Elisa Teyssou

Takahiro Takeda

Vincent Lebon

Séverine Boillée

Brahima Doukouré

Guillaume Bataillon

Véronique Sazdovitch

Cécile Cazeneuve

Vincent Meininger

Eric Leguern

François Salachas

Danielle Seilhean

Dr Stéphanie Millecamps
email: stephanie.millecamps@upmc.fr

Mutations in SQSTM1 encoding p62 in amyotrophic lateral sclerosis: genetics and neuropathology

Keywords: Motor neuron disease, familial and sporadic ALS, Paget's disease, p62, neuropathology, FTLD-ALS

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Introduction

Amyotrophic Lateral Sclerosis (ALS) is the most common adult onset motor neuron disease. Pathological hallmarks detected in spinal motor neurons of most ALS patients include skein like inclusions, which are positive for ubiquitin, ubiquitin-binding protein p62 (also called sequestosome 1, [START_REF] Mizuno | Immunoreactivities of p62, an ubiqutin-binding protein, in the spinal anterior horn cells of patients with amyotrophic lateral sclerosis[END_REF]) and TDP-43 (TAR DNAbinding protein 43, [START_REF] Neumann | Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis[END_REF]), and cystatin C-positive Bunina bodies [START_REF] Okamoto | Bunina bodies in amyotrophic lateral sclerosis immunostained with rabbit anti-cystatin C serum[END_REF]. Recently, SQSTM1 encoding sequestosome 1/p62 protein (440 amino acids) was screened as a candidate gene in a large cohort of ALS patients. Ten different mutations (9 heterozygous missense and one deletion) were identified in 6/340 FALS (1.8%) and 9/206 SALS (4.4%) [START_REF] Fecto | SQSTM1 mutations in familial and sporadic amyotrophic lateral sclerosis[END_REF]. Following, two reports screened SQSTM1 gene in different populations.

Out of 124 SALS and 170 frontotemporal lobar degeneration (FTLD) patients of Italian origin around 3% carried the mutations and this gene was also responsible for 2/54 SALS (3.7%) of Japanese origin [START_REF] Hirano | Mutations in the gene encoding p62 in Japanese patients with amyotrophic lateral sclerosis[END_REF][START_REF] Rubino | SQSTM1 mutations in frontotemporal lobar degeneration and amyotrophic lateral sclerosis[END_REF].

Although expanded hexanucleotide repeats in C9ORF72 occurring in 23-46% of FALS and 4-8% of SALS became the major genetic defects in ALS [START_REF] Dejesus-Hernandez | Expanded GGGGCC Hexanucleotide Repeat in Noncoding Region of C9ORF72 Causes Chromosome 9p-Linked FTD and ALS[END_REF][START_REF] Renton | A Hexanucleotide Repeat Expansion in C9ORF72 Is the Cause of Chromosome 9p21-Linked ALS-FTD[END_REF], mutations in SQSTM1 could account for a substantial part of ALS causing genes making p62 a potential player in ALS. The aim of this study was to confirm the contribution of SQSTM1 to ALS by analyzing 164 French ALS patients (90 FALS and 74 autopsied SALS) in whom all the other ALS genes had been excluded and to analyze the neuropathological features in patients carrying SQSTM1 variants.

Methods

Genotyping. The eight coding exons with exon-intron boundaries of SQSTM1 (ensembl reference sequence: ENSG00000161011) were sequenced in 90 French FALS with mean age of onset of 60 years (SE 1, range 33 to 80 years), mean disease duration of 46 months (SE 5, range 8 to 260 months, including 13 censored data) and 74 SALS French autopsied cases (30 DNA were from blood and 50 DNA were extracted from Liver slices) with mean age of onset of 61 years (SE 1, range 35 to 81 years), mean disease duration of 49 months (SE 5, range 8 to 206 months) (Table 1). Data were censored at the last date of patient visit. Control samples were obtained from sex and age-matched healthy Caucasian individuals of French background. Initial ALS populations comprised 270 FALS with probable or definite ALS [START_REF] Brooks | El Escorial revisited: revised criteria for the diagnosis of amyotrophic lateral sclerosis[END_REF] including 10 autopsied cases and 80 more autopsied SALS cases (30 DNA were from blood and 50 DNA were extracted from Liver slices).

Analyses of C9ORF72, SOD1, TARDBP, FUS, ANG, VAPB, DAO, OPTN and UBQLN2 genes had previously been performed [START_REF] Millecamps | Screening of OPTN in French familial amyotrophic lateral sclerosis[END_REF][START_REF] Millecamps | Phenotype difference between ALS patients with expanded repeats in C9ORF72 and patients with mutations in other ALS-related genes[END_REF][START_REF] Millecamps | Mutations in UBQLN2 are rare in French amyotrophic lateral sclerosis[END_REF][START_REF] Millecamps | Questioning on the role of D amino acid oxidase in familial amyotrophic lateral sclerosis[END_REF][START_REF] Millecamps | SOD1, ANG, VAPB, TARDBP, and FUS mutations in familial amyotrophic lateral sclerosis: genotype-phenotype correlations[END_REF]. Mutation frequencies of C9ORF72, SOD1, TARDBP and FUS have been described System (GE Healthcare). Densitometric analysis was perfomed using Multi Gauge V3.0 (Fujifilm) software using tubulin as internal standard. For each patient, 5 measures (from 5 independent western blot analyses) were recorded for p62 and TDP-43. p62 and TDP-43 levels were compared between patients with SQSTM1 mutation and control SALS patients (with no mutation in the screened genes) using Mann Whitney test (GraphPad Prism 4 software). The significance level for all comparisons was set at 0.05.

Primary antibodies included mouse monoclonal anti-p62 antibody raised against aminoacids 257-437 of the human protein (610833, BD Biosciences), rabbit anti-TARDBP raised against TDP-43 protein (12892-1-AP, ProteinTech) and mouse anti--tubulin B-5-1-2 (Sigma-Aldrich). Peroxidase-conjugated AffiniPure Goat Anti-Rabbit and Anti-Mouse (all with minimal cross-reaction to human proteins) were from Jackson immunoResearch Laboratories. Immunostainings for p62, ubiquitin and TDP-43 were performed on three levels of the spinal cord (cervical thoracic and lumbar) and on the medulla oblongata, frontal isocortex, hippocampus and cerebellum.

RT-PCR.

