Regina Taurines
email: regina.hue@gmx.de

Johannes Thome
email: j.thome@swansea.ac.uk

J Catharina Duvigneau

Sarah Forbes-Robertson
email: forbes-robertson@swansea.ac.uk

Liya Yang
email: liyayangdr@hotmail.com

Karin Klampfl
email: klampfl@kjp.uni-wuerzburg.de

Jasmin Romanos
email: romanos2@kjp.uni-wuerzburg.de

Sabine Mu ¨ller •
email: sabine.mueller@uniklinik-ulm.de

Manfred Gerlach
email: manfred.gerlach@uni-wuerzburg.de

Claudia Mehler-Wex
email: claudia.mehler-wex@uniklinik-ulm.de

Á K Klampfl

Á J Romanos

Á M Gerlach

Á S Forbes-Robertson

Á L Yang

S Mu

Á C Mehler-Wex

Expression analyses of the mitochondrial complex I 75-kDa subunit in early onset schizophrenia and autism spectrum disorder: increased levels as a potential biomarker for early onset schizophrenia

Keywords: Biological marker, Mitochondrial complex I, 75-kDa subunit, Early onset schizophrenia, Autistic spectrum disorder

Searching for a peripheral biological marker for schizophrenia, we previously reported on elevated mitochondrial complex I 75-kDa subunit mRNA-blood concentrations in early onset schizophrenia (EOS). The aim of this study was to further evaluate the utility of this gene as a potential marker for schizophrenia. Both-schizophrenia and autism-are suggested to be neuronal maldevelopmental disorders with reports of mitochondrial dysfunction and increased oxidative stress. Therefore we have investigated the expression levels of mitochondrial complex I 75-kDa subunit mRNA in whole blood of children with autistic spectrum disorder (ASD) and a group of adolescent acute first-episode EOS patients in comparison to matched controls. We have found that compared to the respective controls only the group of EOS patients-and not the ASD group-showed a significantly altered expression of the complex I 75-kDa subunit mRNA. Although further studies are necessary to test for the specificity of this marker, our findings point to the potential use of the mitochondrial complex I as a biomarker for schizophrenia.

Introduction

The severe psychiatric condition ''schizophrenia'' sometimes manifests in children and adolescents as early onset schizophrenia (EOS). The clinical and biological heterogeneity of schizophrenia often hampers a rapid and defin-itive diagnosis. Since diagnostic procedures are only based on descriptive behavioral information, a disease marker in peripheral tissue would help to accelerate diagnosis and monitor treatment course. In several studies [START_REF] Ben-Shachar | Increased mitochondrial complex I activity in platelets of schizophrenic patients[END_REF][START_REF] Ben-Shachar | Cerebral glucose utilization and platelet mitochondrial complex I activity in schizophrenia: a FDG-PET study[END_REF][START_REF] Ben-Shachar | Sp1 expression is disrupted in schizophrenia; a possible mechanism for the abnormal expression of mitochondrial complex I genes, NDUFV1 and NDUFV2[END_REF][START_REF] Dror | State-dependent alterations in mitochondrial complex I activity in platelets: a potential peripheral marker for schizophrenia[END_REF][START_REF] Karry | Mitochondrial complex I subunits expression is altered in schizophrenia: a postmortem study[END_REF] researchers focused their analyses on the complex I of the mitochondrial electron transporter system, due to the repeatedly found abnormalities in cerebral energy metabolism and mitochondrial function in schizophrenia [START_REF] Ben-Shachar | Mitochondrial dysfunction in schizophrenia: a possible linkage to dopamine[END_REF][START_REF] Ben-Shachar | Mitochondria, synaptic plasticity, and schizophrenia[END_REF][START_REF] Buchsbaum | The frontal lobes, basal ganglia, and temporal lobes as sites for schizophrenia[END_REF][START_REF] Buchsbaum | Glucose metabolic rate in normals and schizophrenics during the continuous performance test assessed by positron emission tomography[END_REF][START_REF] Buchsbaum | Relative glucose metabolic rate higher in white matter in patients with schizophrenia[END_REF][START_REF] Cohen | Evidence for common alterations in cerebral glucose metabolism in major affective disorders and schizophrenia[END_REF][START_REF] Gur | Resting cerebral glucose metabolism in first-episode and previously treated patients with schizophrenia relates to clinical features[END_REF][START_REF] Hazlett | Prefrontal cortex glucose metabolism and startle eyeblink modification abnormalities in unmedicated schizophrenia patients[END_REF][START_REF] Hazlett | Abnormal glucose metabolism in the mediodorsal nucleus of the thalamus in schizophrenia[END_REF][START_REF] Haznedar | Cingulate gyrus volume and metabolism in the schizophrenia spectrum[END_REF][START_REF] Iwamoto | Altered expression of mitochondria-related genes in postmortem brains of patients with bipolar disorder or schizophrenia, as revealed by large-scale DNA microarray analysis[END_REF][START_REF] Middleton | Gene expression profiling reveals alterations of specific metabolic pathways in schizophrenia[END_REF][START_REF] Prabakaran | Mitochondrial dysfunction in schizophrenia: evidence for compromised brain metabolism and oxidative stress[END_REF]. Ben-Shachar et al. described a high sensitivity and specificity for the mitochondrial complex I as a potential biomarker of schizophrenia. They found an increased enzymatic activity of the complex I in schizophrenic patients compared to controls [START_REF] Ben-Shachar | Increased mitochondrial complex I activity in platelets of schizophrenic patients[END_REF]. Furthermore a positive correlation between the enzymatic activity and psychotic symptomatology was found [START_REF] Dror | State-dependent alterations in mitochondrial complex I activity in platelets: a potential peripheral marker for schizophrenia[END_REF].

In a recent study we showed increased levels of complex I mRNA expression for the first time in a rare population of adolescent EOS patients [START_REF] Mehler-Wex | Increased mRNA levels of the mitochondrial complex I 75-kDa subunit. A potential peripheral marker of early onset schizophrenia?[END_REF]. In schizophrenia research, many studies pointed to a neurodevelopmental etiology of this disorder [START_REF] Durany | Neurotrophic factors and the pathophysiology of schizophrenic psychoses[END_REF][START_REF] Rapoport | The neurodevelopmental model of schizophrenia: update[END_REF]. There is growing evidence that abnormalities in prenatal or perinatal brain development ultimately result in an increased predisposition to schizophrenia.

Similarly to this disease concept, ASD is supposed to be caused by a neuronal maldevelopment, reflected by cytoarchitectural abnormalities in many brain regions [START_REF] Geschwind | Autism spectrum disorders: developmental disconnection syndromes[END_REF][START_REF] Palmen | Neuropathological findings in autism[END_REF][START_REF] Polleux | Toward a developmental neurobiology of autism[END_REF]. Just like in schizophrenia pathophysiology, a variety of biochemical, anatomical and neuroradiographic studies imply an impairment of brain energy metabolism in ASD [START_REF] Chauhan | Oxidative stress in autism: increased lipid peroxidation and reduced serum levels of ceruloplasmin and transferrin-the antioxidant proteins[END_REF][START_REF] Filipek | Mitochondrial dysfunction in autistic patients with 15q inverted duplication[END_REF][START_REF] Fillano | Mitochondrial dysfunction in patients with hypotonia, epilepsy, autism, and developmental delay: HEADD syndrome[END_REF][START_REF] Graf | Autism associated with the mitochondrial DNA G8363A transfer RNA(Lys) mutation[END_REF][START_REF] Pons | Mitochondrial DNA abnormalities and autistic spectrum disorders[END_REF][START_REF] ¨gu ¨t | Changes in nitric oxide levels and antioxidant enzyme activities may have a role in the pathophysiological mechanisms involved in autism[END_REF].

The aim of our study was to re-assess the relative utility of mitochondrial complex I 75-kDa subunit mRNA levels as a marker for schizophrenia. In order to assess whether the increased 75-kDa subunit mRNA expression generally reflects an abnormal neuronal development or whether it is more closely related to schizophrenia, we have determined the expression of this gene in a group of ASD children, and in parallel in a group of acute first-episode EOS patients. In both groups mRNA levels were assessed in whole blood cells by real-time PCR in comparison to a respective ageand gender-matched healthy control group. As a positive control we have included the previously examined sample group [START_REF] Mehler-Wex | Increased mRNA levels of the mitochondrial complex I 75-kDa subunit. A potential peripheral marker of early onset schizophrenia?[END_REF].

Methods

Subjects

Patients were recruited at the hospital for child and adolescence psychiatry and psychotherapy of the University of Wuerzburg and Ulm. The local ethics committees approved this study and all subjects and parents gave their written informed consent.

Typically developing healthy participants were recruited from primary, secondary or grammar school. They were screened for behavioral problems by the Achenbach Child Behavior Check List (CBCL) [START_REF] Achenbach | Behavioural problems and competencies reported by parents of normal and disturbed children aged four through sixteen[END_REF]. Individuals were excluded from the control group if they suffered from a somatic or neurological disease, were taking medication or exhibited abnormal CBCL scores.

We included into this study 10 patients with acute first-episode, neuroleptic-naı ¨ve EOS and 10 age-and gender-matched controls of our previous report. These samples were re-analyzed in parallel to the other two groups as a positive control. For demographic details please see [START_REF] Mehler-Wex | Increased mRNA levels of the mitochondrial complex I 75-kDa subunit. A potential peripheral marker of early onset schizophrenia?[END_REF].

Our second, independent schizophrenia sample consisted of 1). Schizophrenia diagnoses (see Table 1) were determined by an experienced child psychiatrist according to ICD-10/DSM-IV criteria. Blood was collected in the acute, if possible neuroleptic-naı ¨ve state. Four of the 12 schizophrenic patients were neuroleptic-naı ¨ve, 8 had a short period of drug treatment before the first blood withdrawal (see Table 1). The brief psychiatric rating scale (BPRS) [START_REF] Overall | The brief psychiatric rating scale[END_REF] was performed concomitantly with the blood withdrawal. This group was age-and gender-matched with a group of 12 control children from our pool of controls (for demographic details on age and CBCL scores see Table 1).

