Loïc Guillot

Ralph Epaud

Guillaume Thouvenin

Laurence Jonard

Amira Mohsni

Remy Couderc

François Counil

Jacques De Blic

Rola Abou Taam

Muriel Le Bourgeois

Philippe Reix

Florence Flamein

Annick Clement

Delphine Feldmann
email: delphine.feldmann@trs.aphp.fr

Hôpital Armand Trousseau

New surfactant protein C gene mutations associated with diffuse lung disease

Keywords: children, lung, surfactant protein C (SP-C), SFTPC

Mutations in the surfactant protein C gene (SFTPC) have been recently associated with the development of diffuse lung disease, particularly sporadic and familial interstitial lung disease (ILD). We have investigated the prevalence and the spectrum of SFTPC mutations in a large cohort of infants and children with diffuse lung disease and suspected with surfactant dysfunction. One hundred twenty-one children were first screened for the common SFTPC mutation, p.Ile73Thr (I73T). Ten unrelated patients were shown to carry this mutation. The I73T mutation was inherited in 6 cases, and appeared de novo in 4. The 111 patients without the I73T mutation were screened for the entire coding sequence of SFTPC. Of these, eight (seven unrelated) subjects were shown to carry a novel mutant allele of SFTPC. All these seven new mutations are located in the BRICHOS domain except the p.Val39Ala (V39A) mutation, which is in the surfactant protein C (SP-C) mature peptide. Our results confirm that SFTPC mutations are a frequent cause of diffuse lung disease, and that I73T is the most frequent SFTPC mutation associated with diffuse lung disease.

INTRODUCTION

Diffuse lung disease in children, including interstitial lung disease (ILD) and chronic lung disease, is a rare heterogeneous group of chronic disorders characterized by impaired gas exchange [START_REF] Deutsch | Diffuse lung disease in young children: application of a novel classification scheme[END_REF] . Recent studies suggest that surfactant deficiency plays a role in the pathogenesis [START_REF] Hartl | Interstitial lung disease in children --genetic background and associated phenotypes[END_REF] .

Pulmonary surfactant is a complex mixture of proteins and lipids that prevents collapse at end expiration by reducing surface tension at the air-water interface of the lung alveoli.

Surfactant protein C (SP-C), a hydrophobic protein critically involved in surfactant homeostasis [START_REF] Nogee | Alterations in SP-B and SP-C expression in neonatal lung disease[END_REF] , is expressed by alveolar type II epithelial cells and encoded by a single gene located on chromosome 8 (8p21.3) called SFTPC. Human SFTPC gene is organized into 6 exons (I through V coding, VI untranslated). SP-C is synthesized as a 197-amino acid proprotein (proSP-C) that undergoes multiple processing steps to the mature SP-C peptide of 35 amino acids, to be finally released in the alveoli associated with the other surfactant proteins and phospholipids [START_REF] Nogee | Alterations in SP-B and SP-C expression in neonatal lung disease[END_REF] . Nogee et al. first described a SFTPC mutation in 2001 [START_REF] Nogee | A mutation in the surfactant protein C gene associated with familial interstitial lung disease[END_REF] . The mutation, c.435+1G>A, located at the first base of intron 4 alters the normal donor splice site and results in the skipping of exon 4 and the deletion of 37 amino acids in the carboxy-terminal domain of proSP-C. This mutation was observed in one allele of a patient suffering from chronic interstitial pneumonitis and was inherited following a dominant pattern. To date, 13 SFTPC mutations have been identified [START_REF] Nogee | A mutation in the surfactant protein C gene associated with familial interstitial lung disease[END_REF][START_REF] Nogee | Mutations in the surfactant protein C gene associated with interstitial lung disease[END_REF][START_REF] Thomas | Heterozygosity for a surfactant protein C gene mutation associated with usual interstitial pneumonitis and cellular nonspecific interstitial pneumonitis in one kindred[END_REF][START_REF] Brasch | Interstitial lung disease in a baby with a de novo mutation in the SFTPC gene[END_REF][START_REF] Hamvas | Progressive lung disease and surfactant dysfunction with a deletion in surfactant protein C gene[END_REF][START_REF] Tredano | Mutation of SFTPC in infantile pulmonary alveolar proteinosis with or without fibrosing lung disease[END_REF][START_REF] Stevens | Nonspecific interstitial pneumonia, alveolar proteinosis, and abnormal proprotein trafficking resulting from a spontaneous mutation in the surfactant protein C gene[END_REF][START_REF] Soraisham | Neonatal respiratory failure associated with mutation in the surfactant protein C gene[END_REF] . They always occur in one single allele and have been associated with diffuse lung disease in children and adults. The mutation I73T (c.218 T>C) is the more prevalent mutation; others have been described in only one family. Dominant transmission with variable penetrance has been confirmed in familial cases, but a number of sporadic cases have also been reported with a de novo mutation. The phenotype associated with SFTPC mutations appears to be very variable. Indeed, neonatal forms leading to death in the first years of life as well as childhood and adult forms with chronic respiratory disease have been reported. Single nucleotide polymorphisms (SNPs) have also been detected both in the coding and non-coding sequence, with unclear consequences on proSP-C processing and function [START_REF] Hartl | Interstitial lung disease in children --genetic background and associated phenotypes[END_REF] .

The variable phenotype with unspecific biological findings makes the genetic diagnosis of SP-C inherited disease essential. Thus, the present study aims to investigate SFTPC variations in a large population of 121 children (120 unrelated) with diffuse lung disease and suspected surfactant dysfunction.

MATERIALS AND METHODS

Patients

One hundred and twenty-one newborns/children (60 male, 61 female) diagnosed with diffuse lung disease were recruited over a 5-year period (2002-2007) through a national program on rare lung disease by several university hospital-based pediatric pulmonology practices (Hôpital Armand Trousseau, Paris; Hôpital Necker Enfants Malades, Paris; Groupement Hospitalier Est, Lyon and Centre Hospitalier Universitaire CHU Arnaud de Villeneuve, Montpellier). Seventy four patients originated from Europe, 24 from North Africa, 13 from Africa, 9 from Reunion Island and 1 from French Antilles. Among the 121 patients with diffuse lung disease, 86 had respiratory distress, 59 presenting with neonatal onset and 18 patients died. Clinical observations included family background, physical signs at presentation, chest radiography and computed tomography (CT), and histological lung analysis when available. The protocol was accepted by the Committee for the protection of individuals in Biochemical research as required by French legislation and written informed consent was obtained from the patients or their next of kin included in this study.

The 121 patients where the common 121ins2 SFTPB mutation was initially ruled out were analyzed for the common I73T mutation. Then, the entire SFTPC coding sequence was performed in patients without the I73T mutation.

Mutation Analysis

Genomic DNAs were extracted from patients' blood using an automated BioRobot EZ1 workstation (Qiagen, Hilden, Germany). The common mutation I73T was screened using an allele specific amplification as previously published [START_REF] Cameron | A common mutation in the surfactant protein C gene associated with lung disease[END_REF] and confirmed by the direct sequencing of exon 3. The 5 coding exons of SFTPC and the exon-intron boundaries were analyzed by direct DNA sequencing of PCR products using the following primers: exon 1 sense 5'-ACCCAGGTTTGCTCTTGCT-3', exon 1 antisense 5'-TGAATGGATCTGGATAAGGAAA-3', exon 2 sense 5'-TGTTAGAATCCAGGCCACCT-3', exon 2 antisense 5'-CGTGCCTCTTTCCTTCTAGC-3', exon 3 sense 5'-CTCTTGGGAAAGAGGGAAGC-3', exon 3 antisense 5-'GGGAGAGATGGATGTGGATG-3', exon 4 sense 5'-CTAGTATGACTCCCGTGCCC-3', exon 4 antisense 5'-TGAGGAACAGTGCTTTACAGG-3', exon 5 sense 5'-TCAGCTGAGTCCACTCACTACC-3', exon 5 antisense 5'-GTACCGGTCTGTGAGCTTCC-3'. Sequencing reactions were performed with Big Dye Terminator cycle sequencing kits from Applied Biosytems (Foster city, CA) and run on an ABI Prism 3130 Genetic Analyzer. Sequences were analyzed using SeqScape software and compared with the reference sequence NM_003018.3. Mutations are named following the international nomenclature, starting at nucleotide 1 of the first codon (Human Genome Variation Society: Nomenclature for the description of sequences variation, http://www.hgvs.org/mutnomen/). Multiple protein alignments were performed using the software SIFT (http://blocks.fhcrc.org/sift/SIFT.html) and the search for polymorphisms was carried out using the dbSNP database (http://www.ncbi.nlm.nih.gov/SNP/).

