Johannes Schumacher
email: johannes.schumacherj@uni-bonn.de

Ann Suhl Kristensen

Jens R Wendland

Markus M Nöthen

Ole Mors

Francis J Mcmahon

THE GENETICS OF PANIC DISORDER

Keywords: Panic, anxiety disorders, heritability, comorbidity, trait marker, molecular genetics

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INTRODUCTION

Panic disorder (PD) is one of the anxiety disorders, a group that includes specific and social phobia, agoraphobia (AG), generalized anxiety disorder, separation anxiety disorder, obsessive-compulsive disorder (OCD), and posttraumatic stress disorder. PD is a serious psychiatric disorder (Table 1) with a high lifetime prevalence-between 3.4 to 4.7% in the United States population [START_REF] Kessler | The epidemiology of panic attacks, panic disorder, and agoraphobia in the National Comorbidity Survey Replication[END_REF][START_REF] Kessler | Lifetime panic-depression comorbidity in the National Comorbidity Survey[END_REF]; it is also associated with severe social and occupational impairments. The American Psychiatric Association (APA) defines PD as an episode of abrupt intense fear accompanied by additional physiological or cognitive symptoms (Table 2).

Although PD has no known cause and its underlying etiology is not well understood, studies have consistently shown that neurobiological factors are key to the disease process. Furthermore, PD shows high comorbidity with several other psychiatric and medical conditions, and there is evidence of shared etiological factors with some of these disorders.

For instance, other anxiety disorders, mood disorders, and substance use disorders are frequently observed in patients with PD (Table 1). Currently, a diagnosis of PD is made by collecting patient information, conducting clinical interviews, and observing PD symptoms.

Nevertheless, there is growing hope that the underlying etiology of PD and the biological pathways involved will soon be identified. Studies have consistently observed that genetic inheritance is a strong risk factor for PD. Furthermore, the success of modern technology in analyzing a variety of genetic disorders, has increasingly allowed investigators to identify genetic risk factors, even if their effect sizes are moderate.

In this article, we review current genetic research on PD, including a discussion of the phenotypic aspects and neurobiological concepts that are receiving increased consideration in recent studies. We also discuss the extent to which our understanding of PD is likely to be increased via the results of current and future molecular genetic studies.

PD -FEATURES OF INHERITANCE

HERITABILITY

The familial aggregation of PD has been established by several independent studies and has been shown to be stronger than in other anxiety disorders. Supplementary table 1 shows the results of 19 controlled PD family studies, i. e. in each study also independent non-PD families have been analyzed. The family studies revealed a relative risk (RR) to firstdegree relatives of individuals with PD that ranged from 2.8 [START_REF] Mannuzza | Familial transmission of panic disorder: effect of major depression comorbidity[END_REF] to 14.7 [START_REF] Mendlewicz | Family study of panic disorder: comparison with generalized anxiety disorder, major depression and normal subjects[END_REF]. Furthermore, twin studies (Supplementary Table 2) have confirmed that genetic factors are largely responsible for the familial clustering of PD [START_REF] Bellodi | CO2-induced panic attacks: a twin study[END_REF][START_REF] Kendler | Panic syndromes in a population-based sample of male and female twins[END_REF][START_REF] Kendler | Panic disorder in women: a population-based twin study[END_REF][START_REF] Middeldorp | Familial clustering of major depression and anxiety disorders in Australian and Dutch twins and siblings[END_REF][START_REF] Perna | Panic attacks: a twin study[END_REF][START_REF] Scherrer | Evidence for genetic influences common and specific to symptoms of generalized anxiety and panic[END_REF][START_REF] Skre | A twin study of DSM-III-R anxiety disorders[END_REF][START_REF] Torgersen | Genetic factors in anxiety disorders[END_REF][START_REF] Tsuang | Agoraphobic behavior and panic attack: a study of male twins[END_REF]. In the largest and most informative twin studies-The Vietnam Era Twin Registry [START_REF] Scherrer | Evidence for genetic influences common and specific to symptoms of generalized anxiety and panic[END_REF][START_REF] Tsuang | Agoraphobic behavior and panic attack: a study of male twins[END_REF], the Virginia Twin Registry [START_REF] Kendler | Panic syndromes in a population-based sample of male and female twins[END_REF][START_REF] Kendler | Panic disorder in women: a population-based twin study[END_REF], and the Australian and Netherlands Twin Registries [START_REF] Middeldorp | Familial clustering of major depression and anxiety disorders in Australian and Dutch twins and siblings[END_REF]-more than 1,000 twin pairs were analyzed in each study and it was estimated that additive genetic factors explain approximately 30-46% of the variance in PD. Hettema and colleagues [START_REF] Hettema | A review and meta-analysis of the genetic epidemiology of anxiety disorders[END_REF] combined data from five family and three twin studies selected via defined inclusion criteria. Their analysis yielded heritability estimates of 48% (95% CI 41-54%), with the remaining variance explained predominantly by non-shared environmental factors. Stress, perinatal factors, substance abuse, and life events-e.g., exposure to violence, social isolation, loss-have been proposed as non-shared environmental factors; in contrast, parental separation, poverty, and parenting dimensions such as criticism/rejection or overcontrol are believed to be shared environmental risk factors for PD [START_REF] Hirshfeld-Becker | High risk studies and developmental antecedents of anxiety disorders[END_REF].

MODE OF INHERITANCE

Although inheritance related to a single gene may play a role in some PD family pedigrees (see below), family and twin studies support the view that most PD cases have a complex genetic basis. This notion is corroborated by preclinical studies suggesting that, in animals, anxiety-like behaviors and emotional activity analogous to panic and anxiety are controlled by multiple genes [START_REF] Finn | Genetic animal models of anxiety[END_REF]. However, the number of susceptibility genes, the disease risk conferred by each gene, and the degree of interaction between them, if any, all remain unknown. Furthermore, it must be assumed that the genetic architecture underlying PD differs among cases. For example, the degree of genetic complexity and the pattern of genes involved might differ in familial vs. non-familial cases, in early-vs. late-onset, or when differences in comorbid conditions or gender are considered. Therefore, a detailed documentation of disease course-including longitudinal, neurobiological, and psychophysiological examinations (see below)-may greatly benefit future genetic studies.

PD AND COMORBID DISORDERS

It is well known that PD is associated with a wide variety of other medical and psychiatric conditions. In particular, other anxiety disorders, substance use disorders, and mood disorders (e. g, major depressive disorder (MDD) and bipolar disorder (BD)), are commonly observed in individuals with PD (Table 1). Indeed, the National Comorbidity Survey Replication (NCS-R) report [START_REF] Kessler | Prevalence, severity, and comorbidity of 12-month DSM-IV disorders in the National Comorbidity Survey Replication[END_REF], which is the largest survey of the prevalence and correlates of mental disorders in the US, found that 80% of PD patients had one or more of these disorders. Family and twin studies suggest that some of these comorbid disorders share some genetic liability with PD; however, there is also evidence for that PD has a distinct genetic basis (see below). The following describes those disorders which have been strongest implicated by the NCS-R report within the context of PD. Most of the family studies cited in the following involved the use of healthy or control family samples in addition to the families of affected cases.

PD and Agoraphobia (AG):

PD occurs most often in conjunction with AG; individuals with this disorder fear and avoid settings that are unfamiliar or where they perceive that they have little control. The NCS-R report estimated that approximately 64% of PD patients met diagnostic criteria for AG [START_REF] Kessler | Prevalence, severity, and comorbidity of 12-month DSM-IV disorders in the National Comorbidity Survey Replication[END_REF]. In addition, PD-AG seems to be associated with a more severe disease course; for example, increased number of both panic attacks and other comorbid disorders are more frequent in individuals with PD-AG than in those with PD only [START_REF] Kessler | The epidemiology of panic attacks, panic disorder, and agoraphobia in the National Comorbidity Survey Replication[END_REF][START_REF] Kessler | Prevalence, severity, and comorbidity of 12-month DSM-IV disorders in the National Comorbidity Survey Replication[END_REF].

Whether AG also impacts age of onset (AO) for PD still remains uncertain. Kessler and colleagues [START_REF] Kessler | The epidemiology of panic attacks, panic disorder, and agoraphobia in the National Comorbidity Survey Replication[END_REF] found similar AO in PD and PD-AG groups, but a family study from Iowa reported that individuals with PD-AG had an earlier AO than those with PD only [START_REF] Noyes | Relationship between panic disorder and agoraphobia. A family study[END_REF].

Nevertheless, studies have consistently shown that most of genetic risk factors seem to be shared between PD and AG [START_REF] Noyes | Relationship between panic disorder and agoraphobia. A family study[END_REF][START_REF] Biederman | Effects of parental anxiety disorders in children at high risk for panic disorder: a controlled study[END_REF][START_REF] Biederman | A controlled longitudinal 5-year follow-up study of children at high and low risk for panic disorder and major depression[END_REF][START_REF] Horwath | Is the comorbidity between social phobia and panic disorder due to familial cotransmission or other factors?[END_REF].