The ASD group consisted of 16 male patients (mean age 13.6 years; age range from 8 years, 2 months to 18 years, 0 months). ASD diagnoses were based on the ICD-10/ DSM-IV criteria and determined by an experienced child psychiatrist (see Table 2). Diagnoses were further confirmed by the following measures: The Questionnaire on Behavioral and Social Communication (VSK) [START_REF] Rutter | Social communication questionnaire (SCQ)[END_REF][START_REF] Bolte | FSK-Fragebogen zur Sozialen Kommunikation[END_REF], the Autism Diagnostic Interview-Research [START_REF] Lord | Autism diagnostic interview-revised: a revised version of a diagnostic interview for caregivers of individuals with possible pervasive developmental disorders[END_REF][START_REF] Bolte | Autism Diagnostic Observation Schedule: a standardized observation of communicative and social behavior[END_REF], and the Autism Diagnostic Observation Schedule [37][START_REF] Lord | The autism diagnostic observation schedule-generic: a standard measure of social and communication deficits associated with the spectrum of autism[END_REF][START_REF] Ruehl | Evaluating the comparability of gene expression in blood and brain[END_REF]. For the ASD questionnaire mean scores and cut-offs see Table 2. ASD patients with a known severe somatic or neurological disorder as well as schizophrenia were excluded from the study. For comorbid diagnoses and medication see Table 2. The ASD group was age-and gender-matched with a group of 16 healthy children (for demographic details on age and CBCL scores see Table 2).

Isolation of total RNA

PAXgene blood RNA kit (PreAnalytiX, Hombrechtikon, Switzerland) was used according to the manufacturers instructions to isolate total RNA from blood followed by DNAse treament (RNAse-free DNase Set, Quiagen, Oslo, Norway). We evaluated the RNA purity and yield by UV-spectroscopy.

Reverse transcription

For the reverse transcription we used the RETROscript kit (Ambion, Cambridgeshire, UK) with the moloney murine leukemia virus following the manufacturer' s instructions using oligo(dT) primers.

Real-time quantitative PCR

Primers were designed with Beacon Designer Software (Biosoft International, Stratagene, La Jolla, CA, USA) and synthesized commercially by Invitrogen (Custom Primers, Karlsruhe, Germany). Real-time PCR was carried out at the myIQ (Bio-Rad, Hercules, CA, USA) with AbsoluteTM SYBR Green Fluorescein Mix (Abgene, Epsom, UK); each sample in triplicate. Patient samples and matched controls were run on the same plate in one experiment. We used beta-actin as a housekeeping gene (in accordance with our first protocoll, [START_REF] Mehler-Wex | Increased mRNA levels of the mitochondrial complex I 75-kDa subunit. A potential peripheral marker of early onset schizophrenia?[END_REF], for a better comparability). Primer sequences, annealing temperature and product size of the amplified product are shown in Table 3. PCR conditions were the following: initial enzyme activation cycle 95°C (15 min), denaturation 94°C (20 s), annealing TA (30 s), elongation 72°C (30 s), extra step at 80°C to exclude primer dimers (15 s). 45 cycles of denaturation, annealing, elongation and the extra step were used. For the optimization of the assay conditions and specificity we performed melt curve analysis and gel electrophoresis. We also analyzed controls without reverse transcriptase and no template controls. Our standard curves were performed in triplicates and primer efficiency was determined to be within a suitable range for the use of the 2^DDCT method. Real time quantitative-PCR products were assessed in a 2% agarose gel containing SYBR Save DNA gel stain (Invitrogen) with a 100 bp DNA ladder (Promega).

Data analysis and statistics

To compare gene expression levels we applied the 2^DDCT method using the Biorad iQ5 Optical System Software version 2.0. When comparing age-and gendermatched patient and control groups, two-tailed t-tests were used, after normal distribution was confirmed by Kolmogorov-Smirnov test. A two-tailed type I error rate of 5% was chosen.

For the analysis of the sensitivity and specificity of our potential marker we calculated the 2^DDCT difference of each matched patient-control-pair and determined the cut off that separated best the EOS from the ASD group (by receiver operating characteristic-curves in SPSS).

Results

All patients of our two independent schizophrenia groups had a severe disease. Patients of our previously tested sample [START_REF] Mehler-Wex | Increased mRNA levels of the mitochondrial complex I 75-kDa subunit. A potential peripheral marker of early onset schizophrenia?[END_REF], which we have included as positive control, had a mean initial BPRS value (± standard deviation; SD) of 58.1 ± 10.6. In our new, independent schizophrenia group the mean initial BPRS value was 62.2 ± 16.4 (see Table 1). The two groups did not differ in their mean age ± SD, with 16.5 ± 3.3 years in the first and 16.4 ± 1.4 in the new sample. There were slightly more female patients in the second group (20% in the first versus 33.3% in the new sample). The main diagnosis in both samples was the ICD-10 F20.0, paranoid schizophrenia (80 versus 75%). One difference of the two groups was that in our new, independent sample 4 of the 12 patients were drug-naı ¨ve and 8 were on a short period of neuroleptic medication at the time of blood withdrawal.

The third group we have investigated included ASD patients. In the ASD group, the requested cut-offs of the state of the art standardized autism manuals were clearly exceeded (all data see Table 2).

In our pool of controls we found a correlation between age and 2^DDCT values with a higher 75-kDa/beta-actin ratio in the younger persons (n = 24; r = -0.491, P = 0.015). Therefore our respective control groups were carefully age-matched. Comparing the twelve EOS adolescents of our new, independent schizophrenia sample with age-and gender-matched controls, revealed a significantly increased 75-kDa subunit mRNA expression (ratio to beta-actin; 2^DDCT values: schizophrenia 1.6 ± 0.7 versus controls 0.9 ± 0.8; P = 0.027; for the 2^DDCT values and means of pre-treated and drug-naı ¨ve patients see Fig. 1) similarly to the newly investigated positive control group, where we could reproduce the increased 75-kDa subunit mRNA expression in a different laboratory, by a different investigator (ratio to beta-actin; 2^DDCT values: schizophrenia 2.3 ± 2.1 versus controls 0.8 ± 0.4; P = 0.053). The 2^DDCT values and mean of the two combined EOS samples and two control samples are shown in Fig. 2 (ratio to beta-actin; 2^DDCT values: schizophrenia 1.9 ± 1.2 versus controls 0.9 ± 0.8; P = 0.003). Comparing drug-naı ¨ve with pre-treated EOS patients in the second independent EOS sample [drug-naı ¨ve (n = 4) versus pre-treated EOS (n = 8); P = 0.816; for a graphical presentation of the respective 2^DDCT values see Fig. 1] as well as in the combined EOS sample [drug-naı ¨ve (n = 14) versus pre-treated EOS (n = 8); P = 0.583] we could not detect any 75-kDa gene expression differences.

Comparing the ASD group and age-and gender-matched controls, we could not detect any difference in mRNA levels of the complex I 75-kDa subunit (ratio to beta-actin; 2^DDCT values: ASD 1.3 ± 0.9 versus controls 1.3 ± 1.2; P = 0.880; see Fig. 3), neither when comparing the ASD patients on stimulant medication with the untreated ones (P = 0.460; for a graphical presentation of the respective 2^DDCT values see Fig. 3).

We found a sensitivity of the potential biomarker of 0.82 and a specificity of 0.63.

Discussion

The aim of our study was to further evaluate the utility of mitochondrial complex I 75-kDa subunit mRNA levels as a Fig. 1 Mitochondrial complex I 75-kDa subunit gene expression in the second, independent early onset schizophrenia patients sample (pre-treated and drug-naı ¨ve) and the matched control subjects Fig. 2 Mitochondrial complex I 75-kDa subunit gene expression in the two combined EOS patients samples (pre-treated and drug-naı ¨ve) and the combined control groups Fig. 3 Mitochondrial complex I 75-kDa subunit gene expression in the ASD patients sample (patients with and without stimulants) and the matched control subjects potential biomarker for schizophrenia. In order to assess whether the increased 75-kDa expression generally reflects an abnormal neuronal development or whether it is more closely associated with schizophrenia we have determined the expression of this gene in a group of ASD children, and in parallel in a group of acute first-episode EOS patients. In both groups mRNA levels were assessed in whole blood cells by real-time PCR in comparison to a respective ageand gender-matched healthy control group. As a positive control we have included the previously examined sample group [START_REF] Mehler-Wex | Increased mRNA levels of the mitochondrial complex I 75-kDa subunit. A potential peripheral marker of early onset schizophrenia?[END_REF]. As all EOS and ASD patients showed a strong symptomatology-assessed by the state of the art manuals-suggest the comparison of the 75-kDa gene expression in these two neuronal maldevelopmental disorders for evaluating the utility of this gene as a potential marker for schizophrenia.

In our study we observed an increased 75-kDa subunit expression in our two independent samples of EOS. In the ASD group we were not able to detect any altered gene expression. Our potential marker separated our EOS and ASD groups with a sensitivity of 0.82 and specificity of 0.63. For a final evaluation of specificity further studies on patients with other psychiatric disorders are certainly needed. To increase sensitivity and specificity, probably a set of different biomarkers (such as different gene variants, markers on the level of mRNA and protein expression in combination with functional imaging data etc.) should be used. Such specific biomarkers would allow an early diagnosis and treatment monitoring of schizophrenia.