RESULTS

We screened a cohort of 121 patients with diffuse lung disease for the mutation c.218T>C (p.Ile73Thr, I73T). SFTPC mutation is present in 18 (17 unrelated) patients and seven novel mutations are described.

The I73T mutation was found in 10 patients without neonatal onset, eight presenting with respiratory viral infection. I73T was inherited in 6 cases. In 4 cases, I73T was sporadic and thus absent in both parents. The 111 patients without I73T mutation were screened for all the coding sequence and the exon-intron boundaries of SFTPC. Seven novel SFTPC variations were identified (Table 1). Interestingly, the age of onset was significantly lower in patients with new mutations compared with those carrying the I73T mutation (Table 2). No other significant differences between the two groups of patients were observed.

DISCUSSION

The identification of SP-C alterations has led to significant advances in the diagnosis of diffuse lung disease. Our study on a large cohort of patients confirms that the common mutation I73T in the SFTPC gene is a frequent cause of diffuse lung disease. In addition, we described seven new pathogenic SFTPC variants associated with respiratory diseases occurring during the first months of life.

The mutation I73T has been first reported in one allele of an infant with severe respiratory insufficiency and a histological pattern of nonspecific interstitial pneumonia (NSIP)

and pulmonary alveolar proteinosis (PAP) [START_REF] Brasch | Interstitial lung disease in a baby with a de novo mutation in the SFTPC gene[END_REF][START_REF] Tredano | Mutation of SFTPC in infantile pulmonary alveolar proteinosis with or without fibrosing lung disease[END_REF] . Furthermore, in a cohort of 116 children with ILD, this mutation was present in 7 cases and appeared to be common [START_REF] Cameron | A common mutation in the surfactant protein C gene associated with lung disease[END_REF] . The mutation was inherited or observed de novo. The phenotype associated with I73T was described as eminently variable, ranging from fatal cases to asymptomatic adult carriers [START_REF] Cameron | A common mutation in the surfactant protein C gene associated with lung disease[END_REF][START_REF] Percopo | Variable phenotype associated with SP-C gene mutations: fatal case with the I73T mutation[END_REF] . In our cohort of 121 patients with diffuse lung disease, we found 10 unrelated patients with diffuse lung disease harboring the I73T mutation. This result confirms the high frequency of this mutation in diffuse lung disease.

Besides this frequent mutation, only 12 SFTPC mutations have been previously described. We report in this study seven new mutations. The splice site mutation c.325-1G>A is thought to affect the acceptor splice site of exon 4, and may result in the skipping of exon 4.

Thus, this mutation probably has the same effect than the previously reported mutation c.435+1G>A affecting the donor site of exon 4 [START_REF] Nogee | A mutation in the surfactant protein C gene associated with familial interstitial lung disease[END_REF] . The deletion c.424delC induces the translation of an abnormal protein. The missense mutations V39A, Q145H, L188P, C189Y, and L194P are believed to be pathogenic because they have not been observed in control chromosomes of the same origin. Moreover, the amino acids involved are conserved in mammalian SP-C sequences, and the mutation segregates with the pulmonary disease in familial cases (C189Y and L194P).

The phenotype associated with C189Y appeared to be variable, severe in the first months of life and asymptomatic in adults. In contrast, L194P is associated with pulmonary fibrosis in adults.

The mutation V39A is the first valine of the polyvaline stretch of mature SP-C. The polyvaline stretch is believed to be a crucial actor of the secondary structure of SP-C. Indeed, studies of purified mature SP-C have shown that the polyvaline stretch is highly α-helical and capable of spanning phospholipids bilayers in the liquid crystalline phase 14 .

To date, most of the mutations previously identified in the SP-C gene are localized in the propeptide. In our study, with the exception of the V39A located in the mature peptide, the other new mutations are in the propeptide. Based on functional studies that ascertain the role of SFTPC mutations in the pathogenesis of diffuse lung disease, two mechanisms have been suggested [START_REF] Hartl | Interstitial lung disease in children --genetic background and associated phenotypes[END_REF][START_REF] Stevens | Nonspecific interstitial pneumonia, alveolar proteinosis, and abnormal proprotein trafficking resulting from a spontaneous mutation in the surfactant protein C gene[END_REF] . First, owing to a heterozygous mutation, abnormal proSP-C could disrupt the regular processing of proSP-C within the cell and consequently inhibit SP-C production in the alveoli. This is the case for the I73T and E66K mutations altering intracellular trafficking [START_REF] Brasch | Interstitial lung disease in a baby with a de novo mutation in the SFTPC gene[END_REF][START_REF] Stevens | Nonspecific interstitial pneumonia, alveolar proteinosis, and abnormal proprotein trafficking resulting from a spontaneous mutation in the surfactant protein C gene[END_REF] .

Also, as shown in the Δexon4 and L188Q mutations, aberrant proSP-C forms aggregates that could be toxic for alveolar type II cells and induce an unresolved inflammatory response [START_REF] Bridges | Expression of a human surfactant protein C mutation associated with interstitial lung disease disrupts lung development in transgenic mice[END_REF][START_REF] Mulugeta | A surfactant protein C precursor protein BRICHOS domain mutation causes endoplasmic reticulum stress, proteasome dysfunction, and caspase 3 activation[END_REF][START_REF] Wang | Deletion of exon 4 from human surfactant protein C results in aggresome formation and generation of a dominant negative[END_REF] .

Moreover, in two patients, diffuse lung disease have also been associated with SP-C deficiency without any detectable SFTPC mutation [START_REF] Tredano | Mutation of SFTPC in infantile pulmonary alveolar proteinosis with or without fibrosing lung disease[END_REF] . Thus, Alteration of SP-C metabolism or complete SP-C deficiency is critical in diffuse lung disease pathophysiology.

Interestingly, we found three novel variants, L188P, C189Y and L194P, located in the C-terminus of proSP-C BRICHOS domain (F94-I197)-a highly conserved domain. The BRICHOS domain is composed of about 100 amino acids in the proprotein. It is found in several proteins associated with degenerative and proliferative diseases [START_REF] Sanchez-Pulido | BRICHOS: a conserved domain in proteins associated with dementia, respiratory distress and cancer[END_REF] . The BRICHOS domain is thought to be involved in protein processing and is assumed to have a chaperone-like function. It plays a critical role in proSP-C targeting and processing [START_REF] Mulugeta | A surfactant protein C precursor protein BRICHOS domain mutation causes endoplasmic reticulum stress, proteasome dysfunction, and caspase 3 activation[END_REF][START_REF] Mulugeta | Misfolded BRICHOS SP-C mutant proteins induce apoptosis via caspase-4-and cytochrome c-related mechanisms[END_REF][START_REF] Nerelius | Mutations linked to interstitial lung disease can abrogate anti-amyloid function of prosurfactant protein C[END_REF] . Indeed, in previous studies, two patients have been found to carry a mutation encoding substitution (L188R and L188Q) of the same conserved leucine residue altered in our cohort (L188P). The L188R mutation has been detected in one infant with ILD 5 . On the other hand, the L188Q mutation is detected in large familial pulmonary fibrosis kindred, either in children or adults with cellular interstitial pneumonitis or usual interstitial pneumonitis [START_REF] Thomas | Heterozygosity for a surfactant protein C gene mutation associated with usual interstitial pneumonitis and cellular nonspecific interstitial pneumonitis in one kindred[END_REF] . We have observed L188P in an 18year-old patient who had diffuse lung disease during the first year of life. Unfortunately, familial samples were not available.

Descriptions of the reported new mutations will be the subject of further functional studies, which will improve knowledge on the pathogenesis of associated surfactant diffuse lung disease in children.

TABLE AND FIGURES LEGENDS

a#

  Numbering relative to coding DNA reference sequence NM_003018.3 and starts at translation initiation site (Death at 3 months Two mutations, C189Y and L194P, were inherited (Figure1), whereas the mutations c.325-1G>A and c.424delC were sporadic and absent in the patient's parents and probably appeared de novo. Samples of familial individuals of index cases with V39A, Q145H and L188P were unavailable. The novel variants segregated with the disease in the familial cases (C189Y and L194P) and have not been previously reported as polymorphisms, suggesting that they are pathogenic. In addition, each new variant was not detected in 100 control alleles of the same origin. The mutation c.325-1G>A located at the last base of intron 3 is presumed to abolish the normal acceptor splice site using the GeneSplicer prediction software, and to result in the skipping of exon 4 and the deletion of 37 amino acids in the carboxy-terminal domain of . The mutation c.424delC in exon 4 is a frameshift mutation that induces a premature stop codon at position 185. The variation c.435G>C resulting in the substitution of glutamine 145 by histidine, involved the last base of exon 4. This substitution is supposed to abolish the donor splicing site of exon 4 following the software of splice site prediction, and to result in the skipping of exon 4. The V39A mutation is located in mature SP-C. The six other novel variations have been detected in the BRICHOS domain (Figure.2).