Immunohistochemistry for cystatin C was, in addition, performed on spinal cord and medulla oblongata. After deparaffinization, the 5 µm thick sections were immunolabeled with the antibodies listed in Table 2. For most antibodies, the immunostaining was performed by an automatic slide stainer (Benchmark® XT Ventana® staining system), the slides being pre-treated at 95°C in either CC1 (pH=8) or CC2 (pH=6) proprietary retrieval buffers (Ventana Medical Systems®) (Table 2). The biotinylated secondary antibody was included in the detection kit (Ventana Medical Systems Basic DAB Detection Kit 250-001®). The streptavidin-biotinperoxidase complex was revealed by diamino-benzidine. For ubiquitin immunohistochemistry, the slides were pre-treated by formic acid (FA, 80 %), for 5 to 10 minutes (Table 2). For p62 staining, the slides were microwave pretreated (450W) during 2x10 min in citrate buffer pH 6 (Table 2).

The semi quantitative analysis of the lesions focused on anterior horn motor neurons of medulla oblongata and spinal cord in 45 autopsied ALS patients including patients with SQSTM1 (n=3), C9ORF72 (n=8), SOD1 (n=4), TARDBP (n=2) mutations and other patients with no mutation in any of these genes (n=28).

A consensual definition based on the shape and localization of ubiquitinated inclusions (filamentous or skeinlike, large round and granular) was established by five of us (DS, TT, VL, BD, SM). The semi-quantitative Teyssou et al. ActaNeuropathol 2013 AcceptedManuscript assessment for p62 and TDP-43 in cervical, thoracic and lumbar spinal cord and in medulla oblongata was performed by three of us (TT, VL, BD) who were blinded to the genetic data, following a 4-point ordinal scale (0, none; 1, rare; 2, moderate; 3, severe), a similar scoring approach had been used effectively in several similar comparative pathology studies [START_REF] Brettschneider | Microglial activation and TDP-43 pathology correlate with executive dysfunction in amyotrophic lateral sclerosis[END_REF][START_REF] Brettschneider | Pattern of ubiquilin pathology in ALS and FTLD indicates presence of C9ORF72 hexanucleotide expansion[END_REF][START_REF] Geser | Pathological 43-kDa transactivation response DNAbinding protein in older adults with and without severe mental illness[END_REF][START_REF] Geser | Motor neuron disease clinically limited to the lower motor neuron is a diffuse TDP-43 proteinopathy[END_REF][START_REF] Hart | Distinct TDP-43 pathology in ALS patients with ataxin 2 intermediate-length polyQ expansions[END_REF]. The score means obtained for filamentous, large round, and granular inclusions were compared between patients carrying SQSTM1, C9ORF72, SOD1 mutations and other patients with no mutation in any of these genes using ANOVA (SPSS 11.0 software). If the difference was significant (p<0.05), the groups were compared by pairs using a Tukey's post hoc test analysis. The patient group with mutation in TARDBP (n=2 patients only) was not included in this analysis.

Results

Genetic analysis

In FALS we identified, at the heterozygous state, one missense mutation in exon 8 (the c.1175C>T, p.Pro392Leu) in one patient (case 1) that presented with distal lower limb disease onset at 54 years and deceased after a 133 months disease duration (Fig. 1a,1c). No DNA was available for other affected family members and the segregation could not be further studied (Fig. 1d). In SALS the c.1165+1G>A substitution in intron 7

presumably affecting the exon 7 splicing site (Fig. 2a) was identified in one woman who had a distal lower limb disease onset at 75 years and disease duration of 42 months (case 2). These two patients had concomitant Paget's disease (Table 3).

We also identified, at the heterozygote state, two missense mutations in two SALS patients: the c.259A>G, p.Met87Val in exon 2 (carried by case 3) and the c.304A>G, p.Lys102Glu in exon 3 (carried by case 4, Fig. 1a).

The first patient (case 3) carrying a missense variant showed bulbar onset at 68 and disease duration of 18 months and the second one (case 4) started the disease at 81 years of age which lasted for 10 months (Table 3).

History of Paget's disease was not recorded for these patients. These variants affect aminoacids that are highly conserved in mammals (Fig. 1b). They were not identified in the dbSNP Short Genetic Variations, the 1000

Genomes Project and the NHLBI ESP Exome Variant Server databases and in our analysis of 360 control Caucasian individuals. The sequencing of exon 7 and 8 in this control population identified one single variant, not reported previously: the c.1313C>T, p.Pro438Leu in one 60 year-old man.

To further analyse the effect of the c.1165+1G>A in the intron 7 splicing site variant (carried by case 2), we amplified the p62 cDNA sequence spanning from exons 5 to 8 (Fig. 2c) and sequenced the corresponding fragments. We found that this substitution led to the deletion of part of the exon 7 (r.1052_1165del, Ex7) in the AcceptedManuscript mRNA sequence (Fig. 2b-c) which probably results in the production of a truncated p.Gly351_Pro388del protein of 402 aminoacids as suggested by the additional band present on the western blot analysis (Fig. 2d, case 2).

Quantitative analysis of p62 (for case 2, p62 level is the sum of the two bands) and TDP-43 levels on immunoblots showed that p62 and TDP-43 protein levels were increased in the group of patients with SQSTM1 mutations compared to the control SALS group (Fig. 2e) with a significant difference reached for both stainings (p=0.0129 for p62 and p<0.0001 for TDP-43).

Neuropathology analysis

Autopsy had been performed for 3 of the patients carrying mutations in SQSTM1 (cases 2-4).

The brain of the woman carrying the c.1165+1G>A (p.Gly351_Pro388del) mutation (case 2) weighted 1180g.

Atrophy of the frontal lobe including the precentral gyrus, associated with atrophy of the anterior spinal roots was noticed. Microscopic examination of the spinal cord and medulla oblongata showed mild neuronal loss, cystastin C positive Bunina bodies, TDP-43 and p62 positive skein like and rounded inclusions and TDP-43 positive diffuse granular staining. Pronounced neuronal loss and gliosis were observed in the substantia nigra, without Lewy bodies. In the midfrontal and precentral gyri, fibrillary gliosis was associated with superficial spongiosis. Immunohistochemistry showed numerous TDP-43 positive filamentous inclusions in layer 2, associated to thin neurites (Fig. 4a), resembling Type A pathology according to Mackenzie classification [START_REF] Mackenzie | A harmonized classification system for FTLD-TDP pathology[END_REF].

p62 immunohistochemistry showed a lower number of paranuclear inclusions, and no neurites (Fig. 4c). Despite these neuropathological observations on cortical regions, this patient had no clinical FTD (Table 3).

In the male patient with M87V mutation (case 3), the brain weighted 1345 g before fixation. No cerebral atrophy was noticed, macroscopic abnormalities being limited to atrophy of the anterior spinal roots. At microscopic examination, neuronal loss was severe in the anterior horns of the spinal cord. Immunochemistry showed TDP- 3d-f, 3g-i, supplementary Fig. 1a) and cystastin C positive Bunina bodies. In addition numerous motor neurons contained intracytoplasmic small ubiquitin and TDP-43 positive granules (Fig. 3a,3c). This pattern was not observed with p62 immunohistochemistry (supplementary Fig. 1c). In some rare neurons bigger "seed-like", p62 positive aggregates were observed (Fig. 3b). These seed-like inclusions were also seen with TDP-43 staining (supplementary Fig. 1b). A mild neuronal loss was observed in the substantia nigra without Lewy bodies. There was neither evidence of major neuronal loss in the central motor cortex nor severe degeneration of the corticospinal tracts. A mild superficial spongiosis was observed in the second layer in the midfrontal gyrus.