There are several reports, that peripheral gene expression/activity is correlated with gene expression/metabolic function in the central nervous system (CNS). Sullivan et al. [40] performed a study on the comparability of gene expression in blood and brain. It was found that about half of a set of candidate genes relevant to schizophrenia were expressed in both whole blood and prefrontal cortex tissue. Furthermore it was found, that the complex I activity in platelet mitochondria was positively correlated with the cerebral glucose metabolism in basal ganglia and thalamus, two brain regions implicated in schizophrenia [START_REF] Ben-Shachar | Cerebral glucose utilization and platelet mitochondrial complex I activity in schizophrenia: a FDG-PET study[END_REF]. Findings such as these affirm that it is reasonable and useful to search for disease markers in peripheral tissue as surrogate for central processes, as in our study.

Although this study might be limited by the small sample size, our results are in line with former reports of an increased gene expression and activity of mitochondrial complex I in schizophrenia. Mitochondrial complex I locates at the mitochondrial inner membrane and is composed of 45 different subunits. Our candidate gene, the 75-kDa subunit, is the largest iron-sulfur non-catalytic transmembrane protein of the complex I [START_REF] Ben-Shachar | Increased mitochondrial complex I activity in platelets of schizophrenic patients[END_REF][START_REF] Dror | State-dependent alterations in mitochondrial complex I activity in platelets: a potential peripheral marker for schizophrenia[END_REF]. It has NADH dehydrogenase and oxidoreductase activity and transfers electrons from NADH to the respiratory chain. Functional abnormalities in this subunit might impact on the electron transport of NADH to oxygen and finally on the cellular ATP production, leading to various brain dysfunctions, such as seen in schizophrenia pathophysiology. Ben-Shachar et al. described a high sensitivity and specificity for potential mitochondrial complex I markers in adults with schizophrenia [START_REF] Ben-Shachar | Increased mitochondrial complex I activity in platelets of schizophrenic patients[END_REF][START_REF] Dror | State-dependent alterations in mitochondrial complex I activity in platelets: a potential peripheral marker for schizophrenia[END_REF]. They found an increased enzymatic activity of the complex I in schizophrenic patients compared to controls and patients with affective disorders [START_REF] Ben-Shachar | Increased mitochondrial complex I activity in platelets of schizophrenic patients[END_REF]. Dror et al. [START_REF] Dror | State-dependent alterations in mitochondrial complex I activity in platelets: a potential peripheral marker for schizophrenia[END_REF] observed concordantly, that increased mitochondrial complex I activity was associated with psychotic symptomology, while its decrease was observed in patients with residual schizophrenia. There are also several reports of brain region-dependent altered gene expression of mitochondrial complex I subunits in postmortem tissue of schizophrenic patients. Association studies additionally pointed to a modulating effect of complex I gene variants in schizophrenic psychosis [START_REF] Washizuka | Association of mitochondrial complex I subunit gene NDUFV2 at 18p11 with schizophrenia in the Japanese population[END_REF].

As a possible confounding factor of our results, gene expression of the 75-kDa subunit might be influenced by medication. Most of our ASD patients were on medication, mainly on stimulant drugs. There is little data published on methylphenidate effects on the respiratory chain. In one report chronic methylphenidate administration increased the CNS expression of mitochondrial complex II and IV genes in a tissue dependent way [START_REF] Fagundes | Chronic administration of methylphenidate activates mitochondrial respiratory chain in brain of young rats[END_REF]. Under chronic amphetamine abuse, an increased oxidative stress was repeatedly found (e.g., [START_REF] Frey | Increased oxidative stress in submitochondrial particles after chronic amphetamine exposure[END_REF]), suggesting altered mitochondrial function. Half of our second, independent EOS sample were under a short duration antipsychotic medication at time of blood withdrawal (see Table 1). Under chronic antipsychotic treatment, Casademont et al. [START_REF] Casademont | Neuroleptic treatment effect on mitochondrial electron transport chain: peripheral blood mononuclear cells analysis in psychotic patients[END_REF] monitored a reduction of mitochondrial complex I activity in peripheral blood mononuclear cells in comparison to the drug-naı ¨ve status. There was also a decrease of complex I activity seen when antipsychotics were added, in vitro, to mitochondria isolated from human and rat brains [START_REF] Ben-Shachar | Increased mitochondrial complex I activity in platelets of schizophrenic patients[END_REF][START_REF] Burkhardt | Neuroleptic medications inhibit complex I of the electron transport chain[END_REF][START_REF] Maurer | Inhibition of complex I by neuroleptics in normal human brain cortex paralles the extrapyramidal toxicity of neuroleptics[END_REF]. However, the observation of tissue-specific effects of neuroleptics on complex I activity in vivo (activity reduction in rat brain, but not in muscle) might explain the varying results [START_REF] Barrientos | Biochemical and molecular effects of chronic haloperidol administration on brain and muscle mitochondria of rats[END_REF][START_REF] Prince | Neuroleptic-induced mitochondrial enzyme alterations in the rat brain[END_REF]. Several reports disproved a possible confounding influence of antipsychotics on complex I expression. Whatley et al. [START_REF] Whatley | Mitochondrial involvement in schizophrenia and other functional psychoses[END_REF][START_REF] Whatley | Superoxide, neuroleptics and the ubiquinone and cytochrome b5 reductases in brain and lymphocytes from normals and schizophrenic patients[END_REF] reported that neuroleptics did not modify the complex I NADH-coenzyme Q reductase nor rotenone-resistant NADH cytochrome C reductase activity in postmortem frontal cortex of schizophrenic patients. Ben-Shachar et al. [START_REF] Ben-Shachar | Increased mitochondrial complex I activity in platelets of schizophrenic patients[END_REF] found a similar complex I activity in unmedicated and medicated patients. Also our previous results confirmed these observations, as the increased 75-kDa expression was seen in the neuroleptic-naı ¨ve patients as well as after a three month antipsychotic treatment [START_REF] Mehler-Wex | Increased mRNA levels of the mitochondrial complex I 75-kDa subunit. A potential peripheral marker of early onset schizophrenia?[END_REF]. In our current study we observed the increased 75-kDa subunit gene expression in our new, independent EOS sample in the drug-naı ¨ve state as well as under a short antipsychotic treatment. Observing elevated 75-kDa subunit mRNA levels in the medicated and unmedicated acutely and chronically psychotic state, but not in residual schizophrenia [START_REF] Ben-Shachar | Increased mitochondrial complex I activity in platelets of schizophrenic patients[END_REF][START_REF] Dror | State-dependent alterations in mitochondrial complex I activity in platelets: a potential peripheral marker for schizophrenia[END_REF][START_REF] Mehler-Wex | Increased mRNA levels of the mitochondrial complex I 75-kDa subunit. A potential peripheral marker of early onset schizophrenia?[END_REF] suggests the mitochondrial 75-kDa-subunit as a potential state marker of schizophrenia.

Conclusion

In conclusion, our results strengthen the role of the mitochondrial complex I as a potential peripheral biomarker for schizophrenia. The observed increase in 75-kDa subunit mRNA expression is in line with other reports on altered complex I activity in schizophrenic psychosis and further findings of increased oxidative stress in schizophrenia pathophysiology [START_REF] Prabakaran | Mitochondrial dysfunction in schizophrenia: evidence for compromised brain metabolism and oxidative stress[END_REF][START_REF] Yao | Oxidative damage and schizophrenia. An overview of the evidence and its therapeutic implications[END_REF]. Although further studies are needed to confirm the specificity it might become a convenient supporting peripheral marker for an early clinical diagnosis and treatment of this disease.

FSK 19 . 9 ± 6 . 1 ADOS 2 ± 2 . 1 CBCL

 19961221 sum of communication and social interaction (autism cut-off = 10, autistic spectrum disorder = 7) 11.5 ± 4.4 ADI-R social interaction (cut-off 10) 19.9 ± 8.4 ADI-R communication and language (cut-off for speaking children = 8) 15.0 ± 4.9 ADI-R repetitive behaviours and stereotyped pattern (cut-off = 3) 5.8 ± 3.1 ADI-R abnormal development before the age of 3 years (cut-off = 1) 2.5 ± 1.7 Control subjects (n 5 16, male) Age (years), mean ± SD 14.

  12 patients (8 males, mean age 16.3 years, age range from 13 years, 6 months to 17 years, 11 months; 4 females, mean age 16.6 years, age range from 15 years, 3 months to 17 years, 6 months) with acute first-episode EOS (see Table

Table 1

 1 Demographic statistics of the second, independent EOS sample and the matched control subjects

	EOS patients
	Age/gender
	Male (n)	8
	Age (years), mean ± SD	16.3 ± 1.7
	Female (n)	4
	Age (years), mean ± SD	16.6 ± 1.0
	Schizophrenia ICD 10-diagnoses (n)
	F20.0 paranoid schizophrenia	9
	F20.2 catatonic schizophrenia	1
	F25.0 schizoaffective disorder	2
	Psychiatric co-medication (patients, n)
	Drug-naı ¨ve	4
	On medication	8
	Neuroleptics (olanzapine, quetiapine, clozapine,	6
	chlorprothixen)
	Tranquillizer (lorazepam)	3
	Mood stabilizer (valproic acid)	1
	Others	1
	Initial BPRS (score ± SD)	62.2 ± 16.4
	Control subjects
	Age/gender
	Male (n)	8
	Age (years), mean ± SD	16.0 ± 1.0
	Female (n)	4
	Age (years), mean ± SD	16.6 ± 1.2
	CBCL (score ± SD)
	Male CBCL total	18.4 ± 11.5
	Internalizing	5.4 ± 4.3
	Externalizing	7.4 ± 5.3
	Female CBCL total	7.7 ± 4.7
	Internalizing	4.7 ± 4.6
	Externalizing	1.3 ± 1.2
	BPRS Brief Psychiatric Rating Scale, CBCL Child Behavior Check
	List