Figure 1 :

 1 Figure 1: Pedigree of families with the new SFTPC mutations C189Y and L194P.

Figure 2 :

 2 Figure 2: Localization of new SFTPC Mutations. Schematic representation of SFTPC gene.

Table 1 . Newly discovered SFPTC mutations.

 1

Table 2 . Characteristics of patients with I73T and new SFTPC mutations.

 2

	Total	I73T mutation	New mutations

at onset (mean±sd in months) Viral infection at onset Neonatal respiratory distress

		10	6	4
		8	4	4
		4.0±1.2	5.9±1.9	1.7±0.4*
		8	4	4
		4	0	4
	Symptoms
	Cough	18	10	8
	Tachypnea/dyspnea	18	10	8
	Cyanosis	18	10	8
	Failure to thrive	18	10	8
	*p<0.01, Mann-Whitney

Table 1 :

 1 Newly discovered SFPTC mutations.

Table 2 :

 2 Characteristics of patients with I73T and new SFTPC mutations.

ACKNOWLEDGEMENTS

We thank the patients and their families for their cooperation in this study. We also thank Catherine Meunier, France Michel, Corinne Chauve, Isabelle Sargis, Magali Niasme, and Cristina Das Neves for their expert technical assistance. This work was supported by the « Département de la Recherche Clinique et du Développement » (PHRC 2007, « "Surfactant disorders associated with chronic lung disease in children").

"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 Journal of Medical Genetics and any other BMJPGL products and sublicences such use and exploit all subsidiary rights, as set out in our licence (http://JMG.bmj.com/misc/ifora/licenceform.shtml)."


Thomas W Teasdale

Hazel Emslie

Kirsten Quirk

Jonathan Evans

Jessica Fish

Barbara Wilson
email: barbara.wilson@mrc-cbu.cam.ac.uk

Professor Barbara A Wilson Obe

Alleviation of carer strain during the use of the Neuropage device by people with acquired brain injury

Background: Previous studies have demonstrated the efficacy of a paging system, Neuropage®, in compensating for memory and planning dysfunctions among people with acquired brain injury (ABI, mainly stroke and traumatic brain injury). We here investigated the degree to which this efficacy is accompanied by a reduced experience of strain among their carers.

Method: In a cross-over design, carers of 99 people with ABI completed a questionnaire concerning strain resulting from the injury at three time-points, before the use of Neuropage®, at the end of a 7 week-period of use, and, for one subgroup, a further seven weeks after withdrawal of Neuropage®.

Results: There were significant reductions in strain reported by carers following the period of Neuropage® use (Cohen's d = 0.3 -0.4). This was true whether the carer was a spouse or a parent. The reduced strain among carers continued even after withdrawal of Neuropage®.

Conclusion:

The efficacy of the paging system for people with ABI appears to result in a reduced strain for their carers.

Introduction

The occurrence of disability, mental or physical, in a family member who is not institutionalized and lives at home often places a strain on other members of the family who have thereby become carers [START_REF] Baronet | Factors associated with caregiver burden in mental illness: a critical review of the research literature[END_REF][START_REF] Ferrario | Alzheimer's disease: usefulness of the Family Strain Questionnaire and the Screen for Caregiver Burden in the study of caregiving-related problems[END_REF][START_REF] Chipchase | Factors associated with carer strain in carers of people with multiple sclerosis[END_REF] . Among people who have suffered an acquired brain injury (ABI, e.g. stroke or traumatic brain injury), it is often their difficulties with memory and planning which are the greatest sources of strain to their carers [START_REF] Thomsen | The patient with severe head injury and his family. A follow-up study of 50 patients[END_REF][START_REF] Livingston | Three months after severe head injury: psychiatric and social impact on relatives[END_REF][START_REF] Oddy | Stresses Upon Relatives of Head-Injured Patients[END_REF][START_REF] Oddy | Intervention with families following brain injury: Evidence-based practice[END_REF] , who need to monitor, and perhaps assist in, the performance of everyday activities.

Electronic technology can considerably improve the performance of people with ABI in their daily tasks, thus reducing their dependence on carers [START_REF] Lopresti | Assistive technology for cognitive rehabilitation: State of the art[END_REF] . Particular success has been reported with the Neuropage®, involving an electronic pager worn by the person with ABI and which receives pre-programmed timely reminders to perform tasks -so-called 'target behaviours'which have been specified in advance by that person him-or herself 9;10 . Typical target behaviours include taking medicine, feeding pets, remembering appointments and remembering to take keys when leaving home.

Using the same subjects who entered the initial studies on the efficacy of Neuropage® 9- 11 , the purpose of the present study was to examine whether its use also results in a reduction in the strain experienced by carers.

Method

The design of the study was a cross-over involving subjects alternately allocated in blocks of 10 to one of two groups, with measures taken at three time-points over a 16-week period. At time-point 1, both groups underwent a two-week baseline recording of self-identified target behaviours. During this time a carer completed the postal strain questionnaire (the modified

Instruments

The questionnaire used to assess carer strain was a modified version of the Caregiver Strain Index developed by Robinson [START_REF] Robinson | Validation of a Caregiver Strain Index[END_REF] and concerning "things which other people have found difficult in helping with, after someone comes home from the hospital", e.g. "Helping takes up a lot of time". Our 16-item modified version (mCSI) was tailored towards potential stressors of particular relevance in cases of ABI. Responses were to be made on an 11-point scale in which the extreme ratings were labelled as 0 = 'Never/Not at all' and 10 = 'Always/Very much', a high rating indicated a high degree of strain. A total score was computed averaging the 0-10 response across the 16 items. The internal reliability of the total score was high (Cronbach's Alpha = 0.91).

The study complied with the Declaration of Helsinki and was approved by 11 regional ethics committees around the United Kingdom.

Results

Means and standard errors for the two groups at the three time-points are shown in Figure 1.

- ------------------------------Insert Figure 1 about here -------------------------------The first issue examined was the test-retest reliability of the mCSI itself. For the sevenweek period between time-point 1 and time-point 2 there were no systematic changes in circumstances for the 37 people in Group B. The intra-class correlation between mCSI ratings for the two time-points was 0.86 (p < 0.01), indicating a high level of test-retest reliability. The mean mCSI score did not change significantly for Group B between time-point 1 and time-point 2, (t(36) = 1.46, p >0.05).

Group A reported significantly higher levels of strain at initial baseline than Group B, (t(97) = 2.11, p = 0.04).

The overall effect of the introduction of Neuropage was assessed by combining data from the two groups. For group A, data from time-point 1 was compared to time-point 2 data and for group B, time-point 2 data was compared to time-point 3 data. Thus, in both cases a comparison was made between the immediate pre-Neuropage period with that at the end of the seven weeks of Neuropage usage. A mixed-model analysis of variance was performed with group as a between-subjects factor and time-point as a within-subjects factor. There was no overall difference between groups A and B (F(1,97) = 3.1, p > 0.05) and there was no significant interaction between groups and time-point (F(1,97) = 0.4, p > 0.05). However, the main effect of time-point was highly significant (F(1,97) = 24.1, p < 0.001). The effect sizes for groups A and B can be estimated, using Cohen's d 13 , as 0.33 and 0.39 respectively, which can be considered as small to medium. For both groups, over two-thirds of carers reported lower levels of strain following use of Neuropage® than the median reported strain prior to its use.

In separate matched-pairs t-tests, the reduction in strain was found to be significant for both carers who were spouses (t(58) = 3.3, p = 0.002) and for carers who were parents (t(28) = 2.8, p = 0.01) and the two groups did not differ the degree of reduction (t(86) = 1.1, p>0.05).

A final issue addressed was the extent to which the now demonstrated beneficial effect of Neuropage® was maintained after the device was withdrawn. This was examined by comparing the mCSI ratings for Group A at time-point 2, i.e. during Neuropage® usage, with time-point 3,

i.e. seven weeks after it had been withdrawn. As might be expected from the means shown in Figure 1, there is no significant reversal of mCSI ratings following withdrawal of Neuropage® (t(53) = 0.37, n.s.).