PD and Other Anxiety Disorders:

The NCS-R report suggested that, as with AG, all other anxiety disorders have a high lifetime prevalence in those with PD [START_REF] Kessler | The epidemiology of panic attacks, panic disorder, and agoraphobia in the National Comorbidity Survey Replication[END_REF]. Specific and social phobias, in particular, are among the most common comorbidities in PD (34.3%, 31.1%, respectively) followed by posttraumatic stress disorder (21.6%), generalized anxiety disorder (21.3%), and OCD (8.2%). However, most family studies suggest that risk genes contribute more specifically to each anxiety disorder [START_REF] Noyes | Relationship between panic disorder and agoraphobia. A family study[END_REF][START_REF] Biederman | Effects of parental anxiety disorders in children at high risk for panic disorder: a controlled study[END_REF][START_REF] Horwath | Is the comorbidity between social phobia and panic disorder due to familial cotransmission or other factors?[END_REF][START_REF] Fyer | Panic disorder and social phobia: effects of comorbidity on familial transmission[END_REF][START_REF] Low | Specificity of familial transmission of anxiety and comorbid disorders[END_REF]. However, OCD may be an exception; one family study found evidence for a shared genetic PD-OCD liability [START_REF] Biederman | Effects of parental anxiety disorders in children at high risk for panic disorder: a controlled study[END_REF]. This finding would be consistent with an OCD family study noting that the relatives of OCD patients had increased risk for PD and AG [START_REF] Nestadt | The relationship between obsessive-compulsive disorder and anxiety and affective disorders: results from the Johns Hopkins OCD Family Study[END_REF]. However, a subsequent study suggested that these families may represent a 'distinct genetic subtype' of PD (or OCD) [START_REF] Nestadt | The identification of OCD-related subgroups based on comorbidity[END_REF].

Twin studies have also considered the relationship between PD and other anxiety disorders. Hettema and colleagues [START_REF] Hettema | The structure of genetic and environmental risk factors for anxiety disorders in men and women[END_REF] analyzed data from the Virginia Adult Twin sample using a multivariate modeling approach. They found two genetic factors common across the disorders; the first loaded strongly in PD, AG, and generalized anxiety disorder, whereas the second loaded primarily in specific phobias. Social phobia appeared to be influenced by both factors. Evidence for genetic factors that are shared between anxiety disorders is also provided by studies that have investigated personality traits that are elevated among all anxiety disorders. According to some authors these traits may act as intermediate phenotypes for the observed comorbidity [START_REF] Smoller | The genetic basis of panic and phobic anxiety disorders[END_REF].

PD and Major Depressive Disorder (MDD):

The NCS-R report found that in patients with PD, lifetime risk for MDD was 34.7% [START_REF] Kessler | The epidemiology of panic attacks, panic disorder, and agoraphobia in the National Comorbidity Survey Replication[END_REF]. While most family studies suggest that the majority of the underlying genetic risk is not shared between both disorders [START_REF] Noyes | Relationship between panic disorder and agoraphobia. A family study[END_REF][START_REF] Biederman | A controlled longitudinal 5-year follow-up study of children at high and low risk for panic disorder and major depression[END_REF][START_REF] Crowe | A family study of panic disorder[END_REF][START_REF] Goldstein | Psychiatric disorders in relatives of probands with panic disorder and/or major depression[END_REF][START_REF] Harris | Family study of agoraphobia. Report of a pilot study[END_REF][START_REF] Weissman | The relationship between panic disorder and major depression. A new family study[END_REF], other family studies [START_REF] Maier | The familial relationship between panic disorder and unipolar depression[END_REF] and one twin study [START_REF] Torgersen | Genetic factors in anxiety disorders[END_REF] did find evidence of shared genetic factors between PD and MDD. The hypothesis that some personality traits in PD that are also frequently seen in MDD patients may act as intermediate phenotypes has been raised [START_REF] Hettema | What is the genetic relationship between anxiety and depression?[END_REF],

although several non-genetic factors may also be involved in the comorbidity between PD and MDD. For example, PD-and MDD-specific risk factors might interact on the protein levele. g., via common and/or connected neurotransmitter pathways -or common environmental factors might be present. In addition, some authors have suggested that PD might cause the development of MDD (or vice versa) [START_REF] Hettema | What is the genetic relationship between anxiety and depression?[END_REF].

PD and Bipolar Disorder (BD):

BD has also been associated with PD; lifetime risk of BD appears to be 14.4% in PD patients (NCS-R report) [START_REF] Kessler | The epidemiology of panic attacks, panic disorder, and agoraphobia in the National Comorbidity Survey Replication[END_REF]. However, most family studies are not consistent with the notion that shared genetic factors underlie this comorbidity [START_REF] Biederman | A controlled longitudinal 5-year follow-up study of children at high and low risk for panic disorder and major depression[END_REF][START_REF] Harris | Family study of agoraphobia. Report of a pilot study[END_REF].

PD and Schizophrenia:

The NIMH Epidemiologic Catchment Area Study (ECA) study estimated that schizophrenia occurs more frequently in PD patients [START_REF] Horwath | Agoraphobia without panic: clinical reappraisal of an epidemiologic finding[END_REF]. One family study (Bonn/Mainz) similarly found increased risk of PD in the relatives of schizophrenic patients without PD. However, the schizophrenia risk in PD relatives was not increased compared to controls. The authors concluded that there might be shared genetic factors between both disorders present in only a small subgroup of PD or schizophrenia cases [START_REF] Heun | Relation of schizophrenia and panic disorder: evidence from a controlled family study[END_REF].

PD and Substance Use Disorders:

Most substance use disorders co-occur frequently in individuals with PD. Alcohol abuse/dependence disorder (AD), present in 25% of PD cases [START_REF] Kessler | The epidemiology of panic attacks, panic disorder, and agoraphobia in the National Comorbidity Survey Replication[END_REF], is the most-studied comorbid condition associated with PD. All controlled family studies report increased rates of AD in the relatives of those with PD, even if AD was not present in PD patients [START_REF] Crowe | A family study of panic disorder[END_REF][START_REF] Goodwin | Alcohol use disorders in relatives of patients with panic disorder[END_REF][START_REF] Maier | A controlled family study in panic disorder[END_REF]. However, the reason for this association is difficult to interpret. Some authors have hypothesized that AD develops as an attempt to self-medicate PD and associated anxiety symptoms [START_REF] Swendsen | The comorbidity of alcoholism with anxiety and depressive disorders in four geographic communities[END_REF], whereas other authors have suggested that a specific electroencephalography (EEG) trait, namely low voltage alpha (LV), is an intermediate phenotype for both disorders. This is based on observations that LV occurs more frequently in patients with AD comorbid for an anxiety disorder (including PD) than either AD-only or non-AD subjects [START_REF] Enoch | Relationship of genetically transmitted alpha EEG traits to anxiety disorders and alcoholism[END_REF]. However, this finding remains unreplicated.

According to the NCS report, cigarette smoking is seen in 35.9% [START_REF] Lasser | Smoking and mental illness: A population-based prevalence study[END_REF], and cannabis use/dependence is seen in 20.6%, of individuals with PD [START_REF] Kessler | The epidemiology of panic attacks, panic disorder, and agoraphobia in the National Comorbidity Survey Replication[END_REF]. The most common explanation for these comorbidities is that these may trigger anxiety and, ultimately, PD in susceptible individuals [START_REF] Hollister | Pharmacotherapeutic considerations in anxiety disorders[END_REF][START_REF] Zvolensky | Smoking and panic attacks, panic disorder, and agoraphobia: a review of the empirical literature[END_REF].

PD and Other Medical Disorders:

Several studies have noted an association between PD and other medical disorders. Specifically, respiratory, metabolic, cardiovascular, gastrointestinal, genitourinary, and connective tissue disorders have been reported to be more frequent in PD patients, as have labile hypertension and chronic headaches [START_REF] Harter | Associations between anxiety disorders and physical illness[END_REF]. However, one of the most intriguing findings concerns the so-called 'panic syndrome' initially found in some multiplex PD families [START_REF] Weissman | Potential panic disorder syndrome: clinical and genetic linkage evidence[END_REF], which showed strong linkage to chromosome 13q32 [START_REF] Hamilton | Further genetic evidence for a panic disorder syndrome mapping to chromosome 13q[END_REF]. The syndrome describes the familial co-occurrence and transmission of PD and interstitial cystitis (IC) symptoms, mitral valve prolapses (MVP), chronic headaches, and thyroid disorders. A followup study conducted with an independent sample provided further evidence that this syndrome may represent a distinct PD phenotype [START_REF] Talati | Panic disorder, social anxiety disorder, and a possible medical syndrome previously linked to chromosome 13[END_REF]. That study found that in some families, the first-degree relatives of patients with PD -and also those with social phobia-showed elevated rates of IC, MVP, headaches, and thyroid disorders. Irrespective of first-degree relatives, the frequency of the co-morbid conditions was between 8% (thyroid disorder) and 34% (chronic headaches) in PD patients [START_REF] Talati | Panic disorder, social anxiety disorder, and a possible medical syndrome previously linked to chromosome 13[END_REF] indicating that this syndrome may represent a substantial fraction of PD.