Immunohistochemistry showed some TDP-43 positive inclusions of various shapes, either round or filamentous, spread throughout the cortex, with only a few positive neurites (Fig. 4b), resembling Type B pathology according to Mackenzie classification [START_REF] Mackenzie | A harmonized classification system for FTLD-TDP pathology[END_REF]. p62 immunohistochemistry showed a lower number of inclusions, generally rounded, and no neurites (Fig. 4d). p62 positive glial inclusions were found in both cortex and spinal cord.

The three cases showed numerous p62 and TDP-43 glial positive inclusions in spinal cord. No p62 or TDP-43 inclusion was found in the granular cells of the cerebellum for the three patients.

To determine whether there were differences in ubiquitinated inclusion pathology in cases with or without SQSTM1 variants, we examined spinal cord tissues from 45 ALS patients including the 3 SALS patients carrying SQSTM1 variants (p.Gly351_Pro388del, p.Met87Val and p.Lys102Glu corresponding to cases 2, 3 and 4, respectively), 8 patients with C9ORF72 repeat expansion, 4 patients with SOD1 mutations, 2 patients with TARDBP mutations and 28 other patients devoid of mutation in any of these genes. Several types of ubiquitinated inclusions (stained with p62 and TDP-43) were quantified for these patients (Supplementary Table 1): skein like filamentous inclusions (Fig. 3e-f), large round cytoplasmic inclusions (Fig. 3h-i) and intracytoplasmic diffuse granular aggregates (Fig. 3c). The score means obtained for large round inclusions were compared between patients carrying SQSTM1, C9ORF72, SOD1 mutations and other patients with no mutation in any of these genes using ANOVA. The patient group with mutation in TARDBP was not included in this analysis since we only had n=2 patients. One way ANOVA analyses between the 4 groups of patients revealed that the p62 round inclusion pathology scores were different (p=0.041). Tukey's post hoc test analysis for comparison of the 4 groups by pairs showed that p62 positive rounded inclusions were more numerous in ALS patients with SQSTM1 variant than in patients with no mutation in the screened genes (Tukey's post hoc test, p=0.03) but were not different between SQSTM1 mutation carriers and patients with mutation in another gene

Teyssou et al. ActaNeuropathol 2013 AcceptedManuscript

(SOD1 or C9ORF72). The lack of statistically significant difference between SQSTM1 mutation carriers and patients with C9ORF72 and SOD1 mutation could be due to too small sample sizes. Indeed, there was a significant difference (Mann Whitney's test, p=0.045) between patients with SQSTM1 patients and all the other patients pooled together (devoid of mutation in any of the screened genes and carrying a mutation in SOD1 or C9ORF72). The number of rounded inclusions stained for TDP-43 was not statistically different between the 4 groups of patients (p=0.08). These semiquantitative differences in the numbers of p62 versus TDP-43-positive inclusions may simply reflect differences in the sensitivity of the antibodies employed. Occurrence of filamentous inclusions positive for p62 (p=0.006) or TDP-43 (p=0.01) differed between the 4 groups of patients.

Less TDP-43 positive filamentous inclusion was detected in the SOD1 group than in C9ORF72 (p=0.02) or SQSTM1 (p=0.02) patients which is in accordance with the absence of TDP-43 immunoreactivity previously reported for patients with SOD1 mutation [START_REF] Mackenzie | Pathological TDP-43 distinguishes sporadic amyotrophic lateral sclerosis from amyotrophic lateral sclerosis with SOD1 mutations[END_REF]. There was no difference in the incidence of granular inclusions stained by p62 or TDP-43 antibodies for the four groups of patients.

Discussion

Our SQSTM1 genetic analysis identified 4 variants corresponding to a frequency of 1.1% in FALS and 4% in SALS which is similar to the frequencies previously reported in FALS (1.7%) and SALS (3-4.4%) [START_REF] Fecto | SQSTM1 mutations in familial and sporadic amyotrophic lateral sclerosis[END_REF][START_REF] Hirano | Mutations in the gene encoding p62 in Japanese patients with amyotrophic lateral sclerosis[END_REF][START_REF] Rubino | SQSTM1 mutations in frontotemporal lobar degeneration and amyotrophic lateral sclerosis[END_REF].

We identified two novel SQSTM1 mutations, p.Met87Val and p.Lys102Glu, which are possibly pathogenic. The mutations that we identified in intron 7 and exon 8 have already been described in patients with ALS [START_REF] Fecto | SQSTM1 mutations in familial and sporadic amyotrophic lateral sclerosis[END_REF] or Paget's disease of bones (PDB) [START_REF] Collet | Paget's disease of bone in the French population: novel SQSTM1 mutations, functional analysis, and genotype-phenotype correlations[END_REF][START_REF] Michou | Genetics of Paget's disease of bone[END_REF][START_REF] Michou | Epidemiogenetic study of French families with Paget's disease of bone[END_REF]. PDB is a chronic disease of the skeleton due to focal increased bone turnover affecting 2-3% of individuals older than 55 and 6-7% of individuals over 85 years of age in Caucasian populations [START_REF] Cooper | The epidemiology of Paget's disease in Britain: is the prevalence decreasing?[END_REF]. Manifestations of the disease include bone pain, enlargement, and deformities at the pagetic sites, susceptibility to fractures, deafness and neurologic complications [START_REF] Hamdy | Clinical features and pharmacologic treatment of Paget's disease[END_REF]. Recent studies have confirmed that both genetic and environmental factors are implicated in its etiology [START_REF] Goode | Recent advances in understanding the molecular basis of Paget disease of bone[END_REF]. Seven loci (PDB1 to 7) have been identified and mutations in SQSTM1 gene, mostly located in the c terminus region of the protein encoded by exon 7 and 8, containing the highly conserved ubiquitin binding domain (Fig. 1c), are responsible for the phenotype linked to chromosome 5q35 (PDB3). These UBA domain mutations impair the ability of p62 to bind to ubiquitin [START_REF] Cavey | Loss of ubiquitin binding is a unifying mechanism by which mutations of SQSTM1 cause Paget's disease of bone[END_REF], probably resulting in aberrant NF-B signaling [START_REF] Goode | Recent advances in understanding the molecular basis of Paget disease of bone[END_REF].