Discussion

Before considering these findings in detail, some limitations of the study need to be recognized. First, the information gathered here is indirect, being obtained from relatives completing postal questionnaires. Both the internal and test-retest reliabilities are, however, high, suggesting that the questionnaires were carefully and seriously completed by the relatives, whose motivation to be part of the study was generally very high. A second potential limitation arises in that the relatives were not blind to the study design, being necessarily aware of when Neuropage® was in use. However, is seems unlikely that this knowledge would have led to a bias towards favourable responding in terms of their own strain, if Neuropage® use had not in reality resulted in such a reduction. The mCSI could also be criticized for only including negative items, possibly leading to response bias. A further limitation of the study is the lack of randomisation. The baseline difference between the two groups could have been due to the (albeit non-significant) differences in the two groups' composition, e.g. that Group A contained proportionately more males and more persons with TBI. However, both this difference, and the discrepancy between two group sizes, are not critical since the study design does not hinge upon group comparisons.

Given the already reported extensive evidence for the efficacy of Neuropage® in assisting people with ABI to cope with, in particular, difficulties of memory and planning, 10;11 the present confirmation of a reduced strain for the carers, both parents and spouses, is encouraging. The lack of any systematic change in reported strain in the pre-Neuropage® period for group B -10 -further supports the inference that the reduced strain experienced by carers after its use is indeed a direct consequence of that use, rather than being some other function of the passing of time.

The fact that the people with ABI in this study were on average about five years post-injury also makes it unlikely that the observed improvements over the seven-week period were unrelated to the use of Neuropage®. Ancillary analyses not presented here showed that the strain relief is reported by carers to people with TBI and with CVA and was greater for carers to persons with ABI who showed significant improvement in attaining targets with Neuropage, themselves an overwhelming majority, than for the remaining small minority who did not [START_REF] Fish | Compensatory strategies for acquired disorders of memory and planning: Differential effects of a paging system for patients with brain injury of traumatic versus cerebrovascular aetiology[END_REF] .

In summary, the present results suggest that not only does the use of Neuropage® improve the performance of everyday tasks among people with acquired brain injury, but that this improvement itself can result in a significant and enduring alleviation of strain among their carers.

Figure 1

 1 Figure 1Mean (SD) mCSI in relation to group and time-point

Table 1

 1

	Demographic and medical characteristics
	Group A (Neuropage® 1 st ) Group B (Neuropage® 2nd)
	N	62	37
		_________________________________
		%	%
	Gender
	Male	79	70
	Female	21	30
	Carer
	Spouse	61	57
	Parent	24	38
	Other	15	5
	Type of Injury
	Traumatic Brain Injury	48	39
	Cerebro-Vascular Accident 25	25
	Other (e.g. anoxias, tumours) 27	36
		_________________________________
		Mean (SD)	Mean (SD)
	Age at injury	33 (15)	33 (19)
	Age at program entry	38 (13)	37 (16)

Caregiver Strain Index, mCSI) described below. Group A then used the Neuropage® devices for seven weeks and the target behaviours were measured for the last two weeks, i.e., time-point 2, during which time the mCSI was completed for a second time. Group B were held on a waiting list for the same seven weeks, during the last two weeks of which, i.e. also time-point 2, target behaviour assessment and mCSI data were again collected. At the end of the seven-week period, for Group A the Neuropage® devices were withdrawn, and Group B members correspondingly received them after the seven week waiting period. A further seven weeks followed, again with assessment of the target behaviours and completion of the mCSI during the final two weeks, i.e. time-point 3. Further details of the study design have been reported elsewhere 9;10 .

Participants

Participants in this report were primary carers of the people with ABI who had been recruited to the Neuropage® study, one carer for each person. The original study involved 143 such people, but adequate carer data across the three time-points were only available for 99 (69%), the attrition being primarily owing to people not having carers. Of the 99, 62 had been allocated to Group A and 37 to Group B, the discrepancy arising from the block allocation of subjects and the practical constraints of completing the study. Demographic and medical characteristics of the two groups are shown in Table 1 and there were no significant differences between them.


MD Susanna Melkas

Niku Kj Oksala

Hanna Jokinen

Tarja Pohjasvaara

Risto Vataja

Anni Oksala

Markku Kaste

MD Pekka J Karhunen

Timo Erkinjuntti

K J Niku

H Oksala

T Jokinen

R Pohjasvaara

A Vataja

M Oksala

P J Kaste

T Karhunen

Erkinjuntti

Poststroke dementia predicting survival in longterm follow-up: influence of prestroke cognitive decline and previous stroke

Keywords: cerebrovascular diseases, stroke, dementia, mortality

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

Introduction

Lifetime risk for developing stroke, dementia or both is one in three in subjects at 65 years and older. [START_REF] Seshadri | The lifetime risk of stroke: estimates from the Framingham Study[END_REF] Stroke is associated with over 50% risk of death during 5 years and with over 70% risk of death during 10 years poststroke. [START_REF] Eriksson | Survival and recurrent strokes in patients with different subtypes of stroke: a fourteen-year follow-up study[END_REF][START_REF] Vernino | Cause-specific mortality after first cerebral infarction: a population-based study[END_REF] The median survival time of patients with dementia is also shorter compared to normal population, ranging between three to nine years depending on the study design. [START_REF] Xie | Medical Research Council Cognitive Function and Ageing Study, collaborators. Survival times in people with dementia: analysis from population based cohort study with 14 year follow-up[END_REF] In community based studies the prevalence of poststroke dementia in stroke survivors is about 30%, and in hospital based studies the prevalence ranges from 5.9 to 32% depending on the mean age of the study population, delay between stroke and cognitive assessment and criteria for dementia used. [START_REF] Leys | Poststroke dementia[END_REF] Incidence studies indicate that the risk of poststroke dementia is highest within 6-12 months after stroke and remains doubled even in later, compared to normal population. [START_REF] Leys | Poststroke dementia[END_REF] Poststroke dementia is an important category in the vascular cognitive impairment. [START_REF] Moorhouse | Vascular cognitive impairment: current concepts and clinical developments[END_REF][START_REF] Roman | Vascular cognitive disorder: a new diagnostic category updating vascular cognitive impairment and vascular dementia[END_REF] In addition to vascular burden, poststroke dementia relates also to the degenerative burden of the brain. [START_REF] Snowdon | Brain infarction and the clinical expression of Alzheimer disease. The Nun Study[END_REF] The importance of vascular factors as cause of Alzheimer disease is still under discussion. [START_REF] Roman | A diagnostic dilemma: is "Alzheimer's dementia" Alzheimer's disease, vascular dementia, or both?[comment[END_REF] At least stroke seems to be able to initiate a vascular exacerbation that increases the likelihood of clinical dementia diagnosis in a patient with subclinical Alzheimer disease. [START_REF] Hachinski | National Institute of Neurological Disorders and Stroke-Canadian Stroke Network vascular cognitive impairment harmonization standards[END_REF] Especially in the elderly incident ischaemic cerebrovascular disease relates to clinical expression of dementia associated with Alzheimer disease pathology. [START_REF] Snowdon | Brain infarction and the clinical expression of Alzheimer disease. The Nun Study[END_REF][START_REF] Tyas | Healthy ageing in the Nun Study: definition and neuropathologic correlates[END_REF] Previously, two studies have investigated the influence of poststroke dementia on longterm survival. [START_REF] Tatemichi | Dementia after stroke is a predictor of long-term survival[END_REF][START_REF] Desmond | Mortality in patients with dementia after ischemic stroke[END_REF] Both studies indicate that poststroke dementia is an independent predictor of three years has been studied in another two studies, [START_REF] Henon | Influence of prestroke dementia on early and delayed mortality in stroke patients[END_REF][START_REF] Barba | Previous and incident dementia as risk factors for mortality in stroke patients[END_REF] showing both prestroke dementia and new onset poststroke dementia as independent predictors of survival. Populations in all these studies have included both patients with first ever stroke and those with previous stroke, but possible differences in survival between these two groups were not analysed.

Based on a well defined poststroke cohort with an up to 12 years follow up we investigated the influence of poststroke dementia and related factors on survival after acute stroke.

Methods

Patients

The Helsinki Stroke Aging Memory (SAM) cohort comprised a consecutive series of all Finnish (Caucasian) patients with suspected stroke admitted to Helsinki University Central Hospital (n=1622) between 1 December 1993 and 30 March 1995, as described in detail previously. [START_REF] Pohjasvaara | Comparison of stroke features and disability in daily life in patients with ischemic stroke aged 55 to 70 and 71 to 85 years[END_REF][START_REF] Oksala | Smoking and the platelet fibrinogen receptor glycoprotein IIb/IIIA PlA1/A2 polymorphism interact in the risk of lacunar stroke and midterm survival[END_REF] Patients aged 55-85 years living in Helsinki were included. Patients without ischaemic stroke (n=175), presenting with intracerebral (n=229) or subarachnoid haemorrhage (n=69) were excluded. A total of 642 patients fulfilled the inclusion criteria and were invited to a follow-up visit three months later. Of these, 71 died (11.1%) before the three month follow-up, 82 refused (12.8%) and 3 were lost (0.5%) due to undefined causes.