PD AND PERSONALITY TRAITS

Several studies have focused on personality traits associated with PD. Anxiety sensitivity (AS) and behavioral inhibition (BI) are among the personality traits most often associated with PD, although neuroticism (N) and harm avoidance (HA) have also been implicated in PD as well as in other anxiety disorders and MDD. Some investigators believe that these traits may represent intermediate phenotypes that are more directly influenced by the underlying risk genes than PD itself, and/or that they predate the onset of PD. Furthermore, because these traits are also associated with other anxiety disorders (as well as MDD), they may moderate the comorbidity between these disorders. If shared genetic factors contribute to the observed association between some personality traits and PD, the disease status would represent rather a (quantitative) extreme of dimensions that underlie 'normal' personality.

PD and Anxiety Sensitivity: AS, described as the tendency to interpret sensations of physiological arousal as having harmful physical, psychological, or social consequences [START_REF] Reiss | Expectancy model of fear[END_REF].

The misinterpretation engendered by AS is believed to contribute to a 'fear of fear' cycle, in which anxiety about the sensations further increase arousal. Studies have shown that AS predicts the number, frequency, emergence, and onset of panic attacks as well as the likelihood of developing PD or another anxiety disorder [START_REF] Ehlers | A 1-year prospective study of panic attacks: clinical course and factors associated with maintenance[END_REF][START_REF] Hayward | Predictors of panic attacks in adolescents[END_REF][START_REF] Schmidt | Anxiety sensitivity: prospective prediction of panic attacks and Axis I pathology[END_REF]. However, other studies failed to observe elevated AS in offspring at risk for an anxiety disorder [START_REF] Van Beek | Anxiety sensitivity in children of panic disorder patients[END_REF], confusing the issue of whether AS is associated with PD or represents an intermediate PD trait.

PD and Behavioral Inhibition:

BI is defined as the tendency in childhood to exhibit restraint, withdrawal, and reticence when faced with novel or unfamiliar situations and people [START_REF] Kagan | Biological bases of childhood shyness[END_REF].

Elevated levels of BI have been found in the offspring of parents with PD [START_REF] Battaglia | Physiological and behavioral responses to minor stressors in offspring of patients with panic disorder[END_REF][START_REF] Manassis | Behavioural inhibition, attachment and anxiety in children of mothers with anxiety disorders[END_REF][START_REF] Rosenbaum | Behavioral inhibition in childhood: a risk factor for anxiety disorders[END_REF], although one study failed to support this observation [START_REF] Warren | Maternal panic disorder: infant temperament, neurophysiology, and parenting behaviors[END_REF]. Like AS, BI seems not to be a developmental risk factor for PD only. Instead, other anxiety disorders seem to be predicted by higher BI, especially social phobia [START_REF] Biederman | A 3-year follow-up of children with and without behavioral inhibition[END_REF][START_REF] Biederman | Psychiatric correlates of behavioral inhibition in young children of parents with and without psychiatric disorders[END_REF]. Interestingly, in offspring at risk for PD and MDD, BI was associated with social phobia [START_REF] Hirshfeld-Becker | Behavioral inhibition in preschool children at risk is a specific predictor of middle childhood social anxiety: a five-year follow-up[END_REF]. Similar findings have been obtained at the neurobiological level, which point to common pathways underlying BI and PD. For instance, increased amygdala responsiveness (see below) seems to be associated with both conditions. However, this neurobiological abnormality is not specific for BI and PD, and is also seen in other anxiety disorders [START_REF] Hirshfeld-Becker | High risk studies and developmental antecedents of anxiety disorders[END_REF][START_REF] Mathew | Recent advances in the neurobiology of anxiety disorders: implications for novel therapeutics[END_REF].

PD and Neuroticism: N, a personality trait that reflects an enduring tendency to experience negative emotional states, is one of the three key dimensions of personality according to Eysenck [START_REF] Eysenck | Eysenck Personality Questionnaire Manual[END_REF] and has been included in most theories of personality since its introduction. The association between N and PD has been described in several prospective and family studies [START_REF] Hirshfeld-Becker | High risk studies and developmental antecedents of anxiety disorders[END_REF]. Furthermore, in large studies of twin pairs it has been shown that genetic determinants of N overlap with those of PD. However, these results were restricted to male patients [START_REF] Andrews | The genetics of six neurotic disorders: a twin study[END_REF][START_REF] Martin | Anxiety disorders and neuroticism: are there genetic factors specific to panic?[END_REF].

Most recently, one of the largest studies investigating personality traits in PD has been published and N was the main personality characteristic of patients with PD compared to controls [START_REF] Vohma | Evaluation of personality traits in panic disorder using Swedish universities Scales of Personality[END_REF]. Moreover, the authors of the later study performed a subscale analysis and found that the trait Somatic Trait Anxiety, which describes the tendency to experience autonomic arousal, restless, and tense, was more frequent in PD patients than in controls [START_REF] Vohma | Evaluation of personality traits in panic disorder using Swedish universities Scales of Personality[END_REF].

GENDER EFFECTS

Females are more susceptible to developing PD and show a greater than twofold elevation in lifetime rates compared to males. According to NCS data, the lifetime prevalence of PD is 1.9% in men and 5.1% in women [START_REF] Kessler | Lifetime and 12-month prevalence of DSM-III-R psychiatric disorders in the United States. Results from the National Comorbidity Survey[END_REF]. AO does not differ between genders.

However, whether the higher PD rates in females reflect the presence of gender-specific genetic factors remains under debate. For example, an Australian twin pair study provided evidence that the genetic architecture underlying PD might differ between females and males.

They observed disease correlations in their sister and brother PD twin pairs that were absent in opposite-sex sibling pairs [START_REF] Middeldorp | Familial clustering of major depression and anxiety disorders in Australian and Dutch twins and siblings[END_REF]. Furthermore, another study of these same twin pairs found evidence that genetic determinants of neuroticism (N ) overlapped with those of panic/anxiety among male, but not female, subjects [START_REF] Martin | Anxiety disorders and neuroticism: are there genetic factors specific to panic?[END_REF]. In contrast, the Virginia Twin Registry study failed to observe any evidence of gender-specific PD genetic profiles [START_REF] Hettema | The structure of genetic and environmental risk factors for anxiety disorders in men and women[END_REF]. However, only one study investigated whether parent of origin effects might be present in the familial transmission of PD [START_REF] Haghighi | Parent-of-origin effect in panic disorder[END_REF]; while that study found some preliminary evidence for parent of origin effects, this finding remains unreplicated.

AGE OF ONSET (AO)

The average AO for PD is 23.6 years [START_REF] Kessler | The epidemiology of panic attacks, panic disorder, and agoraphobia in the National Comorbidity Survey Replication[END_REF] and differs compared to other anxiety disorders; although there is variation across studies, the AO of specific phobias typically occurs in middle childhood, social phobia in middle adolescence, and OCD in early adulthood. It is assumed that the link between PD and AO is due to age-specific neuroendocrinology or brain developmental processes. Furthermore, age-specific environmental factors may explain the high prevalence in onset that occurs at 23 years.

However, genetic factors also seem to influence AO in PD. For example, genetic loading seems to be more pronounced in early-onset (EAO) cases of PD (typically defined by an AO of < 20 years). The risk of PD for relatives of EAO patients is higher than that for family members of non-EAO patients [START_REF] Goldstein | Familial aggregation and phenomenology of 'early'-onset (at or before age 20 years) panic disorder[END_REF]. This finding could be consistent with the observation that probands from families with at least one additional PD case tend to have an earlier AO than patients with no other affected relatives [START_REF] Battaglia | Age at onset of panic disorder: influence of familial liability to the disease and of childhood separation anxiety disorder[END_REF].

RISK MARKERS IN PD

Several studies report psychophysiological abnormalities in PD, some of which seem to be associated with increased amygdala responsiveness and amygdalar projections to the striatum, hypothalamus, sympathetic chain, and cardiovascular system. For example, higher electrodermal activity, peripheral vasoconstriction, accelerated heart rate, and neuroendocrine abnormalities have been observed in PD patients [START_REF] Grillon | Anxiety disorders: Psychophysiological Aspects[END_REF]. In addition, offspring at risk for PD display symptoms of higher physiologic arousal, including accelerated heart rates under stress and neuroendocrine abnormalities [START_REF] Battaglia | Physiological and behavioral responses to minor stressors in offspring of patients with panic disorder[END_REF][START_REF] Warren | Maternal panic disorder: infant temperament, neurophysiology, and parenting behaviors[END_REF]. One of the most studied and replicated psychophysiological abnormalities associated with PD is CO2-hypersensitivity, which seems to be specific to PD and which might be useful as an intermediate trait marker (see below).