In France, SQSTM1 mutations represent 13% of PDB patients [START_REF] Collet | Paget's disease of bone in the French population: novel SQSTM1 mutations, functional analysis, and genotype-phenotype correlations[END_REF] corresponding to an estimated frequency of 0.4% for the overall French population. This estimation is in accordance with the single variant we found Teyssou et al. ActaNeuropathol 2013 AcceptedManuscript (c.1313C>T, p.Prol438Leu) in one out of 360 control patients (mutation frequency of 0.3%) after sequencing SQSTM1 exon 7 and 8. However, as shown in both previous studies that have analyzed patients of Caucasian origin [START_REF] Fecto | SQSTM1 mutations in familial and sporadic amyotrophic lateral sclerosis[END_REF][START_REF] Rubino | SQSTM1 mutations in frontotemporal lobar degeneration and amyotrophic lateral sclerosis[END_REF] and now in our study, the frequency of this mutation in SQSTM1 is 10 fold higher in ALS patients therefore arguing in favor of a role of SQSTM1 as a causative or a susceptibility gene in ALS.

The c.1175C>T, p.Pro392Leu has been previously detected in 2/340 FALS and 1/206 SALS but 0/737 control patients [START_REF] Fecto | SQSTM1 mutations in familial and sporadic amyotrophic lateral sclerosis[END_REF]. It is the recurrent mutation identified in Paget's disease patients (PDB), responsible for 46% of familial and 16% of sporadic cases of PDB in the French Canadian population [START_REF] Laurin | Recurrent mutation of the gene encoding sequestosome 1 (SQSTM1/p62) in Paget disease of bone[END_REF]. In addition, mice carrying a proline to leucine mutation at codon 394 of mouse sqstm1 (P394L), corresponding to the P392L SQSTM1 mutation in humans, develop a disease similar to human PDB with focal increases in bone turnover and disruption of the normal bone architecture [START_REF] Daroszewska | A point mutation in the ubiquitin-associated domain of SQSMT1 is sufficient to cause a Paget's diseaselike disorder in mice[END_REF]. No obvious sign of motor neuron disease as impaired motor function or muscular atrophy has been described [START_REF] Daroszewska | A point mutation in the ubiquitin-associated domain of SQSMT1 is sufficient to cause a Paget's diseaselike disorder in mice[END_REF] although no study has assessed motor neuron numbers and shape in the spinal cord of this Paget's disease mouse model.

The c.1165+1G>A substitution in intron 7 affecting the exon 7 splicing was previously identified in a family of Australian descent with Paget's disease (it was called IVS7 + 1 G-A, A390X since it was thought to lead to the production of a truncated protein of 390 amino acids terminating at an inframe stop codon at position +6-9 within intron 7) [START_REF] Hocking | Domain-specific mutations in sequestosome 1 (SQSTM1) cause familial and sporadic Paget's disease[END_REF]. Our results show that this mutation led to the deletion of part of exon 7 in the mRNA sequence (r.1052_1165del) and the production of a truncated p.Gly351_Pro388del protein of 402 aminoacids in the spinal cord. Both pPro392Leu and p.Gly351_Pro388del mutations have been repeatedly identified in French patients with Paget's disease [START_REF] Michou | Epidemiogenetic study of French families with Paget's disease of bone[END_REF]. In contrast to the previously reported ALS patients with SQSTM1 mutations [START_REF] Fecto | SQSTM1 mutations in familial and sporadic amyotrophic lateral sclerosis[END_REF], both ALS patients with mutations in ubiquitin binding domain of SQSTM1 described here (i.e intron 7 for one SALS and exon 8 for one FALS) had concomitant PDB. However coexistence of these two disorders could be underestimated as PDB is often asymptomatic [START_REF] Hamdy | Clinical features and pharmacologic treatment of Paget's disease[END_REF] and its diagnosis is based on radiographic findings.

Coexistence of these two diseases has already been described in some patients with mutations in valosin containing protein (VCP) [START_REF] Johnson | Exome sequencing reveals VCP mutations as a cause of familial ALS[END_REF]. This gene has already been linked to Inclusion Body Myopathy with early-onset Paget's disease of the bone and Frontotemporal Dementia (IBMPFD) [START_REF] Watts | Inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia is caused by mutant valosin-containing protein[END_REF]. In addition, OPTN, another ALS causing gene [START_REF] Maruyama | Mutations of optineurin in amyotrophic lateral sclerosis[END_REF] is also known as a susceptibility gene for PDB [START_REF] Albagha | Genome-wide association study identifies variants at CSF1, OPTN and TNFRSF11A as genetic risk factors for Paget's disease of bone[END_REF]. Interestingly SQSTM1, VCP and OPTN all encode proteins that bind to ubiquitin [START_REF] Dai | Valosin-containing protein is a multi-ubiquitin chain-targeting factor required in ubiquitin-proteasome degradation[END_REF][START_REF] Gleason | Polyubiquitin binding to optineurin is required for optimal activation of TANK-binding kinase 1 and production of interferon beta[END_REF][START_REF] Vadlamudi | p62, a phosphotyrosine-independent ligand of the SH2 domain of p56lck, belongs to a new class of ubiquitin-binding proteins[END_REF]. These three genes that share common functions in protein degradation pathways could confer susceptibility to Paget's disease and motor neuron disease when mutated.

The clinical evaluation of bone integrity and the neuropathological assessment of motor neuron should be explored in ALS and PDB patients, respectively. In the present study, the observation of p62/TDP-43 positive Teyssou et al. ActaNeuropathol 2013 AcceptedManuscript inclusions in spinal motor neurons of case 2 (with c.1165+1G>A, r.1052_1165del, p.Gly351_Pro388del mutation) coupled to the accumulation of p62/TDP-43 proteins that could be detected in the spinal cord of this patient by immunoblot analysis strongly argues in favor of a participating role of this "PDB mutation" in ALS phenotype.

p62 positive inclusions are common in many degenerative diseases including tauopathies and synucleopathies [START_REF] Geetha | Sequestosome 1/p62: across diseases[END_REF][START_REF] Kuusisto | Ubiquitin-binding protein p62 is present in neuronal and glial inclusions in human tauopathies and synucleinopathies[END_REF] and a key features in neuropathological diagnosis and classification of ALS and FTLD, both sporadic and familial [START_REF] Mizuno | Immunoreactivities of p62, an ubiqutin-binding protein, in the spinal anterior horn cells of patients with amyotrophic lateral sclerosis[END_REF][START_REF] Pikkarainen | Neuropathologic features of frontotemporal lobar degeneration with ubiquitin-positive inclusions visualized with ubiquitin-binding protein p62 immunohistochemistry[END_REF]. The cortical pathology observed in the present study in ALS patients with SQSTM1 mutations (cases 2 and 4) confirms again the overlap between motor neuron disease and fronto-temporal lobar degeneration.