Table 3

 3 Primer sequences, annealing temperatures and PCR product sizes

	Name	Gene bank code	Primer sequences 5 0 to 3 0	Annealing	Product
		(NCBI, Gene)		temperature	size
	75-kDa-	BC030833	f-ATGTGCCTTGTTGAAATTGAGAAAG	62°C	482
	subunit = NDUFS1		r-GCATAGGGCTTAGAGGTTAGGG
	Beta-actin	BC002409	f-TGAAGTGTGACGTGGACATCCG	62°C	444
			r-GCTGTCACCTTCACCGTTCCAG

NDUFS1 NADH dehydrogenase (ubiquinone) Fe-S protein 1

Table 2

 2 Demographic statistics of the 16 male ASD patients and the matched control subjects

	ADI-R Autism Diagnostic
	Interview-Research, ADOS
	Autism Diagnostic Observation
	Schedule, ASD autistic spectrum
	disorder, CBCL Child Behavior
	Check List, FSK Fragebogen
	zur Sozialen Kommunikation
	[original version: Social
	Communication Questionnaire
	(SCQ)]

Eur Child Adolesc Psychiatry (2010) 19:441-448


Adrianne Faber
email: adrianne.faber@chello.nl

Luuk J Kalverdijk

• Lolkje

T W De Jong-Van Den Berg

Jacqueline G Hugtenburg

Ruud B Minderaa

Hilde Tobi

Á R B Minderaa

L T W De Jong-Van Den Berg

Co-morbidity and patterns of care in stimulant-treated children with ADHD in the Netherlands

Keywords: Stimulants, Co-morbidity, Attention deficit hyperactivity disorder, Co-medication, Multimodal treatment Oppositional defiant disorder CD Conduct disorder DSM Diagnostic and statistical manual

This study aimed at investigating the use of psychosocial interventions and psychotropic co-medication among stimulant-treated children with attention-deficit hyperactivity disorder (ADHD) in relation to the presence of psychiatric co-morbidity. Stimulant users younger than 16 years were identified in 115 pharmacies and a questionnaire was sent to their stimulant prescribing physician. Of 773 questionnaires sent out, 556 were returned and were suitable for analysis (72%). The results are based on 510 questionnaires concerning stimulant-treated children for whom a diagnosis of ADHD was reported. Of the 510 children diagnosed with ADHD, 31% had also received one or more other psychiatric diagnoses, mainly pervasive developmental disorder or oppositional defiant disorder/ conduct disorder. We found an association between the presence of co-morbidity and the use of psychosocial interventions for the child (P \ 0.001) and the parents (P \ 0.001). In the ADHD-only group, 26% did not receive any form of additional interventions, while psychosocial interventions varied from 8 to 18% in children with ADHD and psychiatric co-morbidity. The presence of diagnostic co-morbidity was also associated with the use of psychotropic co-medication (overall, P = 0.012) and antipsychotics (P \ 0.001). Stimulant-treated youths with ADHD and psychiatric co-morbidity received more psychosocial interventions and psychotropic co-medication than children with ADHD-only. The type of psychosocial interventions and psychotropic co-medication received by the children and their parents, depended on the specific co-morbid psychiatric disorder being present.

Introduction

Stimulants are first-line drugs in the treatment of attentiondeficit hyperactivity disorder (ADHD) [START_REF]Practice parameters for the assessment and treatment of children, adolescents, and adults with attention-deficit/hyperactivity disorder[END_REF][START_REF] Academy | Clinical practice guideline: diagnosis an evaluation of the child with attentiondeficit hyperactivity disorder[END_REF]. The rapid increase of stimulant use among children in the 90s [START_REF] Miller | Prescription of methylphenidate to children and youth, 1990-1996[END_REF][START_REF] Olfson | National trends in the use of psychotropic medications by children[END_REF][START_REF] Reid | Use of psychostimulant medication for ADHD in South Australia[END_REF][START_REF] Sawyer | Use of medication by young people with attention-deficit/hyperactivity disorder[END_REF][START_REF] Schirm | Psychotropic medication in children: a study from the Netherlands[END_REF][START_REF] Zito | Trends in the prescribing of psychotropic medications to preschoolers[END_REF] has raised global concern about the use of stimulants [START_REF] Accardo | What's all the fuss about Ritalin?[END_REF][START_REF] Hoagwood | Treatment services for children with ADHD: a national perspective[END_REF][START_REF] Miller | Prescription of methylphenidate to children and youth, 1990-1996[END_REF][START_REF] Safer | Are stimulants overprescribed for youths with ADHD?[END_REF][START_REF] Sawyer | Use of medication by young people with attention-deficit/hyperactivity disorder[END_REF][START_REF] Zito | Trends in the prescribing of psychotropic medications to preschoolers[END_REF]. One of the concerns is the application of stimulant drugs without other forms of treatment, especially since there has been increasing awareness that many children with ADHD also have other co-morbid psychiatric disorders that warrant special treatment consideration [START_REF] Gillberg | Co-existing disorders in ADHD-implications for diagnosis and intervention[END_REF][START_REF] Kutcher | International consensus statement on attention-deficit/hyperactivity disorder (ADHD) and disruptive disorders (DBDs): clinical implications and treatment practice suggestions[END_REF][START_REF] Taylor | European clinical guidelines for hyperkinetic disorder-first upgrade[END_REF]. Suggested treatment options depend on the primary diagnosis and the presence of co-morbidity. For instance, for children with ADHD and co-morbid oppositional defiant or conduct disorder (ODD/ CD) symptoms, experts recommend a combination treatment of psychostimulant medication and psychosocial intervention such as parent management training and cognitive behavioural therapy [START_REF] Kutcher | International consensus statement on attention-deficit/hyperactivity disorder (ADHD) and disruptive disorders (DBDs): clinical implications and treatment practice suggestions[END_REF]. On the other hand, children with impaired motor skills may benefit from psychostimulant medication combined with treatment by an occupational or physiotherapist [START_REF] Gillberg | Co-existing disorders in ADHD-implications for diagnosis and intervention[END_REF]. Until now, treatment of ADHD with co-existing psychiatric disorders is guided more by physicians' experience than clinical trials, and more trials should be undertaken [START_REF] Taylor | European clinical guidelines for hyperkinetic disorder-first upgrade[END_REF].

The few studies examining treatment patterns among children with ADHD and psychiatric co-morbidity originated from the USA. Two of these studies focused primarily on psychotropic pharmacotherapy patterns. Among a cohort children aged 5-12 years, Boles et al. found that children with ADHD and psychiatric co-morbidity were more likely to use a combination of psychostimulant and other psychotropic medications than children with uncomplicated ADHD (28 versus 12%) [START_REF] Boles | Variations in pharmacotherapy for attention deficit hyperactivity disorder in managed care[END_REF]. A comparable result was found by Radigan et al. among a group of 3-19 year old children [START_REF] Radigan | Medication patterns for attention-deficit/hyperactivity disorder and co-morbid psychiatric conditions in a low-income population[END_REF]. Children with ADHD and two or more co-morbid psychiatric disorders more often used a combination of stimulant and non-stimulant psychotropic medication compared to children with ADHD-only or ADHD and one disorder (37, 28, 19%, respectively). Another study of Robison et al. examined the prevalence of single and combination treatment modalities for children aged 5-18 years with ADHD in relation to co-morbidity [START_REF] Robison | Treatment modalities among US children diagnosed with attention-deficit hyperactivity disorder:1995-99[END_REF]. They reported that children with ADHD without co-morbidity were more likely to receive stimulant medication alone (51%) and less likely to receive additional psychotherapy or mental health counselling (26%) compared to all children with ADHD, with or without co-morbidity (42 and 32%, respectively). Psychotropic co-medication was not taken into account in this study.

We conducted a survey among physicians of a nationwide sample of stimulant-treated children in the Netherlands, to investigate the use of psychosocial interventions and psychotropic co-medication among stimulant-treated children with ADHD in relation to the presence of psychiatric co-morbidity.

Methods

This research was a follow-up of a questionnaire survey among parents of stimulant-treated children in 2003. To obtain a nationwide community sample of stimulant-treated children irrespective of the prescribing physician, pharmacies were used for detecting records of stimulanttreated children in their pharmacy information system. Since all prescriptions from medical doctors are entered in the pharmacy information system, regardless of particular health insurance or reimbursement status of the medication, the pharmacy is a reliable source of detecting medication users [START_REF] Leufkens | Automated pharmacy record linkage in the Netherlands[END_REF].

In the first 3 weeks of May 2003, 115 pharmacies located all over the Netherlands identified current users of methylphenidate and dexamphetamine in their information system and sent a questionnaire to their carer. Current users were defined as children younger than 16 years for whom at least two stimulant prescriptions were dispensed between 1 November 2002 and 1 May 2003, of which at least one prescription was in 2003.

The findings of the parents' questionnaire and the response analysis were subject of another study and were published elsewhere [START_REF] Faber | Recruitment of parents and physicians of stimulant-using children via community pharmacies is successful[END_REF][START_REF] Faber | Parents report on stimulant-treated youths in the Netherlands: initiation and follow-up care[END_REF]. For the present study, it was important that the parents' questionnaire included a request for an informed consent to approach the stimulant prescribing physician. Informed consent was needed from the parents, and also from children if aged 12 years and older. After informed consent was received, a questionnaire and a copy of the informed consent were sent to the physician currently prescribing stimulants for the child. The protocol was approved by the Medical Ethics Board of the University Medical Centre in Groningen.

Questionnaire

The questionnaire was tested among five physicians.