PD and Neurobiological Findings:

Several neuroimaging studies have shown that the amygdala plays a crucial role in processing of fearful material. Incoming stimuli from the thalamus and sensory cortex specifically activate the amygdala, which in turn stimulates various brain areas such as the striatum, hypothalamus, and periaqueductal grey, which are responsible for key panic symptoms. This process is modulated by top-down governance involving information from the frontal, cingulate, and insular cortices as well as the hippocampus. In PD, this anxiety response system is characterized by increased amygdala reactivity or responsiveness [START_REF] Mathew | Recent advances in the neurobiology of anxiety disorders: implications for novel therapeutics[END_REF]. However, amygdala hyperreactivity is also associated with other anxiety disorders as well as in children with BI [START_REF] Hirshfeld-Becker | High risk studies and developmental antecedents of anxiety disorders[END_REF][START_REF] Mathew | Recent advances in the neurobiology of anxiety disorders: implications for novel therapeutics[END_REF]. Additional family and longitudinal studies are needed to clarify whether this functional abnormality might be useful as an intermediate phenotype. Abnormalities related to other brain regions-for instance, altered insular function-have also been reported in PD and other anxiety disorders [START_REF] Mathew | Recent advances in the neurobiology of anxiety disorders: implications for novel therapeutics[END_REF].

PD and CO2-Hypersensitivity: As noted above, several studies have shown that PD is associated with increased CO2-sensitivity. Studies have found that panic attacks can be provoked in a high proportion of PD patients by exposure to CO2 [START_REF] Papp | Carbon dioxide hypersensitivity, hyperventilation, and panic disorder[END_REF]. PD is thus one of the few psychiatric disorders whose symptoms can be provoked. In addition, the healthy relatives of PD patients showed an increased likelihood of PD symptoms after exposure to CO2 [START_REF] Coryell | Hypersensitivity to carbon dioxide as a disease-specific trait marker[END_REF][START_REF] Coryell | Anxiety responses to CO2 inhalation in subjects at high-risk for panic disorder[END_REF][START_REF] Perna | Sensitivity to 35% CO2 in healthy first-degree relatives of patients with panic disorder[END_REF]. Notably, twin studies point to genetic factors underlying this respiratory abnormality [START_REF] Bellodi | CO2-induced panic attacks: a twin study[END_REF]. The findings suggest that CO2-hypersensitivity may be an alternative manifestation of PD in susceptible individuals, thus representing an intermediate phenotype.

Other studies have shown that PD patients exhibit respiratory abnormalities in the absence of CO2 exposure. For example, PD patients showed differences in respiratory rate, tidal volume, and inspiratory flow rate compared to healthy controls; electrodermal and cardiovascular measures did not differ between the two groups [START_REF] Wilhelm | Respiratory dysregulation in anxiety, functional cardiac, and pain disorders. Assessment, phenomenology, and treatment[END_REF]. However, a concept that might integrate these observed respiratory abnormalities is the suffocation false alarm theory [START_REF] Grillon | Anxiety disorders: Psychophysiological Aspects[END_REF], which posits functional and/or structural abnormalities of the respiratory control system located in the brainstem.

PD and Other Vulnerability Marker:

Other agents have been reported to provoke panic attacks more frequently in PD patients compared to healthy controls, including yohimbine, cholecystokinin, and caffeine [START_REF] Krystal | The biological basis of panic disorder[END_REF]. However, compared to CO2, it is less clear whether they represent promising markers for defining intermediate phenotypes.

PD -MOLECULAR GENETIC FINDINGS

In contrast to other neuropsychiatric disorders (e. g, schizophrenia, BD) where genome-wide association studies (GWAS) on large samples have already led to the identification of risk genes, molecular genetic research in PD has, to date, been less successful. Two GWAS have been performed in PD, but the sample sizes may have been underpowered. Most of the genetic studies published to date use linkage or candidate gene methods. The following section summarizes the most promising current molecular findings in PD, which were obtained by genome-wide linkage or association studies. However, since molecular genetic research into PD has not yet led to the identification of any definite riskgene, we provide a brief overview of the most promising findings and refer to the review by Maron et al. [START_REF] Maron | Advances in molecular genetics of panic disorder[END_REF] which summarizes all available molecular genetic findings for PD.

LINKAGE STUDIES

Because of the successful identification of risk genes in several complex disorders via GWAS, the concept of linkage has become less of a focus than in past years. It has become clear that most linkage studies did not provide enough power for the mapping of risk genes with effect sizes usually seen in GWAS. However, linkage results can remain key to the discovery of risk genes that are rare in the general population but exhibit stronger genetic effect sizes. There is hope that such rare, higher-penetrance risk variants exist, given that most of the recently identified GWAS genes explain only a fraction of the heritability seen in complex disorders. Furthermore, the possibility remains that some of the rare higherpenetrance risk genes transmitted in families have already been mapped to chromosomal regions by linkage studies. Those regions may represent ideal candidates for deep or next generation sequencing studies, which are becoming increasingly available and represent a promising tool for the systematic examination of implicated linkage intervals.

To date, six genome-wide linkage studies have been performed in PD. They are listed together with chromosomal regions that showed suggestive linkage evidence in supplementary table 3. However, the most impressive linkage signals-as defined by the level of significance and sample size-have been observed for four chromosomal regions In contrast to other psychiatric disorders-like schizophrenia and BD-no metaanalysis combining different PD linkage samples have been performed so far. However, this approach provides a more powerful tool for the identification of linkage signals associated with genetic effect sizes that are usually too small for their detection by individual studies.

ASSOCIATION STUDIES

Candidate Gene Studies: Almost all association studies have focused on genes whose function can be plausibly related to PD, either by pharmacological or neurobiological studies.

For example, genes that are involved in different neurotransmitter systems (e.g. serotonin, norepinephrine, adenosine, gamma aminobutyric acid (GABA), glutamate), the hypothalamicpituitary-adrenal (HPA) axis, or that encode for different neuropeptides have been repeatedly analyzed (for details see Maron et al. [START_REF] Maron | Advances in molecular genetics of panic disorder[END_REF]). However, as with most other complex disorders, candidate gene studies have not produced robust and replicated association findings in PD. This is complicated by the fact that most studies have used PD samples that were underpowered for the detection of risk variants associated with moderate effect sizes (e. g. >500 cases, >500 controls). In addition, the marker coverage used in most candidate gene studies was not sufficient for capturing the locus-specific pattern of linkage disequilibrium (LD).

Genome-wide Association Studies (GWAS):

Two GWAS have been conducted in PD. One study analyzed 200 PD cases and 200 controls of Japanese descent using the Affymetrix 500K array [81]. The best result was observed for the SNP marker rs860544 at the plakophilin-1 (PKP1) gene on chromosome 1q32 (p=4.60 x 10-08). However, the association could not be replicated in an independent PD sample from the same population (558 PD cases and 566 controls) [82].

The second study analyzed 260 PD cases and 260 controls of German descent using the Illumina 300K array [83]. The most significant result was observed at SNP rs7309727 at the transmembrane-protein-132D (TMEM132D) gene on chromosome 12q24, which reached genome-wide significance on the haplotypic level by incorporating three independent PD samples (>900 cases, >900 controls). In addition, the risk genotypes were associated with a higher mRNA expression of TMEM132D in the frontal cortex. A mouse model of extremes in trait anxiety similarly showed that anxiety-related behavior was positively correlated with Tmem132d mRNA expression in the anterior cingulate cortex. Functional studies have hypothesized that TMEM132D may act as a cell-surface marker for oligodendrocyte differentiation [84].

CONCLUSIONS & FUTURE DIRECTIONS

In the past few years, GWAS have provided extraordinary insights into the genetics of several complex diseases. They have shown that common risk variants can be successfully identified by applying this technology. Nevertheless, they have also demonstrated that the effect sizes of the identified risk genes are usually small, and that only large samples provide enough statistical power for successful mapping approaches. In addition, it has been shown that most of the identified risk genes explain only a fraction of disease heritability, suggesting a role for rarer risk variants or other sources of genetic variation. Indeed, for several disorders-including neuropsychiatric phenotypes-such rare mutations have already been identified (see O'Donovan et al. [85] for a review of schizophrenia).

Based on our current knowledge about complex disorders, applying GWAS to large samples must be considered as the next primary goal for PD research. This approach provides a promising tool for identifying the first risk genes in PD. To our knowledge, no single research has yet collected a sufficiently-sized GWAS sample for PD research; thus only multi-centre collaborations on combined PD samples can provide the statistical power needed for robust association findings.