In contrast to the SQSTM1 Paget's disease linked mutations, those reported to date in ALS patients are distributed throughout the protein (Fig. 1c) suggesting that different domains of the p62 protein endowed with other functions, beside its ubiquitin binding one, could be impaired in ALS. Considering the implication of TDP-43 in ALS and since p62 has been shown to bind to TDP-43 (and to be potentially involved in its degradation), another p62 function that could be impaired by the ALS causing mutations would involve disruption in TDP-43 -p62 interaction, previously suggested to participate in the pathogenesis of TDP-43 proteinopathy [START_REF] Tanji | p62/sequestosome 1 binds to TDP-43 in brains with frontotemporal lobar degeneration with TDP-43 inclusions[END_REF].

Additional evidence have shown that overexpression of p62/SQSTM1 reduces TDP-43 aggregation in an autophagy and proteasome-dependent manner [START_REF] Brady | Regulation of TDP-43 aggregation by phosphorylation and p62/SQSTM1[END_REF]. Our immunoblot findings showing concomitant accumulation of p62 and TDP-43 proteins in spinal cord of SQSTM1 mutation carriers are in line with a shared pathological effect of these two proteins.

Data from p62 deleted mice showed that p62 loss of function results in increased ubiquitin staining in hippocampus and cortex together with accumulation of insoluble ubiquitinated proteins, of hyperphosphorylated tau and neurofibrillary tangles resulting in neurodegeneration and Alzheimer-like phenotype [START_REF] Babu | Genetic inactivation of p62 leads to accumulation of hyperphosphorylated tau and neurodegeneration[END_REF]. It is therefore tempting to hypothesize that a loss of a specific function of p62 protein due to mutations in a specific domain of SQSTM1 could also result in ALS phenotypes.

Previous studies have described that the profile of p62 neuropathology can predict the underlying genetic defect in ALS and FTLD [START_REF] Brettschneider | Pattern of ubiquilin pathology in ALS and FTLD indicates presence of C9ORF72 hexanucleotide expansion[END_REF]. However the number of p62 positive inclusions is not necessarily correlated to the number of TDP-43 aggregates (Al Sarraj et al., 2011). In FTLD-ALS with C9ORF72 mutations, neuronal inclusions are both TDP-43 and p62 positive, except in the cerebellum were inclusions are only p62 positive [START_REF] Al-Sarraj | p62 positive, TDP-43 negative, neuronal cytoplasmic and intranuclear inclusions in the cerebellum and hippocampus define the pathology of C9orf72-linked FTLD and MND/ALS[END_REF].

In patients with SQSTM1 mutations reported here, the granular deposit of TDP-43 and ubiquitin is not positive Teyssou et al. ActaNeuropathol 2013 AcceptedManuscript for p62. These results underline that the relationship between p62 and other accumulating proteins remains largely unknown.

Our study described two novel SQSTM1 mutations identified in ALS patients which are possibly pathogenic and provided neuropathological examination of three mutation carriers. The presence of large round p62 inclusions was consistently observed in these SQSTM1 patients. In mean, there was a higher number of these round p62 inclusions in spinal cord of SQSTM1 carriers compared to the other patients although they were rare in two of the SQSTM1 cases examined. Moreover increased protein levels of p62 and TDP-43 were detected in the spinal cord extracts of SQSTM1 patients. These observations support that these SQSTM1 mutant probably contribute to the ALS phenotype. Overall our study confirmed that SQSTM1 gene mutations could be the cause or a genetic susceptibility factor of the disease in some ALS patients. previously reported in Paget's disease (green), ALS (black) and FTLD (orange). Mutations reported in the present study are indicated in red. Data are compiled from [START_REF] Falchetti | Genetic epidemiology of Paget's disease of bone in italy: sequestosome1/p62 gene mutational test and haplotype analysis at 5q35 in a large representative series of sporadic and familial Italian cases of Paget's disease of bone[END_REF][START_REF] Fecto | SQSTM1 mutations in familial and sporadic amyotrophic lateral sclerosis[END_REF][START_REF] Goode | Recent advances in understanding the molecular basis of Paget disease of bone[END_REF][START_REF] Hirano | Mutations in the gene encoding p62 in Japanese patients with amyotrophic lateral sclerosis[END_REF][START_REF] Michou | Genetics of Paget's disease of bone[END_REF][START_REF] Rubino | SQSTM1 mutations in frontotemporal lobar degeneration and amyotrophic lateral sclerosis[END_REF]. SH2, src homology 2 domain;

Legends of the figures

43 and p62 positive

  skein like and rounded inclusions, numerous TDP-43 positive granules, and cystastin C positive Bunina bodies. A mild neuronal loss was observed in the substantia nigra, without Lewy bodies. No lesion was found at supratentorial level, except diffuse A beta deposits.In the female patient with K102E mutation (case 4), the brain weight was 1066g. No noticeable focal cerebral atrophy was found at macroscopic examination, contrasting with atrophy of anterior spinal roots. At microscopic examination, neuronal loss was mild in the spinal anterior horns and in the XII th nucleus, although Bunina bodies Teyssou et al. ActaNeuropathol 2013 AcceptedManuscript were quite numerous. Immunochemistry showed ubiquitin, TDP-43 and p62 positive rounded inclusions (Fig.

Figure 1 .

 1 Figure 1. SQSTM1 missense mutations identified in ALS patients.

Fig. 1 Teyssou

 1 Fig. 1

  Teyssou et al. ActaNeuropathol 2013AcceptedManuscript elsewhere for FALS[START_REF] Millecamps | Phenotype difference between ALS patients with expanded repeats in C9ORF72 and patients with mutations in other ALS-related genes[END_REF]. Autopsied SALS included 3 patients with C9ORF72 expansion, 2 with SOD1 and one with TARDBP mutation.Sequence of the primers and PCR conditions are available upon request.

	Immunoblotting. Transversal sections of spinal cords were homogenized in 50 mM Tris-HCl pH 8, 150 mM
	NaCl, protease inhibitors (Complete mini tablets, Roche) and 0.5 U/µl Benzonase endonuclease (Merck). The
	samples were incubated at 37°C during 30 minutes. SDS was then added at a final concentration of 2%. Tissue
	extracts were centrifuged at 13,000 rpm for 10 min and protein concentration of supernatants was estimated by
	the bichinconinic acid assay (Sigma Aldrich). Proteins (20 µg) were separated on NuPAGE TM 4-12% Bis-Tris
	Gel (Invitrogen) and electrophoretically transferred to nitrocellulose membranes. Membranes were incubated

overnight with primary antibodies in PBS/ 5% milk/ 0.1% Tween 20, followed by one hour of incubation with appropriate peroxidase-conjugated antibodies. Signals were detected using ECL plus Western Blotting Detection

Acknowledgments

We are grateful to the patients and their families. We thank the Généthon cell and DNA bank (Evry, France) and the CRicm DNA and cell bank (Paris, France) for patients' DNA and the genotyping and sequencing platform facilities of the ICM (Paris, France). This work was financed by the Association pour la Recherche sur la Sclérose latérale amyotrophique et autres maladies du motoneurone (ARSla, France), the Association française contre les myopathies (AFM, France) and the Fondation NRJ-Institut de France (France).