Analysis

Questionnaires were regarded unsuitable for analysis if the questionnaires were returned blank or when the physician reported not being the prescriber. Children were classified based on their diagnosis as reported by their physician. Only children diagnosed with ADHD were included in the present study. When no other diagnosis was reported, the children were classified as ''ADHD-only'', when pervasive developmental disorder (autistic disorder or PDD-NOS) was reported as ''ADHD ? PDD'', when one of these disruptive behaviour disorders was reported as ''ADHD ? ODD/CD'', and when one or more psychiatric diagnoses other than PDD and ODD/CD were reported they were classified as ''ADHD ? other co-morbidity.'' Children with ADHD and both PDD and ODD/CD were excluded from the analysis.

We compared child characteristics and the use of psychosocial interventions and psychotropic co-medication in the four diagnostic groups. Omnibus tests were used to test whether the proportions in the four diagnostic groups differed significantly across each group. Significant Chisquare tests were followed by post hoc comparisons: (1) ADHD-only versus ADHD ? PDD, (2) ADHD-only versus ADHD ? ODD/CD, (3) ADHD-only versus ADHD ? other co-morbidity, (4) ADHD ? PDD versus ADHD ? ODD/CD, (5) ADHD ? PDD versus ADHD ? other co-morbidity, (6) ADHD ? ODD/CD versus ADHD ? other co-morbidity. In general, a P-value \0.05 was considered statistically significant. To adjust for multiple comparisons, post hoc tests were considered significant at P \ 0.01. All analyses were performed using SPSS 11.0.

Results

The 115 pharmacies sent 1,307 questionnaires to parents of stimulant users. Of these, 924 questionnaires (71%) were returned. In 811 of the 924 cases (88%) permission was given to approach the physician, of which 773 contained a valid address. Of the 773 questionnaires sent to physicians, 556 questionnaires were returned (72%). According to the physicians, 512 of the 556 children (92%) were diagnosed with ADHD. Two subjects were diagnosed with ADHD and both PDD and ODD/CD, leaving 510 cases for the present study. Almost half of these questionnaires were filled out by general practitioners (GPs, 47%). Child psychiatrists and paediatricians were responsible for, respectively, 27 and 23%. The mean age of the stimulant-treated children with ADHD in this study was 10.5 years, and the male-to-female ratio was approximately 5.7:1.

Co-morbidity

Of the 510 stimulant-treated children with ADHD, the physician reported only the diagnosis of ADHD in 69%. For the remaining children, the most frequently reported co-morbidities were PDD (53 children, 10.4%), ODD/CD (50 children, 9.8%), followed by learning disorder (28 children, 5.5%), mental retardation (22 children, 4.3%), tic disorder (10 children, 2.0%) and anxiety disorder (5 children, 1.0%). Of the 53 children in the PDD group, 49 were diagnosed PDD-NOS and 4 with autistic disorder. Of the 50 children in the ADHD ? ODD/CD group, 48 children were diagnosed with ODD and 5 with CD; 3 children were reported to have both ODD and CD. For 57 children (11%) co-morbidity other than PDD or ODD/CD was reported.

Type of prescriber

The type of physician prescribing stimulants was associated with the presence of co-morbidity (P \ 0.001). Of the children with ADHD and a co-morbid disorder, 35% received their current stimulant prescriptions from a child psychiatrist, which was almost twice the percentage compared to children with ADHD-only. Stimulant prescriptions for children with ADHD-only were mostly provided by GPs. In 41% of the children with ADHD-only, stimulant prescribing was transferred from a child psychiatrist or paediatrician to a GP after initiation of treatment. For children with co-morbid PDD, ODD/CD or another psychiatric disorder this percentage was lower: 30, 18 and 23%, respectively (P \ 0.01, data not shown). No relationship was found between the presence of co-morbidity and age, gender and time since the first dispensed stimulant prescription was filled (P C 0.42).

Additional interventions

Overall, there is an association between the presence or absence of co-morbidity and attending a specialized school (Table 1). Sixty percent of the children with ADHD and co-morbid PDD were reported to attend a specialized school. This was twice as often compared to children with ADHD-only.

The presence or absence of co-morbidity was also associated with the use of psychosocial interventions aimed at the child (Table 2). Stimulant-treated children with ADHD and co-morbid PDD, ODD/CD or other psychiatric co-morbidity were reported to receive more psychosocial interventions aimed at the child than children with ADHD-only.

Children with ADHD and co-morbid PDD or co-morbid ODD/CD received intensive training, day treatment and treatment in an inpatient clinic more frequently than children with ADHD-only.

The presence of psychiatric co-morbidity was also associated with parent-focused interventions (Table 2). Parent-focused interventions were most frequently offered to parents of children with ADHD ? PDD (89%) or ODD/ CD (84%). Home training was provided when a PDD diagnosis was present in almost a quarter of these families, almost three times more often compared to families of a child with ADHD-only. Of the children in the ADHD-only group, 26% did not receive any psychosocial interventions focused at the child or parents (data not shown). This percentage was only 18 in the ADHD ? other co-morbidity group, 10 in the ADHD ? ODD/CD group and 8 in the ADHD ? PDD group.

Psychotropic co-medication

There is an association between presence or absence of co-morbidity and the use of psychotropic co-medication (Table 2). Use of psychotropic co-medication was most prevalent among stimulant-treated children with ADHD ? PDD (36%), which was significantly more frequent compared to children in the ADHD-only group (18%). Antipsychotic use was the most prevalent co-medication among stimulant-treated children with ADHD ? PDD (17%). This was almost sixfold higher than in the ADHD-only group (3%).

The frequency of melatonin use did not differ significantly between the groups. Clonidine, antiepileptics, antidepressants, and hypnotics/anxiolytics were not frequently used as co-medication.

Discussion

This study among showed that stimulant-treated children with ADHD and co-morbid psychiatric disorders received

Co-morbidity

For 31% of cases psychiatric co-morbidity was reported in this sample of stimulant-treated children with ADHD. Other studies reported higher co-morbidity percentages ranging from 44% in community-derived samples to 87% in clinically-referred children diagnosed with ADHD [START_REF] Mash | Child pathology[END_REF]. In particular, the presence of ODD/CD in our study (10%) was very low compared to, e.g. the 50-60% reported by Gillberg [START_REF] Gillberg | Co-existing disorders in ADHD-implications for diagnosis and intervention[END_REF]. Our low co-morbidity figures may be explained by a number of phenomena. First, low co-morbidity figures may be explained by under-diagnosis of psychiatric disorders because not all children were thoroughly screened by child psychiatrists. Second, may be physicians were not inclined to report diagnoses that do not support the prescription of stimulants since stimulant use was the entry point for this study. In particular, when the current prescriber was a GP it is likely that the first prescriber was a specialist. When the communication between the GP and specialist was less than ideal, the patient's medical record in the GPs documentation could be incomplete. Pervasive developmental disorder as the most frequently mentioned co-morbid disorder, warrants special attention. Within the DSM-IV classification system, ADHD and PDD are exclusionary diagnoses, and therefore it is impossible to give a PDD and an ADHD classification to the same patient. There has been increasing debate about this issue [START_REF] Clark | Autistic symptoms in children with attention deficit-hyperactivity disorder[END_REF][START_REF] Goldstein | The co-morbidity of pervasive developmental disorder and attention deficit hyperactivity disorder: results of a retrospective chart review[END_REF][START_REF] Luteijn | How unspecified are disorders of children with a pervasive developmental disorder not otherwise specified? A study of social problems in children with PDD-NOS and ADHD[END_REF][START_REF]Randomized, controlled, crossover trial of methylphenidate in pervasive developmental disorders with hyperactivity[END_REF][START_REF] Yoshida | The clinical necessity for assessing attention deficit/hyperactivity disorder (AD/HD) symptoms in children with high-functioning pervasive developmental disorder (PDD)[END_REF]. The general point of the critics of the exclusionary criterion is that the overlap in clinical symptoms between ADHD and PDD is evident in practice, and removing the exclusionary criteria may be beneficial for stimulating research and optimizing treatment in these children [START_REF] Luteijn | How unspecified are disorders of children with a pervasive developmental disorder not otherwise specified? A study of social problems in children with PDD-NOS and ADHD[END_REF][START_REF]Randomized, controlled, crossover trial of methylphenidate in pervasive developmental disorders with hyperactivity[END_REF][START_REF] Yoshida | The clinical necessity for assessing attention deficit/hyperactivity disorder (AD/HD) symptoms in children with high-functioning pervasive developmental disorder (PDD)[END_REF]. It seems that many clinicians in the Netherlands have dropped the DSM-IV exclusionary criterion in clinical practice.

Type of prescriber

In the Netherlands, a GP often takes over responsibility for prescribing medication which was initiated by a specialist. We found this was most frequently true for prescribing stimulant medication for children with ADHD-only. However, the majority of the children with psychiatric co-morbidity still received their stimulant prescriptions from a specialist, even in case of children who had been using stimulants for over 2 years. This difference may be explained by the fact that in more complicated cases the specialist remains closely involved with the child's treatment after its initiation.

Additional interventions

In this study, stimulant-treated children with ADHD and co-morbidity and their parents received considerably more treatment modalities than children with ADHD-only.

Similar findings have been reported on the basis of US studies [START_REF] Boles | Variations in pharmacotherapy for attention deficit hyperactivity disorder in managed care[END_REF][START_REF] Radigan | Medication patterns for attention-deficit/hyperactivity disorder and co-morbid psychiatric conditions in a low-income population[END_REF]. Most guidelines recommend the combination of pharmacological and psychosocial interventions [START_REF]Practice parameters for the assessment and treatment of children, adolescents, and adults with attention-deficit/hyperactivity disorder[END_REF][START_REF] Academy | Clinical practice guideline: diagnosis an evaluation of the child with attentiondeficit hyperactivity disorder[END_REF][START_REF] Kutcher | International consensus statement on attention-deficit/hyperactivity disorder (ADHD) and disruptive disorders (DBDs): clinical implications and treatment practice suggestions[END_REF][START_REF] Taylor | European clinical guidelines for hyperkinetic disorder-first upgrade[END_REF]. This is supported by evidence from the MTA study, reporting a somewhat higher efficacy of multimodal interventions in case of ADHD and co-morbid disorders [START_REF] Jensen | ADHD co-morbidity findings from the MTA study: Comparing co-morbid subgroups[END_REF][START_REF] The | A 14-month randomized clinical trial of treatment strategies for attention-deficit/hyperactivity disorder[END_REF]. However, a substantial proportion of the children in our study did not receive additional interventions. It may be that many children with ADHD without co-morbidity and even some children with co-morbidity respond adequately to pharmacological treatment only. However, it is not unlikely that some children receive less than optimal care. This is aggravated by our finding that almost 20% of the stimulant-treated youths were not monitored [START_REF] Faber | Parents report on stimulant-treated youths in the Netherlands: initiation and follow-up care[END_REF], so in those cases the treatment outcome is unknown to the clinician.