Finally, 486 (85.1%) of the living patients were included in the final SAM cohort. [START_REF] Mantyla | Magnetic resonance imaging white matter hyperintensities and mechanism of ischemic stroke[END_REF] Of these, 451 (92.8%) were clinically testable with regard to dementia diagnosis. [START_REF] Pohjasvaara | Comparison of stroke features and disability in daily life in patients with ischemic stroke aged 55 to 70 and 71 to 85 years[END_REF] The study population included no patients with a restroke between the index stroke and the threemonth evaluation. Patient flow and reasons for nonenrollment are shown in the Table1.

The study was approved by the Ethics committee of the Department of Clinical Neurosciences, Helsinki University Central Hospital, Finland. The study was explained to the patients, and informed consent was obtained.

Clinical evaluation

A detailed medical and neurological history was taken as described before, including history of main vascular risk factors. [START_REF] Pohjasvaara | Comparison of stroke features and disability in daily life in patients with ischemic stroke aged 55 to 70 and 71 to 85 years[END_REF] Education was divided in two categories: low (0-6 years of formal education) and high (>6 years of formal education). Smoking habits were scored at admission as non-smokers and smokers (current or former). History of myocardial infarction, cardiac failure, cardiac arrhythmia, atrial fibrillation, arterial hypertension, peripheral arterial disease and diabetes was investigated reviewing all available hospital charts, in addition to structured interview of the subject and a knowledgeable informant. History of hypertension was defined as systolic blood pressure ≥160 mmHg and diastolic blood pressure ≥95 mmHg, according to cut off values used at the time of evaluation. Diabetes was defined as previously documented diagnosis, current use of insulin or oral hypoglycemic medication, or fasting blood glucose >7.0 mmol/L. Stroke severity was assessed using Modified Rankin score (1-5; 3-5 indicating severe stroke). [START_REF] Van Swieten | Interobserver agreement for the assessment of handicap in stroke patients[END_REF] Dementia was diagnosed using the Diagnostic and Statistical Manual of Mental Disorders, 3 rd edition. [START_REF]American Psychiatric Association Committee on Nomenclature and Statistics: Diagnostic and Statistical Manual of Mental Disorders (DSM-III)[END_REF] The clinical mental status examination by the neurologist assessed the following domains: attention; orientation; memory recall; executive functions including abstract thinking, judgment, and problem solving; aphasia; apraxia; agnosia; motor control; constructional and visuospatial abilities; and personal change as detailed previously. [START_REF] Pohjasvaara | Comparison of stroke features and disability in daily life in patients with ischemic stroke aged 55 to 70 and 71 to 85 years[END_REF] The clinical cognitive assessment included also the Folstein MMSE (maximum=30). [START_REF] Folstein | Mini-mental state". A practical method for grading the cognitive state of patients for the clinician[END_REF] In addition, social functioning was assessed using the Index of ADL (activities of daily living), [START_REF] Katz | Progress in development of the index of ADL[END_REF] the IADL scale (instrumental activities of daily living), [START_REF] Lawton | Assessment of older people: self-maintaining and instrumental activities of daily living[END_REF] the Functional Activity Questionnaire, [START_REF] Pfeffer | Measurement of functional activities in older adults in the community[END_REF] the Blessed Functional Activity Scale [START_REF] Blessed | The association between quantitative measures of dementia and of senile change in the cerebral grey matter of elderly subjects[END_REF][START_REF] Erkinjuntti | The Blessed Dementia Scale as a screening test for dementia[END_REF] and the Barthel Index. [START_REF] Mahoney | Functional evaluation: The Barthel Index. A simple index of independence useful in scoring improvement in the rehabilitation of the chronically ill[END_REF] Assessment of prestroke cognitive decline was based on interviews with the patient and a knowledgeable informant. 28 The interviews included structured questions on abnormality in the cognitive domains as well as assessment of social functions before the index stroke. The beginning of the symptoms and their duration was asked for, focusing especially on the period of one year before the index stroke. Based on all available history from the patient and knowledgeable informant, a board certified neurologist (TP) independently judged whether the patient had had prestroke cognitive decline or not. Patients with prestroke cognitive decline included also those with borderline and definite dementia.

Survival data

Longterm survival data and causes of death at 21 September 2006 were obtained from Statistics Finland. The mean (+-SD) follow up time was 7.5 ± 4.0 years with a range between 0.3 and 12.8 years. The death certificate data was reviewed by a forensic pathology specialist (AO) and the causes of death were divided in cardiac, brain associated (differing between infarction, haemorrhage and dementia), infectious, traumatic, cancer or other causes of death.

Data analysis and statistics

SPSS/WIN (version 15.0, SPSS Inc) software was used. Pearson Chi-square test was used to test dichotomous variables and One-way ANOVA to continuous variables. The effect of dementia diagnosis (any poststroke dementia) was first analysed using Kaplan-Meier log rank analysis, as well as the effect of prestroke cognitive decline and previous stroke among the demented patients. The cumulative hazard function was also used and according to these analyses, the proportional hazards assumption was met for each parameter included in further models. In Cox-regression proportional hazards survival analysis, in the forced entry model (Model 1) the potential predictors were used as covariates (age, sex, years of education, in addition to poststroke dementia). Another model (Model 2) was constructed adding poor Rankin score as a covariate. A third model (Model 3) was constructed adding selected variables from the past medical history as covariates, selecting those variables which were indicated as significant predictors of poor longterm survival using Kaplan-Meier log rank analysis. Statistical significance was set at p<0.05.

Role of the funding source

The funding source (declared under Acknowledgements) had no involvement in study design; in the collection, analysis and interpretation of data; in the writing of the report; nor in the decision to submit the paper for publication. The researchers have acted independent of funders.

Results

Demographics, previous medical history and stroke severity

The basic demographics of the population (n=451) are as follows: mean age 72.0 ± 7. (163/403) of the patients had a severe stroke, indicated by Modified Rankin score between 3-5. 12.2% (55/451) of the patients had had a prestroke cognitive decline, and 20.0% (90/451) of the patients had had a previous stroke.

The univariate analysis of association between above mentioned variables and poststroke dementia are presented in Table 2. Patients with poststroke dementia were older (p=0.002), had lower education (p<0.001) and more severe stroke (<0.001) than those without it. The proportion of patients with prestroke cognitive decline was higher than in patients without dementia (32.2 vs 5.4 %, p<0.001).

Influence of poststroke dementia on survival

In Kaplan-Meier log rank analysis, patients with dementia three months after stroke, i.e. with any poststroke dementia (n=115, 25.5%), had shorter median survival after stroke (5.1 years, CI 4.1-6.0) than patients without dementia (n=336, 74.5%, 8.8 years, CI 7.8-9.9, p<0.001).

Among the demented stroke patients, those who had had a prestroke cognitive decline (n=37, 32.2%) had shorter median survival (3.8 years, CI 2.2-5.4) than those without prestroke cognitive decline (n=78, 67.8%, 5.8 years, CI 4.8-6.9, p<0.001). Further, among the demented patients there was no significant difference between survival of patients with their first ever stroke (n=90, 78%, 5.4 years, CI 4.6-6.2) compared to those with at least one previous stroke (n=25, 22%, 4.5 years, CI 2.9-6.1, p=0.676).

To account for potential confounders and to estimate the predictive value of different factors, a multivariate Cox regression proportional hazards analysis was made in three steps, Model 1-3 (Table 3), as described above in Data analysis and statistics. In Model 3 we added selected variables from the past medical history indicated as significant predictors of poor longterm survival using Kaplan-Meier log rank analysis. These predictors were smoking, myocardial infarction, cardiac failure, cardiac arrhythmia and atrial fibrillation (all with p<0.05), and also peripheral arterial disease with borderline significance (p=0.058).