The authors declare that they have no conflict of interest.

Protocols were approved by the Medical Research Ethics Committee of "Assistance Publique-Hôpitaux de Paris" and all participants signed a consent form for the research.

AcceptedManuscript

AID, acidic interaction domain; ZZ, zinc finger region; TRAF6, tumor necrosis factor receptor-associated factor 6 binding domain; PEST, Proline, Glutamic acid, Serine, Threonine rich region; UBA, ubiquitin-associated domain. (d) Pedigree of the patient carrying the c.1175C>T, p.Pro392Leu mutation (case 1). DNA was available for the index cases only (indicated by an arrow). The age at onset, site of onset (in brackets) and age at death (in brackets) are indicated below the patients when the information was available. Amplification of the cDNA product with primers localized in exon 5 and 8 of the SQSTM1 gene resulting in a single fragment of 514 bp in the M87V carrier (case 3), the K102E carrier (case 4) and SALS individuals and in two fragments of 514 and 410 bp in the c.1165+1G>A carrier (Ex7, case 2) (c). Translation of this mRNA product results in a protein that lacks 38 aminoacids encoded by exon 7 (p.Gly351_Pro388del) that could be visualized on immunoblots (lane EX7, case 2, d). For this patient, expression levels of the full length p62 protein are decreased by half. Protein extracts from transversal sections of spinal cord were prepared from patients carrying the c.1165+1G>A (Ex7, case 2), the c.259A>G, p.Met87Val (M87V, case 3) and the c.304A>G, p.Lys102Glu (K102E, case 4) variants and three SALS with no genetic defect in SQSTM1. Protein levels of p62 and TDP-43 were compared in these patients. Samples were standardized using tubulin as internal control. Densitometric analyses of p62 (black) and TDP-43 (grey) protein levels. Data are means ± standard error of the means (s.e.m.) of 5 values (from 5 independent western blot analyses) for each patient. Values for control SALS patients were pooled in a control group. For case 2 with c.1165+1G>A variant (Ex7), p62 level is the sum of the two bands.

Figure 3. Ubiquitinated inclusions in the spinal cord of patients with mutation in SQSTM1.

Spinal cord sections were stained with anti-ubiquitin (a, d, g), anti-p62 (b, e, h) and anti-TDP-43 (c, f, i) antibodies. Skein like filamentous inclusions (d-f) and large round cytoplasmic inclusions (g-i) were detected with all three antibodies. Intracytoplasmic diffuse small granular aggregates were observed with ubiquitin (a) and TDP-43 (c) immunochemistry. This pattern was not found with p62 immunohistochemistry, in which bigger "seed-like", p62 positive aggregates were observed (b). Scale Bar: 10 µm. Double immunofluorescence analyses using p62 (in green) and TDP-43 (in red) antibodies were performed after deparaffinization using a procedure that was previously described [START_REF] Hernandez Lain | Abnormal TDP-43 and FUS proteins in muscles of sporadic IBM: similarities in a TARDBP-linked ALS patient[END_REF]. Antibodies were diluted at 1:100 (p62)

Teyssou et al. ActaNeuropathol 2013 AcceptedManuscript


Saad Zbiri
email: saad.zbiri3@uvsq.fr

Patrick Rozenberg

Carine Milcent
email: carine.milcent@psemail.eu

Home-based postnatal coordinated care after hospital discharge: a PRADO French experiment

Keywords: Postnatal care, Home-based coordinated care, Information, Health professional accessibility, Inequity

Objective: To determine the factors that affect enrollment and full participation (adherence) in the PRADO home-based postnatal coordinated care program in France after hospital discharge.

Methods: A population-based retrospective study was performed using the public health insurance database for the Yvelines district in France. The study population included all affiliated women admitted for delivery and classified as low risk in 2013. These women were eligible for home-based midwifery support after their discharge from the hospital. The enrollment and full participation of the women in home-based postnatal coordinated care were modeled using a simple probit model. Full participation in the home-based postnatal coordinated care was also modeled using a probit Heckman selection model in order to assess the self-selection process of enrollment in the program. The control variables were the characteristics of the patients, the municipalities, and the hospitals.

Results: 2,859 (68.3%) of the 4,189 eligible women chose to participate in the home-based postnatal coordinated care program, of whom 2,496 (59.6% of the eligible women) subsequently took part in the entire PRADO program. On the one hand, enrollment in the home-based postnatal coordinated care was influenced mostly by family context variables including the woman's age at the time of her pregnancy and the number of children in the household, the woman's level of information including prenatal education and prenatal information regarding postpartum care, as well as hospital variables including characteristics and organization of the maternity units. On the other hand, full participation in the homebased postnatal coordinated care was influenced by the accessibility to health professionals, particularly midwives. Furthermore, both the woman's level of information and accessibility to health professionals correlated with the socioeconomic environment.

Conclusion:

Women who become pregnant at a very early or late stage of their life as well as women with low levels of prenatal education and prenatal information regarding postpartum care have a relatively low rate of participation in home-based postnatal coordinated care. A public health policy promoting awareness of prenatal as well as postnatal issues could increase the participation in this coordinated community care. In addition, reducing regional inequality is likely to have a positive impact, as the availability of midwives is a key factor for participation in home-based postnatal coordinated care.

Introduction

The length of hospital stay following delivery has gradually decreased in most high-income countries over the past 40-50 years as a result of medical, economic, social, cultural, and political changes [START_REF] Cegolon | Length of stay following vaginal deliveries: A population based study in the Friuli Venezia Giulia region (North-Eastern Italy)[END_REF][START_REF] Cegolon | Length of stay following cesarean sections: A population based study in the Friuli Venezia Giulia region (North-Eastern Italy), 2005-2015[END_REF]. The main factors underlying this trend of reduced reliance on postnatal care in the hospital setting include increased efforts to reduce the risk of nosocomial infections, stress, and sleeping disorders for women and their newborns [START_REF] Campbell | Length of Stay After Childbirth in 92 Countries and Associated Factors in 30 Low-and Middle-Income Countries: Compilation of Reported Data and a Cross sectional Analysis from Nationally Representative Surveys[END_REF], to improve satisfaction during this period by supporting parental requests to return home soon after the childbirth [START_REF] Bravo | Early postnatal hospital discharge: the consequences of reducing length of stay for women and newborns[END_REF], and to limit the cost to the healthcare system [START_REF] Bowers | Reducing the length of postnatal hospital stay: implications for cost and quality of care[END_REF].