It is unclear whether incorporating sub-or intermediate phenotypes such as CO2hypersensivity will be promising in the initial GWAS. Although those phenotypes are potentially less genetically heterogeneous, it is unclear whether this may outweigh the loss of power due to small sample sizes. In any case, the inclusion of PD sub-and intermediate phenotypes is a promising tool for follow-up analyses after the first PD genes have been identified through GWAS. This strategy will allow detailed disease modeling and will provide important information on the genetic architecture of distinct PD phenotypes. For instance, it would be interesting to analyze how the identified PD genes contribute to AO, disease severity (e.g. number of panic attacks), and specific neuroimaging or psychophysiological abnormalities. In addition, detailed genotype-phenotype studies on stratified samples will help assess whether gender-or family-specific disease architecture exists in PD. Furthermore, identified risk genes can be tested in independent samples with comorbid disorders such as other anxiety or mood disorders, as well as in samples with personality traits (e.g. BI, AS, N, HA) in order to assess the overlap and boundaries between PD and other disorders and conditions on the molecular level.

Finally, the next phase of genetic studies in complex disorders will involve deep sequencing approaches provided by next generation sequencing technology that will enable the detection of rare higher-penetrance risk variants. However, the price per re-sequenced genome, and the complexity of data mining and subsequent analyses pose significant challenges. Therefore, it seems that for the next few years such approaches will be restricted to the genome-wide analysis of a few samples, or of larger samples only in pre-selected chromosomal regions. In PD, this could include the most severely affected patients for genome-wide re-sequencing studies (e.g. extreme EAO cases or patients with an unusually high frequency of panic attacks), as well as cases from families where strong linkage to distinct chromosomal regions has already been reported. For example, families with a 'panic syndrome' have shown linkage to chromosome 13q32 [START_REF] Hamilton | Further genetic evidence for a panic disorder syndrome mapping to chromosome 13q[END_REF], and may represent optimal candidates for upcoming next generation sequencing studies.

Identifying PD genes will finally help establish a better understanding of geneenvironment interactions in order to identify relevant exogenous risk factors. It has long been recognized that environmental factors are of great relevance to the development of PD, but only some of these factors have been implicated. If such factors can be modulated, future PD prevention and individual genetic risk profiling could become possible. Furthermore, genes that accompany the development of PD are naturally of great interest from an evolutionary perspective, since anxiety and fear are 'normal' human emotions with a purportedly strong survival value. Therefore, studying the locus-specific genetic architecture of identified PD genes will be of great interest; such work includes analyzing their LD structure, as well as elucidating their conservation within and across species.

In conclusion, we predict that molecular studies of sufficiently large samples will lead to the identification of PD risk genes in the coming years, and that these discoveries will contribute to our understanding of the underlying neurobiology of anxiety-related disorders and behaviors. In addition, these findings will most likely impact the nosology of anxiety disorders, and lead to the development of improved prevention strategies. The panic attacks are not due to the direct physiological effect of a substance or a general medical condition The panic attacks are not better accounted for by another mental disorder, such as other anxiety disorders

  . The Yale Study of 19 extended US pedigrees with PD by Kaabi and colleagues [77] found the strongest linkage signal on chromosome 4q32-q34 (multipoint p-value of 5.4 x 10-06 at D4S413 (158 Mb)). Notably, Camp and colleagues [78] obtained results in the same linkage region in a sample of 87 extended Utah pedigrees with recurrent early-onset MDD and anxiety disorders (including PD); the male-specific analysis produced a LOD score of 2.60 at D4S2631 (156 Mb). In the Columbia Study of 120 multiplex US pedigrees with PD, Fyer and colleagues [79] observed their best linkage signal on chromosome 15q21 (NPL score of 3.44 at D15S822 (24 Mb)). Strong PD linkage signals were also reported on chromosome 9q31 in a study of 25 extended pedigrees from Iceland (LOD score 4.18 at D9S271 (102 Mb)) [80], and on chromosome 13q32 in 60 multiplex pedigrees from the US with panic syndrome that included IC, MVP, migraine, and thyroid problems (LOD score 3.57 at D13S793 (96 Mb)) [45].

Table 2

 2 Panic Disorder (PD): Clinical characteristics adopted from the Diagnostic and Statistical Manual of Mental Disorders 4 th ed. (DSM-IV) Recurrent panic attacks -defined as a discrete period of intense fear or discomfort -in which four (or more) of the following symptoms developed abruptly and reached a peak within 10 minutes • Palpitations, pounding heart, accelerated heart rate • Nausea or abdominal distress • Sweating • Feeling dizzy, unsteady, lightheaded, faint • Trembling or shaking • Derealization of depersonalization • Sensations of shortness of breath or smothering • Fear of losing control or going crazy • Feeling of choking • Fear of dying • Chest pain or discomfort At least one of the attacks has been followed by 1 month (or more) of one (or more) of the following • Persisting concern about having additional attacks • Worries about the implications of the attack or its consequences • A significant change in behavior related to the attacks

ACKNOWLEDGMENTS

The authors thank all the members of the Panic International Consortium (PAN•I•C).

Ioline Henter provided invaluable editorial assistance and critical review during the preparation of the manuscript.

FUNDING

This study was supported by the National Genomic Network of the 'Bundesministerium für Bildung und Forschung' (BMBF), by an NIH/DFG Research Career Transition Award to JS, by the Alfried Krupp von Bohlen und Halbach-Stiftung to MMN, and by the Intramural Research Program of the National Institute of Mental Health, National Institutes of Health (IRP-NIMH-NIH) to FJM and JRW.

COMPETING INTERESTS

The authors report no biomedical financial interests or potential conflicts of interests.

77 Kaabi B, Gelernter J, Woods SW, Goddard A, Page GP, Elston RC. Genome scan for loci predisposing to anxiety disorders using a novel multivariate approach: strong evidence for a chromosome [START_REF] Mendlewicz | Family study of panic disorder: comparison with generalized anxiety disorder, major depression and normal subjects[END_REF]

LICENCE FOR PUBLICATION STATEMENT

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

TABLES

Table 1 Panic Disorder (PD): Basic characteristics potentially relevant for genetic studies

• Lifetime prevalence: 3.4-4.7% (females 5.1%, males 1.9%)

• Estimated heritability: 48%

• Complex genetic inheritance in most cases

• Average age of onset: 23.6 years

• Comorbidity with other psychiatric disorders: 80%

• Other anxiety disorders: 8.2-64.0%

• Major depressive disorder (MDD): 34.7%

• Bipolar disorder (BD): 14.4%

• Schizophrenia: 17.3%

• Alcohol abuse/dependence disorder: 25.0%

• Cannabis abuse/dependence disorder: 20.6%

• Smoking: 35.9%

• Associated personality traits: Anxiety sensitivity (AS), behavioral inhibition (BI), neuroticism (N), harm avoidance (HA)

• Neuroimaging findings: Amygdala hyperactivity

• Psychophysiological findings: Respiratory abnormalities (incl. CO2-hypersensivity)


MD Adriano Chiò

Andrea Calvo

Cristina Moglia

Letizia Mazzini

Gabriele Mora

Parals

Phenotypic heterogeneity of ALS: a population-based study

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

employees) on a worldwide basis to the BMJ Publishing Group Ltd to permit this article (if accepted) to be published in JNNP and any other BMJPGL products and sublicences such use and exploit all subsidiary rights, as set out in our licence.

Introduction

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder of adult life characterized by the progressive involvement of lower and upper motor neurons at bulbar and spinal level. In 5-10% of patients a positive family history for ALS can be detected. However, in most patients the cause of ALS remains unknown. It is a generally accepted notion that the clinical spectrum of ALS includes different phenotypes marked by a varying involvement of spinal and bulbar upper and lower motor neurons. [START_REF] Ravits | Focality of upper and lower motor neuron degeneration at the clinical onset of ALS[END_REF][START_REF] Ravits | ALS motor phenotype heterogeneity, focality and spread. Deconstructing motor neuron degeneration[END_REF] Accordingly, eight distinctive clinical phenotypes are recognized in literature -classic, bulbar, flail arm, flail leg, pyramidal, respiratory, pure lower motor neuron (PLMN) and pure upper motor neuron (PUMN). [START_REF] Wijesekera | Natural history and clinical features of the flail arm and flail leg ALS variants[END_REF][START_REF] Pringle | Primary lateral sclerosis: clinical features, neuropathology and diagnostic criteria[END_REF][START_REF] Tartaglia | Differentiation between primary lateral sclerosis and amyotrophic lateral sclerosis: examination of symptoms and signs at disease onset and during follow-up[END_REF][START_REF] Visser | Disease course and prognostic factors of progressive muscular atrophy[END_REF][START_REF] Sabatelli | Natural history of young-adult amyotrophic lateral sclerosis[END_REF][START_REF] Gordon | Clinical features that distinguish PUMN, upper motor neuron-dominant ALS, and typical ALS[END_REF][START_REF] Shoesmith | Prognosis of amyotrophic lateral sclerosis with respiratory onset[END_REF] A discussion has recently arisen concerning the possibility that different ALS presentations have different aetiologies or underlying factors -whether genetic, environmental or both -that modify the phenotype. [START_REF] Rosenfeld | What's in a name? Lumping or splitting ALS, PLS, PMA, and the other motor neuron diseases[END_REF] However, at present no studies have been carried out to assess and compare all ALS phenotypes using an epidemiological approach.