In Model 1 (Table 3), poststroke dementia and advanced age were independent predictors of poor longterm survival (p<0.001). Adding stroke severity to the model (Model 2, Table 3), poststroke dementia remained associated with increased risk of death (p=0.002), in addition to advanced age (p=0.002) and poor Rankin score (p<0.001). Finally in Model 3 (Table 3), in general (p=0.002) and especially brain infarction (p=0.001) were associated with poststroke dementia. In Kaplan-Meier analysis with brain associated death as endpoint patients with poststroke dementia had shorter survival (6.8 years, CI 4.6-8.9) compared to patients without dementia (11.1 years, CI 10.2-11.9, p<0.001). In multivariate Cox regression analysis using brain associated cause of death as endpoint and adding covariates in three steps (Table 5), poststroke dementia remained as independent predictor of poor survival (p<0.001), in addition to advanced age (p<0.001) and poor Rankin score (p=0.001). Unexpected, in this population myocardial infarction in previous medical history seems to be associated with reduced risk for brain associated death (HR 0.49, CI 0.26-0.94, p=0.033).

Discussion

The present large (N=451) well defined poststroke cohort with an up to 12 years follow-up showed that in patients with acute stroke, dementia is a significant predictor of poor longterm survival and death from brain associated causes. Prestroke cognitive decline but not previous stroke had an additional negative influence on survival.

This study is the longest follow-up confirming that poststroke dementia relates to poor survival. Previously Tatemichi et al. [START_REF] Tatemichi | Dementia after stroke is a predictor of long-term survival[END_REF] (N=251, follow-up 5 years) and Desmond et al. [START_REF] Desmond | Mortality in patients with dementia after ischemic stroke[END_REF] (N=453, follow-up 10 years) came to same conclusion with comparable study designs and demographics. Similarly to our study in these two studies the result remained significant after controlling with major confounders. The study of Barba et al. [START_REF] Barba | Previous and incident dementia as risk factors for mortality in stroke patients[END_REF] (N=251, follow-up 28.7 months) including also intracranial haemorrhages showed similar poor poststroke survival.

The effect of poststroke dementia in the present study is somewhat weaker than that found in previous studies with longterm follow-up: HR 1.5 in the present study, compared with RR 3.1 in the study of Tatemichi and RR 2.4 in the study of Desmond, all after adjusting for major confounders.

Previous reports on prestroke dementia, its frequency and effects on survival [START_REF] Henon | Influence of prestroke dementia on early and delayed mortality in stroke patients[END_REF][START_REF] Barba | Previous and incident dementia as risk factors for mortality in stroke patients[END_REF][START_REF] Appelros | What causes increased stroke mortality in patients with prestroke dementia?[END_REF] can be compared with our findings with prestroke cognitive decline. Additional negative influence of prestroke dementia on the poststroke survival, in line with our finding, was reported in two of these studies, [START_REF] Barba | Previous and incident dementia as risk factors for mortality in stroke patients[END_REF][START_REF] Appelros | What causes increased stroke mortality in patients with prestroke dementia?[END_REF] but not in one. [START_REF] Henon | Influence of prestroke dementia on early and delayed mortality in stroke patients[END_REF] First ever stroke is of interest because stroke alone is associated with shortened survival. [START_REF] Eriksson | Survival and recurrent strokes in patients with different subtypes of stroke: a fourteen-year follow-up study[END_REF][START_REF] Vernino | Cause-specific mortality after first cerebral infarction: a population-based study[END_REF] However its influence has not been analysed separately in previous studies on poststroke survival. In the present study 21.7% of patients had had a previous stroke but previous stroke did not have an influence on survival (4.5 years with previous stroke vs. 5.4 years with first ever stroke).

In our study there was an association between poststroke dementia and brain associated cause of death, more specifically brain infarction. Previous studies have not found this association. [START_REF] Desmond | Mortality in patients with dementia after ischemic stroke[END_REF][START_REF] Henon | Influence of prestroke dementia on early and delayed mortality in stroke patients[END_REF] Our result relies on the high validity of determination of causes of death in Finland. The cause of death has been verified by autopsy in 30% of all deaths in Finland in the past two decades (Statistics Finland, www.tilastokeskus.fi) which is rather high proportion compared to other European countries. In addition, the death certificates of all deceased, whether subjected to autopsy or not are reviewed by the district forensic physician. In the present study all death certificate data was in addition reviewed by a specialist in forensic medicine. The official cause of death has been demonstrated to be an accurate means for evaluating disease specific mortality in Finland. [START_REF] Makinen | Assessment of causes of death in a prostate cancer screening trial[END_REF] There was no specific association between poststroke dementia and dementia as a cause of death in our analysis. This probably has to do with the general restraint of using dementia as the primary cause of death otherwise than when there is clear evidence for primarily degenerative, progressive dementia, in particular Alzheimer disease. Fatal progression of vascular dementia is often associated with stroke recurrence, and in those cases the cause of death is brain infarction. Another observation is that in this population previous myocardial infarction seemed to be associated with reduced risk for brain associated death. A further analysis was done with multivariate Cox regression analysis using cardiac death as endpoint.

The analysis showed tendency, although not significant (p=0.095), that previous myocardial infarction is associated with increased risk for cardiac death. The result indicates that in patients with cardiac disease the cardiac mortality dominates irrespective of stroke, but further investigation is needed before we know if the result is generalisable.

A potential weakness of our study is the possibility of selection bias, as the cohort was formed three months after the index stroke. Attrition over three months may limit generalisation of the results. Therefore, we retrospectively obtained additional data on stroke related deaths in Helsinki University Hospital district during the collection of the cohort from an independent organisation (Statistics Finland, www.tilastokeskus.fi) . In this retrospective data, up to 64 percent of stroke related deaths occurred in women. While the proportions of both sexes in the present study were equal, this suggests that some women may have died before hospital assessment at three months. Due to exclusion of patients, the true survival rate may be underestimated. Another potential weakness relates to the definition of prestroke cognitive decline, which we based on detailed clinical history instead of a more reliable assessment with standardised IQCODE as did Henon et al. [START_REF] Henon | Influence of prestroke dementia on early and delayed mortality in stroke patients[END_REF] and Barba et al. [START_REF] Barba | Previous and incident dementia as risk factors for mortality in stroke patients[END_REF] No formal validation was carried out on our definition procedure, but the diagnosis relies on clinical competency and excessive efforts to find out relevant background information.

As noted above, cut off values for hypertension in our study are not consistent with current definitions. Previous studies have used cut off values comparable with our study. [START_REF] Desmond | Mortality in patients with dementia after ischemic stroke[END_REF][START_REF] Barba | Previous and incident dementia as risk factors for mortality in stroke patients[END_REF] Hypertension has not been an independent predictor of survival in these studies either.

Significance of hypertension according to current criteria remains to be examined in the future longterm follow ups of poststroke cohorts.

Strengths of our study are the well defined, large and consecutive cohort and the longterm follow up (12 years). The cohort has allowed analysis of the effect of prestroke cognitive decline and the effect of previous stroke on survival. The data on survival and causes of death is comprehensive with negligible amount of unresolved deaths. In addition, the diagnosis of dementia is similar with diagnosis in previous studies, allowing direct comparisons.

Since poststroke dementia significantly worsens the prognosis after stroke, especially in patients with prestroke cognitive decline, it is important to recognise patients with this condition to be able to offer them optimal comprehensive care.

  addition to poststroke dementia (HR 1.53, CI 1.15-2.04, p=0.003), advanced age (HR 1.07, CI 1.05-1.09, p<0.001), poor Rankin score (HR 1.91, CI 1.47-2.48, p<0.001), smoking (HR 1.35, CI 1.02-1.77, p=0.035), cardiac failure (HR 1.61, CI 1.18-2.19, p=0.003) and atrial fibrillation (HR 1.89, CI 1.05-3.42, p=0.035) remained as independent predictors of poor longterm survival.

Figure 1 .

 1 Figure 1. The effect of poststroke dementia on overall poststroke survival (endpoint all cause death) in Stroke Aging Memory cohort. Kaplan-Meier log rank analysis.

Figure 2 .

 2 Figure 2. The effect of prestroke cognitive decline on survival of patients with poststroke dementia (endpoint all cause death) in Stroke Aging Memory cohort. Kaplan-Meier log rank analysis.

Figure 3 .

 3 Figure 3. The effect of previous stroke on survival of patients with poststroke dementia (endpoint all cause death) in Stroke Aging Memory cohort. Kaplan-Meier log rank analysis.

Table 1 .

 1 Patient flow in the Helsinki Stroke Aging Memory Study. Reasons for nonenrollment Consecutive patients with suspected stroke N=1622 Not fulfilling the current criteria for stroke n=175 Intracerebral haemorrhage n=229 Subarachnoidal haemorrhage n= 69 Patients with ischaemic stroke n=1149 Age under 55 or over 85 years n=346 Not living in the city of Helsinki n=158 Not speaking Finnish language n=3Fulfilling inclusion criteria, invited to 3 month follow-up n=642 Death before the 3 month follow-

  Figure 1

  Figure 2

  Figure 3

Table 2 .