A consensus has not been reached to date regarding the appropriate length of postpartum stay in the hospital. The World Health Organization (WHO) recommends that all women should remain in the hospital at least 24 hours after childbirth. This recommendation is mainly intended for low-income countries, as it allows identification of any serious complications that require emergency care [START_REF]WHO recommendations on postnatal care of the mother and newborn[END_REF]. However, postpartum care is an important preventive support that should not be limited to the immediate postpartum period. It allows monitoring and treatment of complications in the mother and/or the newborn, the provision of support to facilitate the woman's transition to going home, counseling regarding infant feeding, parent education processes for newborn care, and health promotion indications [START_REF] Chalmers | WHO principles of perinatal care: the essential antenatal, perinatal, and postpartum care course[END_REF]. Although it varies considerably, the average length of the hospital stay for low-risk mothers and newborns recommended in several high-income countries is approximately three days [START_REF]Sortie de maternité après accouchement : conditions et organisation du retour à domicile des mères et de leurs nouveau-nés[END_REF][START_REF] Sellwood | Review of NICE guidelines on routine postnatal infant care[END_REF][START_REF] Grullon | The safety of early postpartum discharge: a review and critique[END_REF].

The current trend of healthcare services to encourage early hospital discharge of women following delivery has led to a number of concerns. These are centered on the notion that hospitals may not be providing sufficient appropriate services or infrastructure support to women as a result of this trend of early discharge from the hospital after childbirth. Reduction in the length of stay underscores the need for home-based postnatal coordinated care to support women who are discharged from the hospital before they have received adequate education regarding being a new parent. Several learned societies in the Western World have suggested that postnatal management should incorporate well-established home-based postnatal coordinated care after discharge from the hospital [START_REF]Sortie de maternité après accouchement : conditions et organisation du retour à domicile des mères et de leurs nouveau-nés[END_REF][START_REF] Sellwood | Review of NICE guidelines on routine postnatal infant care[END_REF][START_REF] Brown | Early postnatal discharge from hospital for healthy mothers and term infants[END_REF]. However, while hospital care is provided to all admitted patients, some women may end up not continuing with the postpartum coordinated care once they have been discharged.

Our study objective was to identify the determinants of the use, both in terms of enrollment and full participation (adherence), of home-based postnatal coordinated care after being discharged from the hospital. We focused not only on patient-level variables including household characteristics and prenatal and postnatal care characteristics but also on factors related to the hospitals and to the healthcare system including the accessibility to health professionals. Our study tested the hypothesis that specific patient, municipality, and/or hospital characteristics can impact the initial enrollment and/or full participation in homebased postnatal coordinated care. As a first step, we considered the initial enrollment in the program as a random process. As a second step, we considered a selection model where we first modeled the patient decision for enrollment in the program; Secondly, we identified the determinants impacting the women's full participation in the program.

Materials and methods

Data source

This was a French population-based retrospective study of women eligible for coordinated postnatal care after discharge from the hospital. Since 2010, the single-payer public health insurance system has provided home-based coordinated care to support women once they have been discharged from the hospital after having given birth. First, a public health insurance advisor at the maternity ward assists the woman with getting in touch with a midwife. If the woman is interested in participating in the home-based postpartum coordinated care program (PRADO), two visits are scheduled between day 0 (day of delivery) and day 7, and the first visit has to be on the first day after the woman's return home. The hospital discharge under the PRADO program depends on the approval of the maternity medical team (doctor or midwife).

The data were from three linked administrative datasets for the year 2013. The main dataset was extracted from the public health insurance dataset of the Yvelines district, which contains all of the insured people of the district (100% of the population). The data comprised the information regarding the women's individual demographic, socioeconomic, and location data, as well as the characteristics of the prenatal and the postnatal care of the women, and information regarding the hospital of admission. The second dataset, compiled by the French National Institute of Statistics, was extracted from population census data and it provided information regarding the socioeconomic situation of all French municipalities. The third dataset, compiled by the Ministry of Health, provided information regarding the accessibility to health professionals for all French municipalities. The three datasets were merged using the denomination and the code of the municipally of residence of the women.

Study population

The study population consisted of all women affiliated with the public health insurance agency of the Yvelines district and who were eligible for home-based postnatal coordinated care (N = 4,189). To be eligible, women had to be at low clinical risk and admitted to a hospital for delivery: women aged 18 years and older, without any co-morbidity or complications, giving birth by vaginal delivery at full term to a singleton infant not requiring maintenance in a medical environment nor a particular feeding mode.

Study variables

Our variables included individual characteristics of the women including characteristics of their household and of their prenatal and postnatal care, municipality characteristics, and the hospital characteristics and organization.

The patient-level variables considered were age (five age brackets: 18 to 23 years, 24 to 29 years, 30 to 35 years, 36 to 41 years, and 42 years or older), the number of children in the household (four categories: 1, 2, 3, and 4 children or more), the type of healthcare coverage (two categories: policyholder and person eligible for benefits, referred to here as the beneficiary), the number of antenatal visits (three categories: 0 to 5, 6 or 7, and 8 visits or more), prenatal follow-up by a gynecologist (two categories: yes and no, "yes" if at least one antenatal visit), prenatal follow-up by a general practitioner (two categories: yes and no, "yes" if at least one antenatal visit), prenatal follow-up by a midwife (two categories: yes and no, "yes" if at least one antenatal visit), prenatal hospital follow-up (two categories: yes and no, "yes" if at least one antenatal visit), prenatal community follow-up (two categories: yes and no, "yes" if at least one antenatal visit), the number of obstetric ultrasounds (three categories: 0 to 1, 2 or 3, and 4 ultrasounds or more), prenatal education (two categories: yes and no), prenatal information regarding postnatal care (two categories: yes and no), and postnatal hospital readmission of the woman or the newborn until day 7 (two categories: yes and no).

The municipality-level variables considered were the location of the municipality (urban, rural), household deprivation as assessed by the median annual income (four categories at regular intervals based on the variable values and not the variable distribution in the population: least deprived (from 37,300 to 46,100 euros), less deprived (from 28,500 to 37,299 euros), more deprived (from 19,700 to 28,499 euros), and most deprived (from 10,900 to 19,699 euros), accessibility to a gynecologist as assessed by the index of spatial accessibility (ISA) (four categories at regular intervals based on the variable values: lowest (1 to 4.7), low (4.8 to 8.5), high (8.6 to 12.3), and highest (12.4 to 16)), accessibility to a general practitioner as assessed by the ISA (four categories at regular intervals based on the variable values: lowest (7 to 31.9), low (32 to 56.9), high (57 to 81.9), and highest (82 to 106)), and accessibility to a midwife as assessed by the ISA (four categories at regular intervals based on the variable values: lowest (1 to 2.4), low (2.5 to 3.9), high (4 to 5.4), and highest (5.5 to 7)).