Psychotropic co-medication

We found an association between the presence of psychiatric co-morbidity and the use of psychotropic co-medication among stimulant-treated children with ADHD. This is in line with the few earlier reports from the USA [START_REF] Boles | Variations in pharmacotherapy for attention deficit hyperactivity disorder in managed care[END_REF][START_REF] Radigan | Medication patterns for attention-deficit/hyperactivity disorder and co-morbid psychiatric conditions in a low-income population[END_REF]. However, Radigan et al. reported an increased use of alpha agonists, antipsychotics, antidepressants, antiepileptics and anxiolytics among Medicaid children with ADHD and a co-morbid psychiatric disorder [START_REF] Radigan | Medication patterns for attention-deficit/hyperactivity disorder and co-morbid psychiatric conditions in a low-income population[END_REF], while we found only an increased use of antipsychotics among children with ADHD and co-morbid PDD. Part of this difference may be explained by different prescribing practices in the USA and the Netherlands.

The more frequent use of antipsychotics among children with ADHD and PDD may be understood as confirmation that the presence of PDD symptoms in children with ADHD has practical implications for treatment. After all, several studies have reported a positive effect of antipsychotics in reducing aggressive behaviour in children with PDD and disruptive behaviour disorders [START_REF] Buitelaar | A randomized controlled trial of risperidone in the treatment of aggression in hospitalized adolescents with subaverage cognitive abilities[END_REF][START_REF] Delbello | Phenomenology and epidemiology of childhood psychiatric disorders that may necessitate treatment with atypical antipsychotics[END_REF][START_REF]Research Units on Pediatric Psychopharmacology Autism Network (2001) Risperidone in children with autism and serious behavioral problems[END_REF]. In contrast to reports from the USA, we found antipsychotics to be the most prevalent co-medication instead of antidepressants. In our study, co-medication with antidepressants was reported in less than 2%, compared to 4-7% in the USA [START_REF] Martin | Multiple psychotropic pharmacotherapy among child and adolescent enrollees in Connecticut Medicaid managed care[END_REF][START_REF] Olfson | National trends in the use of psychotropic medications by children[END_REF][START_REF] Zito | Psychotropic medication patterns for youths with attention deficit hyperactivity disorder[END_REF]. This lower use of anti-depressants may be partly explained by differences in timing of the study periods, but is probably more likely to be explained by cross cultural differences in medical practice, including the attention paid to internalizing versus externalizing disorders.

Limitations

Children with ADHD who did not receive stimulants were not included in our study and therefore the findings of our study cannot be generalized to all children with ADHD. Another limitation of our study is that no information was available on how children were assessed, so no statements could be made about the appropriateness or the relative weight of the diagnoses.

Our two-step recruitment procedure implied that physicians could only be approached if the responding parents and their child gave their consent, which may have led to selection bias. No association was found between giving consent and the child's age, gender or the type of prescribing physician, but physician response rate differed significantly for the type of prescribing physician, being highest for paediatricians (83%) and lowest for child psychiatrists (62%) [START_REF] Faber | Recruitment of parents and physicians of stimulant-using children via community pharmacies is successful[END_REF].

Physicians were the only information source in this study. This may possibly have resulted in an underestimation of psychosocial interventions, because physicians may not be aware of all interventions in non-medical settings, for example at school. However, in the Netherlands mental health interventions are not delivered through the school system like in the USA. Individual children can receive didactic interventions such as remedial teaching within the school system, but individualized behaviour management programs are not available on regular schools or are always part of an elaborate behaviour management program put in place through health care professionals, with teachers as ''co therapists.'' Using the current prescriber of stimulant medication as the only information source also may have lead to information bias. GPs taking over prescribing responsibility may have less information and knowledge about psychosocial interventions than psychiatrists or paediatricians. This may have resulted in an underestimation of interventions mentioned by GPs.

Conclusion

Multimodal treatment of youth with ADHD is a common medical practice in the Netherlands, particularly for youth with co-morbid psychiatric disorders. Whether this reflects what is known about the effectiveness of multimodal interventions for children with ADHD and co-morbidity and their parents is unknown and might be subject for further research. The use of psychotropic co-medication for youth has led to some concerns, specifically about the safety of combining stimulants with antipsychotics for children diagnosed with ADHD and PDD.

  Common and simple interventions at regular schools that do not require medical consultation, such as altered seating and home-schoolnotes were not qualified as psychosocial interventions in this study. Interventions aimed at parents to teach them specialized child-management techniques were classified as parent-focused interventions. Individual parent training or group-based parent training was distinguished. Home training is an intensive form of family training in the home setting, sometimes with the use of video recording. Other forms of therapy or courses for parents were classified as ''other training/course.''

	interventions. Training which took a maximum of 2 h per
	week was classified as non-intensive, whereas intensive
	treatment programs took more than 2 h per week. Physio-
	therapy implied physical exercises for motor problems and
	all dietary interventions (e.g. elimination diet) were clas-
	sified under ''diet.'' Treatment for children admitted to a
	hospital or residential centre for mental disorders was
	defined as inpatient treatment. Treatment for children
	attending day hospital programs was defined as day treat-
	ment. Special education services were limited to children
	who attend specialized schools.
	The
	resulting questionnaire contained open-ended and multiple
	choice questions about stimulant treatment, psychiatric
	diagnoses, co-medication and several types of non-phar-
	macological psychosocial interventions focused at the child
	and/or the parents. Only psychosocial interventions deliv-
	ered by specifically trained mental health professionals
	(e.g. psychologists, psychiatrists, psychotherapists, social
	workers) were included. Psychosocial interventions
	directly aimed at the child, such as individual or group-
	based cognitive behavioural therapy, social skills and
	self-regulation training, were classified as child-focused

Table 1

 1 Characteristics of stimulant-treated children diagnosed with ADHD-only, ADHD with PDD, ADHD with ODD/CD and ADHD with other psychiatric co-morbidity

	ADHD-only	ADHD ? co-morbidity

Table 2

 2 Characteristics of additional interventions and psychotropic co-medication among stimulant-treated children diagnosed with ADHDonly, ADHD with PDD, ADHD with ODD/CD and ADHD with other psychiatric co-morbidity

	ADHD-only ADHD ? co-morbidity

Eur Child Adolesc Psychiatry (2010) 19:159-166

Acknowledgments This study was funded by the Dutch National Health Insurance Board. Financial disclosures: Luuk Kalverdijk, Hilde Tobi and Adrianne Faber received a limited grant from Janssen-Cilag. Ruud Minderaa received a limited grant from Eli Lilly and is an advisor in the field of ADHD for Janssen-Cilag and Eli Lilly in Europe.

Open Access This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.


Sylvia Elisabeth Reitter

Thomas Waldhoer

Michaela Mayerhofer

Ernst Eigenbauer

Cihan Ay

Paul Alexander Kyrle

Prof. Dr Ingrid Pabinger
email: ingrid.pabinger@meduniwien.ac.at

; Waldhoer

Thomas † Mayerhofer

Ay Cihan

Alexander

Long-term survival of patients with a history of venous thromboembolism

Keywords: Venous thromboembolism, thrombosis, pulmonary embolism, survival, mortality

Introduction

Venous thromboembolism (VTE) is a serious and often life-threatening disease, which presents as deep venous thrombosis and/or pulmonary embolism. Patients with symptomatic VTE have a considerable risk of recurrent VTE, which persists for many years [START_REF] Palareti | Predicting the risk of recurrence of venous thromboembolism[END_REF].

In developed countries the incidence of VTE ranges between 1-2 per 1000 persons per year. [START_REF] Anderson | A population-based perspective of the hospital incidence and case-fatality rates of deep vein thrombosis and pulmonary embolism. The Worcester DVT Study[END_REF][START_REF] Nordstrom | A prospective study of the incidence of deep-vein thrombosis within a defined urban population[END_REF] The incidence of a first-time VTE increases exponentially with age: ranging from a very low rate (0.05 per 1000 persons per year) among children aged 15 years to a rate of 5 per 1000 persons per year among individuals older than 80 years of age. [START_REF] Kyrle | The risk of recurrent venous thromboembolism in men and women[END_REF] Two-thirds of first-time episodes of deep venous thrombosis (DVT) occur in connection with external factors, such as surgery, immobilization or trauma [START_REF] Kyrle | Deep vein thrombosis[END_REF]. In women, pregnancy, puerperium and oral contraceptives (OC) represent further risk factors. In addition, demographic risk factors for first VTE as well as for recurrent events are known, such as advanced age [START_REF] Schulman | Post-thrombotic syndrome, recurrence, and death 10 years after the first episode of venous thromboembolism treated with warfarin for 6 weeks or 6 months[END_REF][START_REF] Eichinger | Symptomatic pulmonary embolism and the risk of recurrent venous thromboembolism[END_REF], obesity [START_REF] Garcia-Fuster | Long-term prospective study of recurrent venous thromboembolism in patients younger than 50 years[END_REF], cancer and male sex [START_REF] Kyrle | The risk of recurrent venous thromboembolism in men and women[END_REF]. Some clinical factors are known to increase the risk of recurrence, e.g. first manifestation as pulmonary embolism (PE) [START_REF] Eichinger | Symptomatic pulmonary embolism and the risk of recurrent venous thromboembolism[END_REF].