 2 The association of poststroke dementia with demographic parameters, previous medical history and stroke severity.

		No dementia		Any poststroke dementia
	Variable	Valid n	Mean/n	(SD/%) Valid n	Mean/n	(SD/%) p value
	Mean age	336	70.4	(7.6)	115	72.9	(7.6)	0.002
	Female sex	336	162	(48.2)	115	59	(51.3)	0.567
	Low education (<6yr )	331	83	(25.1)	112	54	(48.2)	<0.001
	Smoking (current/former)	335	164	(49.0)	115	60	(52.2)	0.551
	Myocardial infarction	336	64	(19.0)	115	22	(19.1)	0.984
	Cardiac failure	335	69	(20.6)	115	28	(24.3)	0.399
	Cardiac arrhythmia	336	85	(25.3)	115	29	(25.2)	0.986
	Atrial fibrillation	335	59	(17.6)	115	26	(22.6)	0.238
	Arterial hypertension	336	161	(47.9)	115	53	(46.1)	0.734
	Peripheral arterial disease	336	39	(11.6)	115	14	(12.2)	0.871
	Diabetes	336	78	(23.2)	115	33	(28.7)	0.239
	Poor Rankin score	305	100	(32.8)	98	63	(64.3)	<0.001
	Prestroke cognitive decline	336	18	(5.4)	115	37	(32.2)	<0.001
	Previous stroke	336	65	(19.3)	115	25	(21.7)	0.579
	Dichotomous variables: Pearson Chi-square test
	Continuous variables: One-way ANOVA

Table 3 .

 3 Cox regression analysis on the association of multiple risk factors and poststroke dementia with poor longterm survival (all cause death endpoint) among patients with ischaemic stroke (Stroke Aging Memory cohort).

		Model 1, forced entry		Model 2, forced entry		Model 3, forced entry
	Variable	HR	95% CI	p value	HR	95% CI	p value	HR	95% CI	p value
	Age	1.08	1.06-1.09	<0.001	1.07	1.12-2.06	0.002	1.07	1.05-1.09	<0.001
	Female sex	0.77	0.68-0.97	0.028	0.80	0.62-1.03	0.089	0.88	0.66-1.16	0.352
	Low education <6 years	1.07	0.83-1.37	0.609	0.95	0.72-1.25	0.707	0.92	0.69-1.21	0.544
	Poor Rankin score	-			1.85	1.43-2.39	<0.001	1.91	1.47-2.48	<0.001
	Smoking (current/former)	-			-			1.35	1.02-1.77	0.035
	Myocardial infarction	-			-			1.08	0.78-1.49	0.661
	Cardiac failure	-			-			1.61	1.18-2.19	0.003
	Cardiac arrhythmia	-			-			0.73	0.42-1.25	0.246
	Atrial fibrillation	-			-			1.89	1.05-3.42	0.035
	Peripheral arterial disease	-			-			1.09	0.75-1.58	0.663
	Poststroke dementia	1.71	1.33-2.20	<0.001	1.56	1.18-2.06	0.002	1.53	1.15-2.04	0.003

Table 4 .

 4 The association of poststroke dementia with causes of death in Stroke Aging Memory cohort.

			No dementia	Any poststroke
			n=219*		dementia n=100
	Variable	Valid n	n	%	n	%	p value
	Cardiac	91	68	(31.1)	23	(23.0)	0.140
	Brain	108	62	(28.3)	46	(46.0)	0.002
	Infarction	87	47	(21.5)	40	(40.0)	0.001
	Haemorrhage	11	8	(3.7)	3	(3.0)	0.767
	Dementia	10	7	(3.2)	3	(3.0)	0.926
	Infectious	15	9	(4.1)	6	(6.0)	0.459
	Traumatic	11	8	(3.7)	3	(3.0)	0.767
	Cancer	45	35	(16.0)	10	(10.0)	0.154
	Other	40	28	(12.8)	12	(12.0)	0.844
	Pearson Chi-square test

*The cause of death unknown for 9 subjects, all with no dementia.

Table 5 .

 5 Cox regression analysis on the association of multiple risk factors and poststroke dementia with poor longterm survival (brain associated death endpoint) among patients with ischaemic stroke (Stroke Aging Memory cohort).

		Model 1, forced entry		Model 2, forced entry		Model 3, forced entry
	Variable	HR	95% CI	p value	HR	95% CI	p value	HR	95% CI	p value
	Age	1.07	1.04-1.11	<0.001	1.08	1.04-1.11	<0.001	1.07	1.04-1.11	<0.001
	Female sex	1.10	0.74-1.64	0.641	1.00	0.65-1.55	0.998	0.85	0.52-1.39	0.513
	Low education <6 years	0.93	0.60-1.42	0.721	0.76	0.47-1.23	0.266	0.77	0.47-1.26	0.295
	Poor ranking score	-			2.17	1.36-3.45	0.001	2.15	1.34-3.46	0.001
	Smoking (current/former)	-			-			0.75	0.46-1.25	0.267
	Myocardial infarction	-			-			0.49	0.26-0.94	0.033
	Cardiac failure	-			-			1.03	0.58-1.83	0.914
	Cardiac arrhythmia	-			-			1.05	0.42-2.58	0.925
	Atrial fibrillation	-			-			1.43	0.53-3.89	0.482
	Peripheral arterial disease	-			-			1.31	0.72-2.41	0.378
	Poststroke dementia	2.22	1.48-3.35	<0.001	2.36	1.49-3.73	<0.001	2.33	1.46-3.73	<0.001

Acknowledgements

This study was supported by grants from the Clinical Research Institute, Helsinki University Central Hospital and the Medical Research Fund of Helsinki University Central Hospital. We thank Statistics Finland for their expert assistance in the data collection.

Copyright

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Competing interest statement

All authors declare that the answer to the questions on your competing interests form are all No and therefore have nothing to declare.

Details of contributors

The authors have contributed to the manuscript as follows: S Melkas designed the research, analysed and interpreted the data, performed statistical analysis and drafted the manuscript.


Dr Alain Furby

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Anthony Béhin

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Late-onset cervicoscapular muscle atrophy and weakness after radiotherapy for Hodgkin's disease: a case series

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Introduction

The classical treatment of Hodgkin's disease (HD) is based on chemotherapy plus radiation therapy (RT). [START_REF] Hoppe | Hodgkin's lymphoma: the role of radiation in the modern combined strategies of treatment[END_REF][START_REF] Liao | Mantle irradiation alone for pathologic stage I and II Hodgkin's disease: long-term follow-up and patterns of failure[END_REF][START_REF] Glossmann | New treatments for Hodgkin's disease[END_REF] Late neurological adverse events after extended-field RT include brachial or lumbar plexopathy [START_REF] Wadd | Brachial plexus neuropathy following mantle radiotherapy[END_REF][START_REF] Malow | Neuralgic amyotrophy in association with radiation therapy for Hodgkin's disease[END_REF] and chronic progressive myelopathy. [START_REF] Béhin | Complications of radiation therapy on the brain and spinal cord[END_REF] A few patients were found to develop severe weakness and atrophy of cervical and shoulder girdle musculature that developed several years after mantle irradiation. [START_REF] Johansson | Late side-effects are common after treatment of Hodgkin's disease. Muscular atrophy following radiotherapy is a neglected risk[END_REF][START_REF] Portlock | Nemaline myopathy: a possible late complication of Hodgkin's disease therapy[END_REF][START_REF] Rowin | Late appearance of dropped head syndrome after radiotherapy for Hodgkin's disease[END_REF][START_REF] Aggarwal | Radiation induced myopathy following mantle field radiation for hodgkin's disease: a report of 4 cases[END_REF] The clinical presentation usually includes a dropped head syndrome. The pathophysiology of this rare post-RT complication is still unclear. In this study we report detailed clinical, electrophysiological and pathological findings in six original cases sharing the same condition of late-onset cervicoscapular muscle atrophy and weakness following mantle irradiation for HD.

Case reports

The six patients had a medical history of HD (stage I to stage III) treated by chemotherapy followed by extended-field RT, including mantle irradiation, and were considered to be in complete remission. Details are given in table 1. They all were referred for weakness and atrophy of cervical paraspinal and shoulder girdle musculature. The mean delay between irradiation and first symptoms was 19.7 years (ranged from five to 30). Neck pain was reported by four patients. Patients did not complain of cramps, fasciculations, myokimia, or any other abnormal spontaneous muscle activity, as well as of any sensory deficit.