The aim of this study was to evaluate the clinical characteristics and the outcome of different ALS phenotypes in a large population-based setting.

Methods

The Piemonte and Valle d'Aosta Register for ALS (PARALS) is a prospective register collecting all cases of ALS in the Piemonte and Valle d'Aosta regions of Italy (total population at the 2001 national census, 4,332,842; total area 28,692 sqkm). The register was established in 1995 and is still in operation. Epidemiological data regarding the 1995-2004 period have recently been published. [START_REF] Chiò | Epidemiology of ALS in Italy: a 10-year prospective population-based study[END_REF] Case collection. The main sources of cases were the neurology departments of the two regions. Investigators used an ad hoc questionnaire to collect patients' demographic data, disease history, neurological and laboratory findings, and treatments. Diagnostic EMG examination was performed in all patients according to standard procedures. The secondary sources for case collection were: the Piemonte and Valle d'Aosta Central Regional Archives; and the mortality coding from the Italian Bureau of Statistics. Clinical records of cases found through secondary sources were obtained, and relevant clinical information for each case was analyzed in order to verify if the patient met the eligibility criteria; all living patients were contacted by phone and visited by one of the neurologists involved in the study.

Diagnostic criteria.

The diagnosis of ALS was based on the original El Escorial diagnostic criteria (EEC) [START_REF] Brooks | Escorial World Federation of Neurology criteria for the diagnosis of amyotrophic lateral sclerosis: Subcommittee on Motor Neuron Diseases/Amyotrophic Lateral Sclerosis of the World Federation of Neurology Research Group on Neuromuscular Diseases and the El Escorial Clinical Limits of Amyotrophic Lateral Sclerosis workshop contributors[END_REF] although from 2000 cases were also classified according to El Escorial revised criteria. [START_REF] Brooks | El Escorial revisited: revised criteria for the diagnosis of amyotrophic lateral sclerosis[END_REF] Patients with PUMN 4,5 and PLMN [START_REF] Visser | Disease course and prognostic factors of progressive muscular atrophy[END_REF] were prospectively included in the register; they were not considered in the original epidemiological paper, [START_REF] Chiò | Epidemiology of ALS in Italy: a 10-year prospective population-based study[END_REF] but are included in this study. A clinical follow-up of each patient was performed at regular intervals (2 to 4 months). A standard form was used for collecting clinical information at each follow-up visit. The presence of frontotemporal dementia (FTD) was determined using an internally generated questionnaire administered to caregivers during the follow-up visits and was based on Neary's criteria. [START_REF] Lund | Clinical and neuropathological criteria for frontotemporal dementia[END_REF][START_REF] Neary | Frontotemporal lobar degeneration: a consensus on clinical diagnostic criteria[END_REF] Phenotypic classification. We classified the patients into the eight recognized phenotypes of ALS -classic, bulbar, flail arm, flail leg, pyramidal, respiratory, PLMN and PUMN. The classification was based on clinical data gleaned from all available sources (clinical charts, clinical notes of the collaborating centres, including the standard forms used for the register) which were prospectively collected during the patients' follow-up. It was established according to the clinical and EMG picture at diagnosis and revised during the follow-up.

Classic (Charcot's) phenotype. Classic ALS was characterized by onset of symptoms in upper or lower limbs, with clear but not predominant pyramidal signs.

Bulbar phenotype. These patients had a bulbar onset with dysarthria and/or dysphagia, tongue wasting, fasciculation and no peripheral spinal involvement for the first 6 months after symptoms onset. Pyramidal signs were not required to be evident in the first 6 months, but needed to be evident thereafter.

Flail arm phenotype. [START_REF] Wijesekera | Natural history and clinical features of the flail arm and flail leg ALS variants[END_REF] Patients in this group were characterized by progressive predominantly proximal weakness and wasting in the upper limbs. In this category we also included patients with pathological deep tendon reflexes (DTRs) or Hoffman sign in the upper limbs at some point during the disease, but without hypertonia or clonus. Functional involvement had to be confined to the flail limbs for at least 12 months after symptoms onset.

Flail leg phenotype. [START_REF] Wijesekera | Natural history and clinical features of the flail arm and flail leg ALS variants[END_REF] Patients were characterized by progressive distal onset of weakness and wasting in the lower limbs. In this category we also included patients with pathological DTRs or Babinski sign in the lower limbs at some point during the disease, but without hypertonia or clonus. Patients with wasting and weakness beginning proximally in the legs without distal involvement at presentation were classified as classic ALS.

Pyramidal phenotype (predominant-upper motor neuron ALS). [START_REF] Sabatelli | Natural history of young-adult amyotrophic lateral sclerosis[END_REF][START_REF] Gordon | Clinical features that distinguish PUMN, upper motor neuron-dominant ALS, and typical ALS[END_REF] These patients had clinical manifestations dominated by pyramidal signs, mainly severe spastic para/tetraparesis, associated with one or more of the following signs: Babinski or Hoffmann sign, hyperactive reflexes, clonic jaw jerk, dysarthric speech, pseudobulbar affect. Spastic paresis could be present at the beginning or in the fully developed stage of the disease. These patients showed at the same time clear-cut signs of lower motor neuron impairment from onset of the disease, as indicated by muscle weakness and wasting and by the presence of chronic and active denervation at the EMG examination in at least two different sites.

Respiratory phenotype. [START_REF] Shoesmith | Prognosis of amyotrophic lateral sclerosis with respiratory onset[END_REF] These patients had prevalent respiratory impairment at onset, defined as orthopnoea or dyspnoea at rest or during exertion, with only mild spinal or bulbar signs in the first 6 months after onset. These patients showed signs of upper motor neuron involvement.

PLMN. [START_REF] Visser | Disease course and prognostic factors of progressive muscular atrophy[END_REF] These patient had clinical and electrophysiological evidence of progressive LMN involvement. From this category we excluded patients with motor conduction block(s) on extensive standardized nerve conduction studies, clinical UMN signs, history of disease that mimic motor neuron disease, family history of inherited SMA and deletion in the SMN1 gene or expansion of CAG repeat in the androgen receptor gene. Neuroimaging studies were performed to rule out structural lesions. PUMN. [START_REF] Pringle | Primary lateral sclerosis: clinical features, neuropathology and diagnostic criteria[END_REF][START_REF] Tartaglia | Differentiation between primary lateral sclerosis and amyotrophic lateral sclerosis: examination of symptoms and signs at disease onset and during follow-up[END_REF] These patients had clinical signs of UMN involvement, i.e. severe spastic para/tetraparesis, Babinski or Hoffmann sign, hyperactive reflexes, clonic jaw jerk, dysarthric speech, pseudobulbar affect. From this category we excluded patients with clinical or electromyographical signs of LMN involvement, according to El Escorial criteria, during the follow-up, history of disease that mimic motor neuron disease, family history of spastic paraparesis/tetraparesis and mutation of genes related to hereditary spastic paraplegia (SPG3A, SPG4, SPG6, SPG7 and SPG20).

Statistical Methods. Ninety-five percent confidence intervals (CIs) were calculated assuming a Poisson distribution. [START_REF] Schoenberg | Calculating confidence intervals for rates and ratios[END_REF] Comparison between means were made with the analysis of variance (ANOVA). The effect of age and gender on ALS phenotypes was assessed with multivariate analysis of variance (MANOVA), including age, gender and the interaction between age and gender as dependent variables. Survival was calculated to death/tracheostomy or censoring date (December 31 st , 2009), using the Kaplan-Meier method and compared with the log-rank test.

Multivariable analysis was performed with Cox's proportional hazards model (stepwise forward)

(see E-Table 2 for a list of the variables included in the model).

A p level <0.05 was considered significant. Data were processed using SAS statistical package (Cary, NC; version 8.2). No patients were lost to follow-up.

Standard Protocol Approvals, Registrations, and Patient Consents.

The study design was approved by the Ethical Committee of the coordinating centre. Patients' consent was not required since this study did not modify the routine clinical practice. However, databases were managed according to the Italian law for the protection of privacy.

Results

A total of 1351 patients were diagnosed with ALS in the period 1995-2004 in Piemonte and Valle d'Aosta. [START_REF] Chiò | Epidemiology of ALS in Italy: a 10-year prospective population-based study[END_REF] Clinical phenotype was established in 1332 patients (98.6%). Clinical phenotype was established blindly by three of the authors (AC, LM, GM). When the classification differed, a discussion was performed to reach a consensus. In 19 patients (1.4%) no consensus was reached mainly due to lack of full clinical details; these cases were excluded from the analysis. These patients did not differ in terms of demographic or clinical characteristics from those with full clinical details.