Estimates on mortality after VTE vary considerably. Short-term mortality (30 and 90 days after VTE) varies from less than 3 to 5 % for DVT and from 6 to 23 % for PE. Less is known about long-term survival rates. Long-term survival rates reported thus far vary from 61 to 75 %. [START_REF] Anderson | A population-based perspective of the hospital incidence and case-fatality rates of deep vein thrombosis and pulmonary embolism. The Worcester DVT Study[END_REF][START_REF] Kniffin | The epidemiology of diagnosed pulmonary embolism and deep venous thrombosis in the elderly[END_REF]. Factors influencing the survival rate in thrombosis patients are as follows: time since onset of thrombosis [START_REF] Naess | Incidence and mortality of venous thrombosis: a population-based study[END_REF], age [START_REF] Douketis | The risk for fatal pulmonary embolism after discontinuing anticoagulant therapy for venous thromboembolism[END_REF], idiopathic thrombosis [START_REF] Naess | Incidence and mortality of venous thrombosis: a population-based study[END_REF][START_REF] Douketis | The risk for fatal pulmonary embolism after discontinuing anticoagulant therapy for venous thromboembolism[END_REF] and, most importantly, cancer [START_REF] Naess | Incidence and mortality of venous thrombosis: a population-based study[END_REF][START_REF] Flinterman | Recurrent thrombosis and survival after a first venous thrombosis of the upper extremity[END_REF]. Studies comparing survival rates of patients with a history of thrombosis to the survival of the general population are scarce and show considerable variability [START_REF] Naess | Incidence and mortality of venous thrombosis: a population-based study[END_REF][START_REF] Flinterman | Recurrent thrombosis and survival after a first venous thrombosis of the upper extremity[END_REF][START_REF] Heit | Predictors of survival after deep vein thrombosis and pulmonary embolism: a population-based, cohort study[END_REF].

The aim of our study was to investigate the long-term survival rate in patients with a history of DVT and/or PE who were referred to our department for coagulation testing, and to evaluate the relative survival of these patients in comparison to the general Austrian population. We also assessed long-term survival according to the etiology of VTE (spontaneous or following a triggering event) and to the site of thrombosis.

Patients and methods

Patients

All patients with a history of VTE who were investigated at our out-patient department for routine thrombophilia testing between September 1, 1994 and December 31, 2007, were included in the study. All VTE events had occurred at least three months prior to their first visit at our department and not earlier than 1980. VTE had always been confirmed by objective methods, such as phlebography, plethysmography, duplex ultrasonography, perfusion ventilation lung scan or computed tomography. We included neither patients with active cancer at time of referral, nor patients with asymptomatic PE, nor patients with an isolated visceral vein thrombosis (e.g. thrombosis of the Vv. mesentericae or V. portae) in our study. The minimum age of our patients at time of inclusion was 14 years.

Overall, 4958 patients with a history of DVT or PE were seen at our outpatient department within the study period. 1749 patients were not included in the analysis due to the following reasons: 297 were not eligible due to active cancer, 936 due to acute VTE (VTE < 3 months ago), 214 because their first venous thromboembolic event occurred before 1980, 40 due to isolated visceral thrombosis, 262 because their VTE was diagnosed only clinically or PE was asymptomatic.

Regarding treatment definitions, short-term oral anticoagulation (OAC) was defined as treatment with vitamin-K-antagonists after the first DVT or PE for up to 6 months after the event, long-term treatment was defined as treatment beyond 18 months after the initial DVT or PE.

All VTEs were classified as secondary or idiopathic. An event was regarded as secondary, when at least one of the following factors was present: trauma, surgery, marked immobility (e.g. cast, prolonged bed confinement due to an acute illness, long-distance travel), pregnancy (peri-and postpartal) or Caesar-ean section within the last 6 weeks or use of oral contraceptives. An event was classified as idiopathic when none of the above-mentioned factors was recorded in the patient history.

Methods

A retrospective survival analysis of the above-mentioned patients for the period September 1, 1994 to December 31, 2008 was performed. The present study was approved by the ethics committee of the Medical University of Vienna.

Patient data was derived from the detailed and structured medical records made at each patient's visit to our outpatient department. A physician reviewed all medical records in order to confirm the cases. Patients for whom the information on the duration of OAC following their first VTE was incomplete were contacted by telephone or mail to obtain the missing information.

In addition to patient file information, data on the mortality of our patients was obtained from the Austrian Central Death Register, which is recorded, maintained and updated by Statistics Austria [14]. Causes of death were coded according to the International Classification of Diseases (ICD versions 9 and 10, respectively). Data on the overall mortality of the general Austrian population was also obtained from Statistics Austria.

Statistical analysis

The survival of our patients was estimated and summarized using the Kaplan-Meier method [START_REF] Kaplan | Nonparametric-estimation from incomplete observations[END_REF].

After adjustment for age, gender and calendar period, the patient survival rates were compared with those of the general Austrian population using relative survival methods [START_REF] Dickman | Regression models for relative survival[END_REF]. Calculations were made in SAS and SAS macros provided by Dickman et al. [START_REF]Estimating and modelling relative survival using SAS[END_REF].

Regarding the delineation of cumulative survival and cumulative relative survival rates, the overall considered time period covers 14 years. However, we have chosen a period of only 10 years for calculating the given cumulative (relative) survival rates and for the p-values, because only few events occurred thereafter, and the confidence intervals beyond this point were wide, making a clear interpretation difficult.

Results

Patient characteristics

In total, 3209 patients were included in the present study, of whom 40 % were men (n = 1280). The mean age of our patients at time of inclusion, i.e. when visiting our department for coagulation testing, was 46.2 years (range 14 -89 years). Table 1 displays the characteristics of all included patients.

Median time interval between the first DVT or PE and the investigation at our department was 14 months (interquartile range (IQR) 6 -71 months). For 97 % of all patients (n = 3101) information was available regarding duration of anticoagulation following their initial VTE. Of these patients, 1682 (52.4 %) had short-term anticoagulation (< 6 months) with vitamin-K-antagonists or low molecular weight heparin, 881 (27.5 %) had anticoagulation between 6 and 18 months and only 538 patients (16.8 %) had long-term anticoagulation with vitamin-K-antagonists for > 18 months after the first DVT or PE. At the time of study inclusion, 1178 patients (36.7 %) had OAC therapy.

The median follow-up time between the first visit at our clinic for routine coagulation screening and 12/2008 or death was 6.6 years.

Survival in the patient population

During the entire analysis period, i.e. between September 1, 1994 and December 31, 2008, a total of 169 patients died, of whom 47 % (n = 79) were men. Regarding the specific causes of death, see table 2. The mean age at death was 69 years (IQR 59 -81 years).

Regarding death due to VTE, the eight cases of VTE were definite PE in six cases and possible PE (sudden death of undetermined cause) in two cases. The six cases of bleeding were intra-cerebral haemorrhages in three cases, and bleeding of an ulcus duodeni, coagulopathy of unknown origin and rupture of an aortic aneurysm in one case each.

Myocardial infarction, cardiac insufficiency, atherosclerosis, cardiomyopathy and hypertensive heart disease were grouped to "cardiovascular causes / heart failure".

"Pulmonary" causes of death (apart from PE) were chronic obstructive pulmonary disease and emphysema. "Renal" causes were (chronic) renal insufficiency and nephritic syndrome.

Cumulative survival rates are depicted in Figures 1a -1d. The cumulative survival in the total study population was 0.97 after 5 years and 0.87 after 10 years. The cumulative survival rate of men indicated a significant decrease when compared to that of women (p = 0.01), the probability of survival in men and women was 0.96 and 0.97 after 5 years and 0.91 and 0.93 after 10 years. Regarding triggering factors, the lowest survival was found in patients with spontaneous VTE (cumulative survival after 10 years = 0.88, 95% CI 0.85 -0.90), as opposed to 0.94 (95% CI 0.91 -0.96) in patients with VTE following trauma or surgery; in those with VTE following another triggering event it was 0.94 (95% CI 0.88 -0.97), whereas in patients under oral contraceptives or hormone therapy or in patients with VTE during pregnancy or peripartal the cumulative survival was 0.99 (95 % CI 0.96 -0.99).

With respect to the site of VTE, the lowest cumulative survival rates were found in patients with PE (cumulative survival = 0.91 after 10 years, 95 % CI 0.86 -0.95) and with VTE in other localisations with or without DVT of the leg (cumulative survival = 0.89, 95 % CI 0.85 -0.93), while it was 0.93 (95 % CI 0.91 -0.94) in patients with DVT of the leg only and 0.93 (95 % CI 0.86 -0.97) in those with a combination of PE and DVT of the leg or VTE in other localisations.

Duration of anticoagulation (long-term vs. short-term) did not have an influence on the cumulative survival rates (p = 0.96).

Relative cumulative survival

The cumulative relative survival of all our patients in comparison to the general Austrian population was 1.02 (95 % confidence interval (CI) 1.00 -1.03). When stratified by gender, the cumulative relative survival of female patients equalled that of the general population with a cumulative relative survival rate of 1.00 (95 % CI 0.98 -1.01), whereas in male patients the cumulative relative survival rate was 1.05 (95 % CI 1.03 -1.08).

With respect to triggering factors, the cumulative relative survival of patients with spontaneous VTE was 1.02 (95 % CI 0.99 -1.04, figure 2a). In women whose thrombosis was triggered either by oral contraceptives or hormone replacement therapy or pregnancy, a cumulative relative survival rate of 0.97 (95% CI 0.97 -1.01, Figure 2b) was found. In patients with VTE following trauma or surgery, the cumulative relative survival was 1.03 (95% CI 1.00 -1.05, figure 2c). Patients with other triggering events had a relative survival that was not significantly different from the general population's (1.04, 95 % CI 0.99 -1.06, Figure 2d).