Clinical examination showed severe weakness and atrophy of splenius capitis, supraspinatus, infraspinatus, trapezius, sternocleidomastoid and deltoid muscles, leading to a dropped head syndrome in four cases (Fig. 1). Cranial nerves were normal and patients had neither swallowing difficulties nor dysarthria. Stretch reflexes and sensory examination were normal, except a symmetrical, distal hypoesthesia in the lower limbs (with absent achillean stretch reflexes) attributed to diabetic polyneuropathy in patient No 3.

Laboratory investigation was unremarkable in all patients, regarding blood cell count, electrolyte dosage, C reactive protein, serum protein immunoelectrophoresis, thyroid function test, and serologies for human immunodeficiency virus 1 and 2, and B and C hepatitis virus.

Serum creatin kinase level was slightly elevated in four cases (mean = 395 UI/l). Cerebral spinal fluid examination showed elevated protein concentration (0.71g/l) in one patient and was normal in 2 others. Cervical spinal magnetic resonance imaging and whole-body positron emission tomography for lymph node and metastatic examination disclosed no abnormalities.

Cervical computed tomography scan performed in 3 patients revealed diffuse muscle atrophy with fatty changes in paraspinal muscles.

In all the patients, weakness may have progressed but did not extend beyond the initially involved muscle groups over several years (one to nine years). Treatment by oral steroids (1 mg/kg) was administered for three months in one patient (N°2), without any clinical benefit.

Pathological studies

A biopsy of the deltoid muscle was performed in the four patients in whom this muscle was clinically affected by wasting and weakness (see Table 1). Variation in muscle fiber size was mild in two patients (N°1, 6) and more pronounced in one patient (N°2), with a combination of hypertrophic and atrophic fibers from one sample to another (Fig. 1). A few nuclear clumps were observed. Many fibers had a markedly disorganized architecture: some were split; others had a whorled or annular aspect. A moderate increase in the number of internalized myonuclei was found. Endomysial connective tissue was slightly increased, and some areas were infiltrated by adipocytes. Some samples showed a predominance of muscle fibers of type 1.

We did not find type grouping, target/targetoid changes, necrotic fibers, inflammatory infiltrates, rods or amyloid deposits. Blood vessels were unremarkable. Immunostaining for dystrophin, sarco-and dystroglycans, dysferlin and caveolin was negative.

Discussion

In 1998, Johansson et al. [START_REF] Johansson | Late side-effects are common after treatment of Hodgkin's disease. Muscular atrophy following radiotherapy is a neglected risk[END_REF] first pointed out that neck and shoulder girdle muscles could present wasting and weakness in long-term HD survivors as a late complication of mantle irradiation. The following report was that of Portlock et al. [START_REF] Portlock | Nemaline myopathy: a possible late complication of Hodgkin's disease therapy[END_REF] who described histological features of nemaline myopathy in trapezius muscle of a patient who had undergone mantle irradiation for HD 16 years before. Additional cases of dropped head syndrome occurring as a late complication of mantle irradiation for HD were reported in one paper (3 cases) [START_REF] Rowin | Late appearance of dropped head syndrome after radiotherapy for Hodgkin's disease[END_REF] and an abstract (4 cases). [START_REF] Aggarwal | Radiation induced myopathy following mantle field radiation for hodgkin's disease: a report of 4 cases[END_REF] The common clinical feature in all these patients, including our six patients, was a severe wasting confined to neck and shoulder girdle musculature, leading to a dropped head syndrome in most cases. It develops as a pure motor syndrome without clinical sensory manifestations, except cervical pain that preceded weakness by several months or years in some patients. Contracture of anterior cervical muscles was also described, [START_REF] Rowin | Late appearance of dropped head syndrome after radiotherapy for Hodgkin's disease[END_REF] but not observed in our patients. Absence of paresthesias, hypesthesia, or sensory nerve conduction abnormalities and preserved stretch reflexes can rule out the hypothesis of a plexopathy, another rare complication of mantle irradiation. [START_REF] Wadd | Brachial plexus neuropathy following mantle radiotherapy[END_REF][START_REF] Churn | Early onset of bilateral brachial plexopathy during mantle radiotherapy for Hodgkin's disease[END_REF] The pathophysiology of cervico-scapular muscle weakness and wasting following mantle irradiation in HD could result from primary muscle damage or anterior horn or root lesions at upper cervical level. In the present series, EMG changes were myopathic in one patient, neurogenic in two patients and included a combination of myopathic MUPs with neurogenic recruitment in two others. In the literature, both myopathic and neurogenic EMG alterations have also been reported. [START_REF] Rowin | Late appearance of dropped head syndrome after radiotherapy for Hodgkin's disease[END_REF] Regarding histopathological studies, the abnormalities were non-specific in the four patients in whom we performed muscle biopsy, but they were more in line with predominant non-inflammatory myopathic changes. Such a finding has been previously reported, [START_REF] Aggarwal | Radiation induced myopathy following mantle field radiation for hodgkin's disease: a report of 4 cases[END_REF] as well as the presence of rod/nemaline bodies 8 or features of muscle denervation. [START_REF] Rowin | Late appearance of dropped head syndrome after radiotherapy for Hodgkin's disease[END_REF] Both myopathic and neurogenic processes may underlie the pathophysiological changes, because cervico-scapular muscles are located within the field of mantle irradiation as well as upper cervical anterior horns and roots. In addition, the potential neuromuscular toxicity of chemotherapeutic drugs could play a sensitization role. However, myopathic and neurogenic processes may be not equally involved in all patients and territories. Following our observations, myopathic features seemed to be predominant regarding histopathological findings in deltoid muscles, while definite neurogenic signs could be found in needle EMG examination of neck and paraspinal muscles. Nevertheless, myopathic alterations were clearly predominant in three patients of this series.

Irradiation has been considered to cause relatively little or no direct damage to mature muscle fibers and in fact myositis is an infrequently reported adverse effect of RT. [START_REF] Welsh | Radiation myositis[END_REF] However, high radiation doses are known to dramatically alter the proliferation of muscle precursor cells and were used in experimental animal models to inhibit spontaneous muscle regeneration. [START_REF] Alameddine | Regeneration of skeletal muscle fibers from autologous satellite cells multiplied in vitro. An experimental model for testing cultured cell myogenicity[END_REF] Fajardo et al. [START_REF] Fajardo | Vascular lesions following radiation[END_REF] hypothesized that radiation could induce primarily vascular lesions due to high susceptibility to radiation of capillaries and vessels. These lesions may result from an imbalance in the cross talk between endothelial cells and vascular smooth muscle cells. [START_REF] Milliat | Influence of endothelial cells on vascular smooth muscle cells phenotype after irradiation: implication in radiation-induced vascular damages[END_REF] A secondary increase in collagen content and a proliferation of fibrous tissue may occur in the irradiated tissue, [START_REF] Wegrowski | Modification of collagen and noncollagenous proteins in radiation-induced muscular fibrosis[END_REF] leading to progressive deterioration over several years following RT. These processes are delayed, evolving many years after RT completion and they are generally dose-and time-dependent.

In conclusion, clinicians should be aware that weakness and wasting can occur in neck and shoulder girdle musculature of long-term HD survivors who had previously undergone mantle irradiation. Whatever the underlying pathophysiology, the lesions appear to be irreversible as other delayed complications of RT. This observation supports the current development of involved-field RT protocols based on limited rather than extended fields of irradiation to reduce the risk of late complications.

Legends of figure and table

  Nerve conduction studies were normal in the four limbs, including no proximal conduction block or any sign of demyelinating neuropathy. A reduction of compound muscle action potential amplitude in upper trapezius muscle to spinal nerve stimulation was observed in patient N°3. Needle electromyography (EMG) showed abnormal muscle activity at rest (fibrillation potentials or complex repetitive discharges) in three patients (N°2, 3, 4). During contraction, myopathic EMG findings (motor unit potentials (MUPs) of reduced amplitude and duration) were found in paraspinal and deltoid muscles in one patient (N°1). Two patients (N°4, 5) showed neurogenic EMG changes in splenius capitis, upper trapezius, supraspinatus and infraspinatus muscles. A combination of myopathic MUPs with neurogenic recruitment was predominantly found in paraspinal, neck and deltoid muscles in two patients (N°2, 3). In the last patient (N°6), needle EMG remained normal, but the examination was incomplete and did not study the most affected muscles.

  Figure 1. A. Clinical presentation (patient N°6): severe wasting of cervicoscapular muscles

Table 1 :

 1 Figure 1