Epidemiological and clinical characteristics of ALS phenotypes. The clinical features of

ALS phenotypes are reported in Table 1 and their mean annual incidence rates and gender rate ratios are shown in Table 2.

1. Classic phenotype. This is the commonest ALS phenotype in men and the second commonest in women, with a men to women rate ratio of 1.65:1. Its age at onset is 62.8 years (SD 11.3), with a peak of the age-specific incidence rate in the 7 th decade in both genders (Figure 1A). Four percent of cases with this phenotype have FTD. Its median survival time is 2.6 years (Supplemental Table 1), with a 10-year survival rate of 13.0%.

Bulbar phenotype.

Bulbar ALS has the same incidence in the two genders (1/100,000 population), with a men to women rate ratio of 0.98:1. The peak of the age-specific incidence rate is in the 8 th decade in both genders (Figure 1B). Nine percent of bulbar patients have FTD, the highest figure among ALS phenotype. The median survival time of bulbar ALS is the second worst (2.0 years); only 3.4% of patients survived up to 10 years.

Flail arm phenotype.

This phenotype is relative rare and more common in men (incidence rates, 0.28 in men and 0.07 in women), with a men to women rate ratio of 4.00:1. Its mean age at onset is 62.6 years (SD 11.8). In this phenotype FTD is very rare (1.4%). Flail arm phenotype is relatively benign, with a median survival time of 4.0 years and a 10-year survival rate of 17.4%. In these patients, ALS remains restricted to upper limbs for a mean of 20 months after the onset.

Flail leg phenotype.

This phenotype has a similar incidence in the two genders, with a men to women rate ratio of 1.03:1. The mean age at onset is the second highest (65.0 years) and the peak of age-specific incidence rate is in the 8 th decade (Figure 1D). FTD is present in 4% of patients with this phenotype. The median survival time (3.0 years) and the 10-year survival rate (12.8%) of this phenotype are similar to those of classic ALS. In these patients, ALS remains restricted to upper limbs for a mean of 16 months after the onset.

Pyramidal phenotype.

Patients with this phenotype have a quite young age at onset (58.3 years). Gender are equally represented, with a men to women rate ratio of 1.04:1. The peak of the age-specific incidence rate is in the 7 th decade (Figure 1E). FTD is rather uncommon (2.5%). The median survival time is 6.3 years and the 10-year survival rate is 31.9% (E-Table 1).

6.

Respiratory phenotype. This is the rarest phenotype (annual incidence rate: men, 0.06/100,000; women, 0,01/100,000). Its median survival time is 1.4 years and no patient with this phenotype survived up to 10 years.

PLMN. PLMN has a quite low incidence rate and is twice more frequent in male gender

(men to women rate ratio, 2.04:1). Patients with PLMN are younger than those with any other ALS phenotype (56.2 years) with a peak of the age-specific incidence rate in the 7 th decade among men and in the 6 th among women (Figure 1F). No patient with PLMN phenotype developed FTD. PLMN patients have a longer survival than any other phenotype besides PUMN (median survival time, 7.3 years).

PUMN.

PUMN has a relatively low incidence rate (0.12 in both genders) and mean age at onset (58.9 years). The peak of age-specific incidence rate is in the 6 th decade in both genders (Figure 1G). The median survival time of PUMN is the longest among ALS phenotypes (13.1 years), with a 10-year survival rate of 71.1%.

Relative frequency of ALS phenotypes through the age-groups.

The relative frequency of ALS phenotypes showed an intriguing trend with age: among men (Figure 2A) there was a decline in frequency of flail arm, pyramidal and classic phenotypes with increasing age, and an increase of bulbar (from 10% to 51%) and flail leg (from 0 to 12%) phenotypes; among women (Figure 2B), the decline in frequency of pyramidal (from 37.5% to 6%) and classic (from 37.5% to 12%) phenotypes was even more pronounced, as well as the rise in frequency of the bulbar phenotype (from 6% to 72%).

Influence of age and gender on ALS phenotypes.

According to the results of the multivariate analysis of variance (MANOVA), 54% of total variance of ALS phenotypes was explained by age and gender.

ALS phenotypes survival.

The survival curves of ALS phenotypes are compared in Figure 3. The outcome of the different phenotypes differed significantly (p<0.0001). At multivariable analysis (Cox model) (Supplemental Table 2), the variables independently related to outcome were age at onset (p<0.0001), bulbar phenotype (p<0.0001), PUMN (p<0.0001), pyramidal phenotype (p<0.0001), respiratory phenotype (p<0.0001), FTD (p=0.0008), and flail arm phenotype (p=0.008).

Discussion

This is the first comprehensive survey directly comparing all ALS phenotypes in a large epidemiological setting with a prospective design. The advantage of this large population-based study is that it includes detailed and standardized phenotypic information, thus avoiding the referral bias that is inherent to clinic-based cohorts. [START_REF] Logroscino | Incidence of amyotrophic lateral sclerosis in Europe[END_REF] The main finding of our study is that ALS clinical phenotypes carry highly distinctive clinical, demographic and prognostic characteristics. In brief, bulbar phenotype is typical of older patients, has similar incidence rates in the two genders, and carries the worst survival. At the opposite extreme, pyramidal phenotype and PUMN are typical of younger patients and have the most benign outcome. Flail arm phenotype is rare, more frequent in men and has a relatively good prognosis, while flail leg phenotype, which is the third more common phenotype, has an equal incidence in both genders and a slightly worse outcome. Classic phenotype, at last, has the highest incidence among men, and carries an intermediate outcome.

The different clinical and demographic characteristics of ALS phenotypes translates in different probabilities of having a presentation with a specific phenotype at different ages. As shown in Figure 2, bulbar phenotype represented less than 10% of cases under 39, to increase up to more than 50% over 80, with a more marked trend among women, whereas classic phenotype steadily decreased going from the younger to the older decades. The influence of age on ALS clinical features had been also reported in a clinical-based series of ALS patients in Japan. [START_REF] Atsuta | Age at onset influences on wide-ranged clinical features of sporadic amyotrophic lateral sclerosis[END_REF] The male predominance generally reported in ALS [START_REF] Logroscino | Incidence of amyotrophic lateral sclerosis in Europe[END_REF][START_REF] Talman | Clinical phenotypes and natural progression for motor neuron disease: analysis from an Australian database[END_REF] is true for only four phenotypesclassic, flail arm, respiratory and PLMN. The two genders were almost equally represented among the other phenotypes. Therefore, when considering incidence rates and not the absolute figures, the predominance of women in the bulbar phenotype described in clinical series appears to be simply related to the older age at onset of this phenotype. ALS phenotype bears a strong influence on disease outcome. Bulbar and respiratory phenotypes carried the worst prognosis, [START_REF] Chiò | Prognostic factors in ALS: A critical review[END_REF] while pyramidal phenotype had by far the longest survival. [START_REF] Sabatelli | Natural history of young-adult amyotrophic lateral sclerosis[END_REF] Interestingly, the survival time of the pyramidal phenotype of ALS was shorter than that of PUMN, but longer than all other ALS phenotypes, supporting the notion that it represents an intermediate form between classic ALS and PUMN. [START_REF] Gordon | Clinical features that distinguish PUMN, upper motor neuron-dominant ALS, and typical ALS[END_REF][START_REF] Gordon | The natural history of primary lateral sclerosis[END_REF] Also flail arm phenotype had a relatively good outcome, with a median survival time of four years. [START_REF] Wijesekera | Natural history and clinical features of the flail arm and flail leg ALS variants[END_REF][START_REF] Katz | Brachial amyotrophic diplegia: a slowly progressive motor neuron disorder[END_REF] The outcome of classic and flail leg phenotypes were similar, at variance with the findings of a previous paper reporting a better prognosis for flail leg phenotype. [START_REF] Wijesekera | Natural history and clinical features of the flail arm and flail leg ALS variants[END_REF] Cox's multivariable analysis confirmed the independent role of ALS phenotype on survival and suggests that this classification should be used for patients' stratification in clinical trials, for example utilizing the minimization method. [START_REF] Altman | Treatment allocation by minimization[END_REF] FTD was present in about 5% of patients, mainly those with bulbar phenotype. However, many ALS patients have evidence of frontotemporal behavioural dysfunction that may not satisfy Neary criteria for FTD and subclinical syndromes have not been considered in the present paper. [START_REF] Strong | The syndromes of frontotemporal dysfunction in amyotrophic lateral sclerosis[END_REF] Our findings support the idea that ALS is a clinically heterogeneous disease. The heterogeneity of motor phenotypes in ALS has been recently analyzed considering three symptom dimensions -body region of onset, relative mix of upper motor neuron and lower motor neuron involvement, and rate of progression, 2 and hypothesizing that the initial trigger of ALS is a stochastic process, with an apparently random but focal initiation. According to our findings, however, the focal initiation of ALS is not entirely random, since it is strongly determined by patients' age and gender, as indicated by the fact that these two factors explained about 50% of the clinical variance.

