prior to disease onset. [START_REF] Desport | Hypermetabolism in ALS: correlations with clinical and paraclinical parameters[END_REF][START_REF] Desport | Factors correlated with hypermetabolism in patients with amyotrophic lateral sclerosis[END_REF][START_REF] Dupuis | Evidence for defective energy homeostasis in amyotrophic lateral sclerosis: benefit of a high-energy diet in a transgenic mouse model[END_REF] Resting energy expenditure is increased in patients with ALS. Also, lower premorbid BMI has been previously found to be associated with ALS suggested to be a proxy for increased premorbid physical activity. [START_REF] Scarmeas | Premorbid weight, body mass, and varsity athletics in ALS[END_REF] These results therefore indicate that lipid profiles in ALS are a reflection of either increased premorbid physical activity or hypermetabolism. A recent review concludes that clinical and experimental studies have shown that abnormal energy homoeostasis has a role in ALS and altered lipid levels have been put forward as an explanation for energy imbalance in ALS. [START_REF] Dupuis | Energy metabolism in amyotrophic lateral sclerosis[END_REF] Another previously studied biochemical indicator associated with vascular disease is homocysteine, which has atherogenic and pro-thrombotic properties. [START_REF] Eikelboom | Homocyst(e)ine and cardiovascular disease: a critical review of the epidemiologic evidence[END_REF] Our study in 303 patients and 1350 controls showed lower homocysteine levels among patients, in contrast to the increased fasting homocysteine levels in 62 patients with ALS compared to 88 controls in a previous study. [START_REF] Zoccolella | Elevated plasma homocysteine levels in patients with amyotrophic lateral sclerosis[END_REF] Homocysteine levels may be influenced by vitamin B6 and B12 levels.

However, as vitamin B deficiencies lead to hyperhomocysteinemia, the corrected homocysteine levels in our patients would be even lower if they had vitamin B6 or B12 deficiencies. This factor does not, therefore, interfere with our conclusion that homocysteine levels in our patients are lower than in controls and discrepancies between studies are most probably attributable to differences in controls populations (outpatients in a tertiary clinic versus a prospective cohort in our study).

A consideration is that data for patients and controls in the blood sample study were ascertained in different ways. Non-fasting blood samples were collected from patients with Sutedja 12 ALS and from female controls, but fasting blood samples from male controls. This does not, however, explain the observed difference in levels of cholesterol and homocysteine between patients and controls; if results were influenced, we would expect an even larger difference in blood levels between patients and controls. Moreover, blood samples of patients were collected after onset of disease making a distinction between cause and consequence difficult.

It would be interesting to go back in the patient records, well before the onset of ALS, to determine if the lipid markers were always in the favourable range. All blood samples were, however, collected at the first visit -usually a relatively early stage of the disease -and stratification for disease duration did not show differences between samples taken at an early stage and at a late stage. Also lipid controls were not age-matched, but we adjusted for this in our analyses.

In the questionnaire study we took into account onset of first weakness and only scored selfreported risk factors which occurred prior to disease onset. The finding of lower cholesterol levels in patients at diagnosis seem to agree with the data from self-reports of more frequent use of cholesterol-lowering agents and the low BMI preceding disease onset.

In the questionnaire study, a limitation is possible recall bias in the patient group. However, there was no indication of overreporting vascular risk factors in the patient group. Also, using acquaintances as controls could have contributed to overmatching which would bias the risk estimate towards the null. If the results were influenced, we would expect an even stronger association between ALS and use of cholesterol lowering medication. Moreover, the prevalence of vascular risk factors in our control group did not show major differences compared to the figures reported for the Dutch population in the national registry. This suggests that the control group (which also consisted of population-based individuals randomly selected by the general practitioner) is representative of the general population.

Sutedja 13

The present study does not show cardiovascular risk factors and disease to be associated with ALS, but the association of ALS with a favourable lipid profile may support the hypothesis that an increased metabolism, as part of a complex genetic profile, independent of activity levels or diet, may play a role in the pathogenesis of ALS. Determining causation, however, requires more research.

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Fonds (PBF)

and the Catharijne Stichting. We would like to thank H. Kersten and M. Kersten for their generous support as well as J.R. van Dijk and the Adessium foundation.