In a further analysis the relative survival of patients presenting with different sites of venous thromboembolism was evaluated (see figure 3). Relative survival in patients with PE was 1.03 (95 % CI 0.98 -1.06, Figure 3a), in those with DVT of the leg it was 1.02 (95 % CI 1.00 -1.04), Figure 3b), in patients with combined PE and DVT of the leg or VTE of another localisation it was 1.01 (95 % CI 0.94 -1.05, Figure 3c), and in the group with other localisations it was 1.01 (95 % CI 0.96 -1.05, Figure 3d).

Discussion

We aimed to evaluate long-term survival following an episode of VTE. Our data are based on a cohort of more than 3200 patients, in whom survival after an initial episode of VTE was investigated over a long period of time. Our data show that long-term survival of patients without cancer, who survived the acute phase of VTE, is comparable to survival rates of the general Austrian population.

As expected, the cumulative survival rate of men was lower compared to women, and patients with idiopathic VTE had a less favourable prognosis for survival than those with a triggering event. When pa-tients were compared to the general population, in none of the analysed subgroups (different sites of VTE, idiopathic vs. secondary VTE) a reduced cumulative relative survival rate was found. However, in comparison to the general population it turned out that men even had a tendency towards a longer relative survival, whereas the cumulative relative survival in women equalled that of the general Austrian female population.

Plausible explanations for the tendency towards a better survival in our group of male patients compared to the average Austrian male population might be a change of lifestyle and more emphasis on healthconsciousness. The acute event of VTE might stimulate health-consciousness and the awareness of illness, thereby leading to a change of lifestyle, e.g. cessation of smoking, change in diet, weight reduction, as well as to more frequent medical check-ups. This may have a larger impact in men, because women have in general been found to be more health-conscious, also consulting their physician for medical attention and undergoing routine check-ups more regularly. This fact was documented in the Austrian micro census that revealed that women of all age groups consult general practitioners more often than men. [START_REF]Austrian Micro Census[END_REF] A study in Sweden convincingly showed that changes in lifestyle, such as those mentioned above, influence fibrinolytic parameters in patients with VTE and even lower the recurrence rates. [START_REF] Schulman | Influence of changes in lifestyle on fibrinolytic parameters and recurrence rate in patients with venous thromboembolism[END_REF] Such life-style changes might have an even stronger impact on other frequent diseases, such as arteriosclerotic heart disease or cancer. We presume that a higher attention to symptoms associated with life-threatening diseases and changes in life style could reasonably explain this rather unexpected improved long-term survival in our male patient cohort.

Recently, Naess et al. [START_REF] Naess | Incidence and mortality of venous thrombosis: a population-based study[END_REF] performed a population-based study in Norway, investigating the mortality of patients with VTE. They found that in patients with VTE and without cancer the mortality rate gradually approached that of the general population during the first year of the event. The mortality rate was, as expected, higher in the acute phase following VTE, i.e. during the first months. The reported overall high long-term mortality rate was found to be mainly related to cancer.

Another recent study published by Flinterman et al. [START_REF] Flinterman | Recurrent thrombosis and survival after a first venous thrombosis of the upper extremity[END_REF] reflects our findings as well. When excluding patients with malignancy, the standard mortality ratio (SMR) was 1.1 for all patients; 0.5 for men and 4.7 for women. The median follow-up period was three years. This study was conducted among patients with a venous thrombosis of the upper extremity, so these data are not directly comparable with the results from our study; nonetheless, they point into the same direction. Although the location of VTE differs from our cohort, the comparison with the general population showed a similar survival of the patient cohort and, interestingly, there was also a notable difference between the SMR of men versus women, in the way that men had a much better SMR than women.

A population-based study performed by Heit et al. [START_REF] Heit | Predictors of survival after deep vein thrombosis and pulmonary embolism: a population-based, cohort study[END_REF][START_REF] Heit | The epidemiology of venous thromboembolism in the community[END_REF] in Olmsted County among 2218 patients with DVT or PE compared the mortality of patients with VTE to the mortality among the general population. In this survey, the median follow-up time was 6.6 years. Patients who survived the first year had a survival notably worse than expected; namely, 71.6 % instead of the expected 80.3 % after eight years. The cohort studied by Heit et al. had their first VTE between 1966 and 1990. It has to be mentioned that diagnosis and treatment of VTE have been considerably improved in the last 2-3 decades. In the acute phase a relatively high percentage of deaths (37 %) occurred upon diagnosis or VTE was even diagnosed post mortem. In these respects this study differs from ours, as it was not our aim to study the death rate in the acute phase, neither did we include data from post-mortem examinations of the population. Furthermore, in contrast to our study, patients with cancer were also included in the study in Olmsted County. These facts might well account for the difference between the study by Heit et al. and the present study. Douketis et al. [START_REF] Douketis | The risk for fatal pulmonary embolism after discontinuing anticoagulant therapy for venous thromboembolism[END_REF] evaluated the survival rate in two cohorts of patients (one Italian and one Canadian, in total 2052 patients), data of these cohorts were combined. Similar to our results, patients with spontaneous thrombosis had a worse survival, whereas there was not much difference in survival with regard to the site of the venous thromboembolic event. The overall reported mortality rate of 14 % during an average follow-up period of 54 months after discontinuation of anticoagulant therapy is higher than in our cohort. However, patients included in this study in mean were 14 years older than the patients of our cohort (62 versus 46 years), which most likely accounts for the higher mortality rate. Unfortunately, in this study neither a comparison between male and female patients nor a comparison to the general population was made. Especially the latter would have been of major importance in order to compare the reported death rate to our study.

Another study by Schulman et al. [START_REF] Schulman | Post-thrombotic syndrome, recurrence, and death 10 years after the first episode of venous thromboembolism treated with warfarin for 6 weeks or 6 months[END_REF] showed a death rate of 28.5 % after 10 years of follow-up, which was 43 % higher than expected from population data. Patients included in this study were older than those in our study (mean age above 60 years) and the acute phase following VTE was also included in the assessment.

In a study by Ridker et al. [21], which did not include the acute phase following VTE, but evaluated the survival of patients following a median of 6.5 years of full-dose anticoagulation therapy, a death rate of 2.4 % after a mean follow-up of 2.1 years was observed. This is quite comparable to our mortality rate of around 4 % after five years when considering the longer follow-up period in our cohort. Also, the median age of 53 years for patients included in the study by Ridker et al. is close to the median age in our cohort. Ridker et al. only included patients with an idiopathic VTE, which was not the case in our study. Therefore, we would actually expect an overall lower risk profile in our cohort. No direct comparison with the general population was performed in this study.

White et al. [START_REF] White | Death due to recurrent thromboembolism among younger healthier individuals hospitalized for idiopathic pulmonary embolism[END_REF] retrospectively reviewed data of more than 3400 patients below the age of 55 after an initial episode of idiopathic PE. Recurrence and mortality rates were determined over a mean period of 3.2 years. During the follow-up period, 3.4 % of the patients died. This corresponds to the death rate of 5.3 % found in our patient cohort.

Our study has several limitations. Certainly, the most important limitation is the retrospective design.

However, the fact that we included all consecutive patients who visited our department for routine thrombophilia testing during a given time period, can, on the other hand, be considered a special feature of our study. Furthermore, we are unable to draw any conclusions concerning short-term survival, as our study does not cover the acute phase following VTE. Anyway, this was not the objective of our study. It is probable that some deaths may not have been recorded due to patients' relocation abroad. However, the Austrian population relocates only rarely, and the Austrian Death Register covers the whole nation. Furthermore, if some deaths have been overlooked due to this reason, this would also have affected the female group. Therefore, it can be concluded that the relocation abroad does not account for the difference noticed between men and women. The fact that we did not include patients with malignancies in our study, of course, also accounts for a better survival. However, this effect might rather be expected in the first couple of years of follow-up, whereas differences in mortality in comparison with the general population were observed after 5 years of observation. Cancer was the most frequent cause of death in our patient population, reflecting the causes of death that can be expected in the age group of patients after 50 years of age. We would like to point out that our entire patient cohort consists of outpatients.

Therefore, there might be some selection bias. Chronically ill or bed-ridden patients might not have been included to a representative extent. This, of course, is also reflected by the relatively young age at the time of inclusion (mean 46.2 years).

In conclusion, our results indicate that after the critical initial period, VTE does not seem to impair longterm survival of outpatients without active malignancy. This is most probably due to the current improved diagnostic and treatment modalities of recurrent VTE, which are most effective and safe. When deciding on long-term anticoagulation, the risk of fatal bleeding should be thoroughly balanced against the risk of fatal recurrent VTE, which -even though it might definitively occur, as also seen in our patient population -yet seems to have no major impact on the overall survival of the cohort of patients with a history of VTE in comparison to that of the general population after overcoming the critical initial time period.

Figure 1 :

 1 Figure 1: Cumulative survival

Figure 2 :

 2 Figure 2: Cumulative relative survival and triggering events

Figure 3 :

 3 Figure 3: Cumulative relative survival figures and localisation

Table 1 Patient characteristics Site of VTE

 1

	Patient number/
	Total patient	Percentage % men
	number

Table 2 Causes of death Cause of death Number of patients (%)

 2

	(Total number n=169)

Acknowledgements

We thank Tanja Altreiter, MA (Clinical Division of Haematology and Haemostaseology, Department of Medicine I, Medical University Vienna) for proof-reading of the manuscript.

Study supervisor: Pabinger

Disclosure of Conflicts of Interests

The authors state that they have no conflicts of interest.