A previous study analyzed ALS prognostic groups using a latent class clustering. [START_REF] Ganesalingam | Latent cluster analysis of ALS phenotypes identifies prognostically differing groups[END_REF] The authors identified five classes, of which the first included most patients with classic, flail arm and flail leg phenotypes, the second, which carries the worst prognosis, includes most bulbar onset patients, and the fourth and fifth include patients with mixed phenotypes but with a milder clinical course.

The reasons of the influence of these two factors on ALS clinical presentation remain presently unknown. In the pre-clinical model of ALS, the SOD1 transgenic mouse, male rodents carrying the human mutated gene have an earlier age at onset, an early progress of locomotor rating scores, and a shorter survival than their female counterparts, with a clear gender-related phenotypic dimorphism. [START_REF] Veldink | Sexual differences in onset of disease and response to exercise in a transgenic model of ALS[END_REF][START_REF] Suzuki | Sexual dimorphism in disease onset and progression of a rat model of ALS[END_REF] This dimorphism has been related to gonadal hormones. [START_REF] Groeneveld | Ovariectomy and 17beta-estradiol modulate disease progression of a mouse model of ALS[END_REF] However, in human ALS, data on reproductive factors and sexual hormones are scarce and contradictory, [START_REF] Rudnicki | Estrogen replacement therapy in women with amyotrophic lateral sclerosis[END_REF][START_REF] Popat | Effect of reproductive factors and postmenopausal hormone use on the risk of amyotrophic lateral sclerosis[END_REF] not allowing to draw any definitive conclusion.

A strong effect of age and gender on clinical phenotype has been reported also in Parkinson's disease (PD), [START_REF] Haaxma | Gender differences in Parkinson's disease[END_REF] where the later age at onset, the more frequent presentation with tremor and the milder clinical course in female have been explained with the higher initial dopamine levels in the substantia nigra, which delay the moment of reaching a critical threshold of striatal dopamine depletion. Unfortunately, in ALS there are no studies assessing the age-and gender-related diversities in motor neuron pool and the receptor function at upper and lower motor neuron level.

Genetic factors may play a role in determining the range of ALS phenotypes, although to date no genes have been shown to have a definite effect on phenotype. Patients with SOD1 mutations show a phenotypic heterogeneity even within the same mutation, [START_REF] Andersen | Phenotypic heterogeneity in motor neuron disease patients with CuZn superoxide dismutase mutations in Scandinavia[END_REF] although some specific missense mutations carry a consistently worse (i.e. A4V, G41S) or better prognosis (i.e. H46R, G93C). Patients with FUS mutations are also quite heterogeneous, but some mutations seem to carry more defined phenotypes: the R514S and R521C missense mutations are characterized by a predominantly proximal and axial phenotype [START_REF] Chiò | Two Italian kindreds with familial amyotrophic lateral sclerosis due to FUS mutation[END_REF][START_REF] Lai | FUS mutations in sporadic amyotrophic lateral sclerosis[END_REF] and the P525L missense mutation is characterized by a very young age at onset (<30 years), with a bulbar presentation and a short duration. [START_REF] Chiò | Two Italian kindreds with familial amyotrophic lateral sclerosis due to FUS mutation[END_REF][START_REF] Kwiatkowski | Mutations in the FUS/TLS gene on chromosome 16 cause familial amyotrophic lateral sclerosis[END_REF] A role of environmental factors on ALS phenotypic expression cannot be ruled out. The overall higher frequency of ALS in men could be related to occupational exposures to metals and toxins, while the increase of cigarette smoking, an established exogenous risk factor for ALS, [START_REF] Armon | Smoking may be considered an established risk factor for sporadic ALS[END_REF] could explain the progressive reduction of the gender gap reported in recent ALS epidemiological studies. [START_REF] Logroscino | Incidence of amyotrophic lateral sclerosis in Europe[END_REF] We conclude that our epidemiological data strongly support that recognized ALS phenotypes have different clinical, demographic and outcome characteristics, and can be recognizable even in a population-based setting. The pathophysiological bases of the varying pattern of motor neuron degeneration, partly related to age and gender, are still largely unknown. A better understanding of the factors that influence the phenotypic expression of ALS would be important for identifying the underlying biochemical, genetic and environmental mechanisms of the disease. Moreover, the categorization of ALS patients according to clinical phenotypes should be considered for improving the design of clinical trials and to better focus the communication of diagnosis and outcome with the patients and their families.

Table 1 .

 1 Mean age at onset, mean time delay from onset to diagnosis and frequency of frontotemporal dementia

	Phenotype	Number of	Mean age at onset	Median age at	Mean diagnostic	Median	Cases with
		cases (%)	(SD), years	onset (interquartile	delay (SD), months	diagnostic	FTD (%)
				range) § (SD)		delay
						(interquartile
						range) § (SD)
	Classic	404 (30.3%)	62.8 (11.3)	64.6 (56.1-70.6)	10.9 (9.6)	8 (5-13)	16 (4.0%)
	Bulbar	456 (34.2%)	68.8 (9.7)	69.9 (62.9-75.0)	9.8 (7.0)	8 (5-12)	41 (9.0%)
	Flail arm	74 (5.5%)	62.6 (11.8)	63.3 (54.8-72.2)	12.8 (11.0)	9 (5-15)	1 (1.4%)
	Flail leg	173 (13.0%)	65.0 (9.6)	65.6 (58.5-71.2)	13.1 (10.1)	11 (7-17)	7 (4.1%)
	Pyramidal	120 (9.1%)	58.3 (13.5)	60.1 (49.2-68.3)	15.9 (13.4)	12 (6-22)	3 (2.5%)
	Respiratory	14 (1.1%)	62.2 (8.6)	62.0 (58.3-65.3)	6.4 (4.3)	5 (3-9)	-
	PLMN	38 (2.9%)	56.2 (11.3)	55.2 (45.7-61.3)	15.5 (12.4)	14 (10-19)	-
	PUMN	53 (4.0%)	58.9 (10.9)	56.5 (48.3-62.6)	15.9 (14.3)	15 (10-19)	2 (3.8%)
	Overall ALS	1332	64.3 (11.3)	65.3 (59.7-71.8)	10.8 (10.4)	9 (5-14)	70 (5.4%)
			p=0.0001 *		p=0.0001 *		p=0.0001 **

Table 2 .

 2 ALS phenotypes. Overall and men vs. women mean annual crude incidence rates (/100,000 population), 95% confidence intervals (CIs), and gender incidence rate ratios ALS phenotypes: incidence rates according to age group for males vs. females.Respiratory phenotype is not shown due to the low number of cases. PUMN, pure upper motor neuron phenotype; PLMN, pure upper motor neuron phenotype. Relative frequency of ALS phenotypes according to age. A, men; B, women. The percentage is calculated on the incidence rates. PUMN, pure upper motor neuron phenotype; PLMN, pure upper motor neuron phenotype.

	Phenotype	Overall,	Men,	Women,	Men to Women
		incidence rate	incidence rate	incidence rate	incidence rate
		(CI)	(CI)	(CI)	ratio
	Classic	0.94 (0.85-1.04) 1.17 (1.03-1.32)	0.71 (0.61-0.83)	1.65:1
	Bulbar	1.05 (0.96-1.15) 1.04 (0.91-1.19)	1.06 (0.94-1.20)	0.98:1
	Flail arm	0.17 (0.13-0.21) 0.28 (0.21-0.36)	0.07 (0.04-0.12)	4.00:1
	Flail leg	0.40 (0.34-0.47) 0.40 (0.32-0.50)	0.39 (0.31-0.48)	1.03:1
	Pyramidal	0.28 (0.23-0.34) 0.28 (0.21-0.36)	0.27 (0.21-0.35)	1.04:1
	Respiratory	0.03 (0.02-0.05) 0.06 (0.03-0.10)	0.01 (0-0.03)	6.00:1
	PLMN	0.08 (0.06-0.11) 0.11 (0.07-0.17)	0.05 (0.03-0.08)	2.04:1
	PUMN	0.12 (0.09-0.16) 0.12 (0.08-0.18)	0.12 (0.08-0.17)	0.98:1
	Overall ALS	3.07 (2.89-3.25) 3.46 (3.23-3.71)	2.68 (2.44-2.90)	1.29:1

Figure 3. Tracheostomy-free survival, according to ALS phenotypes. Yellow, PUMN; red, PLMN; light blue, pyramidal ALS; grey, flail arm; violet, classic ALS; green, flail leg; blue, bulbar; cyan, respiratory. Crosses are censored patients.

Acknowledgements

Adriano Chiò, Letizia Mazzini, and Gabriele Mora had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. This paper was supported in part by grants from Regione Piemonte (Ricerca Finalizzata 2002, grant 12944; Ricerca Scientifica Applicata 2004, grant A317), and Compagnia di San Paolo (grant 2003.0078).