The hospital-level variables considered were funding of the hospital (two categories: public and private), hospital university status (two categories: teaching and non-teaching), level of care of the maternity ward (three categories: no neonatology unit, neonatology unit, and neonatal intensive care unit), staffing levels for obstetricians as measured by the number of obstetricians in full-time equivalents (FTEs) per 100 deliveries, staffing levels for midwives as measured by the number of midwives in FTEs per 100 deliveries, the day of delivery (two categories: working day and non-working day), and the day of hospital discharge (two categories: working day and non-working day).

The index of spatial accessibility (ISA) used here was developed by the French Ministry of

Health (indicateur d'accessibilité potentielle localisée (APL))

. This index provides a measure of the spatial adequacy between supply and demand for primary care at a local level. It aims to improve the usual indicators of accessibility to care. It is captured, on the supply side, by the density of health professionals while taking into account their practice rates in the health area and, on the demand side, by the geographical distance between patients and the professionals in the zone defined above while considering differences in patient age groups and in the observed rates of attendance by professionals [START_REF] Vergier | Déserts médicaux : comment les définir ? Comment les mesurer ? Les Dossiers de la Drees[END_REF]. This index is based on the "twostep floating catchment area" method and it is interpreted as a density [START_REF] Luo | An enhanced two-step floating catchment area (E2SFCA) method for measuring spatial accessibility to primary care physicians[END_REF].

Statistical analysis

We studied the main factors that affect the use of home-based postnatal coordinated care. As a first model, we assessed the probability of choosing to participate in home-based postnatal coordinated care. Using a probit regression, we estimated the probability of the initial enrollment in the PRADO program for all of the eligible women as a function of the household and prenatal care characteristics, the municipality characteristics, as well as the hospital characteristics and organization. Enrollment was defined as a dummy variable equal to 1 with enrollment in the PRADO program and equal to 0 when the woman did not wish to participate in the PRADO program.

We then estimated the probability of full participation in home-based postnatal coordinated care. Full participation in the coordinated program was defined as agreeing to partake in the two home-based postnatal visits, with the first visit in the first 24 hours after being discharged. Full participation was defined as a dummy variable equal to 1 with full participation in the PRADO program and equal to 0 when the woman did not partake in the entire PRADO program. Only one woman was available for the first visit for the program but then declined further participation. As the independent variables, we considered a vector of explanatory variables with household characteristics, prenatal care variables, postnatal hospital readmission, and the municipality characteristics including indicators of the accessibility to health professionals. First, we studied the population of women enrolled in this postpartum care program. In the sample, 68% of the patients eligible for this program decided to participate. We thus estimated a probit regression for the probability of full participation in the PRADO program (second model). The results obtained here were conditional on the decision to participate. In case the selection process to enroll or not to enroll was non-exogenous, we used a Heckman probit model to assess the probability of full participation in the PRADO program (third model) [START_REF] Heckman | Sample selection bias as a specification error[END_REF]. We assumed that the hospital characteristics affect the decision to participate but that they do not affect full participation once the woman has decided to participate in the program [START_REF] Puhani | The Heckman correction for sample selection and its critique[END_REF].

Finally, when relevant, we measured the correlation between any determinant factor(s) that significantly impacted the initial enrollment and/or full participation in the home-based postnatal care program and the socioeconomic environment as measured by the median annual income of the municipality.

All probit models were performed using the cluster-robust variance that accounted for the dependence between the observations at the municipality level. The results are reported as coefficients with their 95% confidence intervals (CIs). The significance levels are two-sided with a probability threshold of p < 0.05. The analyses were performed using Stata software (Stata Corporation, College Station, TX, USA) [START_REF] Statacorp | Stata Statistical Software: Release[END_REF].

Ethics statement

The extraction and analysis of the data for scientific research were conducted with permission from the public health insurance agency of the Yvelines district, which is the entity responsible for this data. Informed consent was not required as the study was based on routinely collected de-identified administrative data, as regulated by French law. The data used for this study were reported to the National Data Protection Authority.

Results

A total of 4,189 eligible women were included in the analysis. Of these, 2,859 women chose to participate in the home-based postnatal coordinated care after their discharge from the hospital, thus amounting to 68.3% of the total (95% CI [66.8;69.6]). Of the women who opted for the home-based postnatal coordinated care, 2,496 participated in the full PRADO program, thus amounting to 87.3% (95% CI [86.0;88.5]) of the women who chose to participate and 59.6% (95% CI [58.1;61.1]) of the total number of eligible women. Table 1 presents the descriptive statistics.

Table 2 shows the determinants of the initial enrollment in the home-based postnatal coordinated care (PRADO) for all of the eligible women. The results are reported in terms of coefficients. We found several groups of observable factors that were significantly associated with enrollment in the home-based coordinated postnatal care program. The first group of factors was the household characteristics: women with a pregnancy at a very early or late stage of their life, those having three children or more, and women who were not health insurance policyholder but were eligible for benefits (referred to here as a beneficiary) had a lower probability of enrollment in the PRADO program. These variables, especially healthcare coverage variable, may capture socioeconomic aspects such as occupation or employment status. The second group of factors was the prenatal and postnatal information collected: participation in a prenatal education program and participation in a communication program for prenatal information on postpartum care. These programs aimed at providing information are organized and presented by the medical staff during the pregnancy. We found that the women who attended a prenatal education program and those who had received information regarding postnatal care were more likely to enroll in the PRADO program. The third group of factors was the hospital unit characteristics: women admitted to private hospitals, non-teaching hospitals, those with a high level of care (based on the presence of a specific neonatology care unit), and those with a high level of staffing of both obstetricians and midwives (as measured by the FTE appointments per 100 deliveries over the average) had a relatively low probability of enrollment in the program. Furthermore, women who delivered on non-working days and those who were discharged on working days were more likely to enroll in the PRADO program.

These results highlight the conditions that affect the initial enrollment of women in the PRADO program. As a result, the conditions for home-based postnatal coordinated care enrollment were: 1-the household's intrinsic characteristics, 2-the extent to which the woman had been informed regarding the delivery and the post-delivery period, 3-the hospital characteristics and organization.

The impact of accessibility to a gynecologist was only observed for women living in areas with a high level of accessibility to a gynecologist. This variable may capture other aspects of the women or the municipality.

Table 3 provides the probit model estimates of the full participation in the home-based postnatal coordinated care for the sample of women who opted for the PRADO program. The results are reported in terms of coefficients. A prenatal education program impacted the probability of full participation. Indeed, the women who participated in a prenatal education program were more likely to fully participate in the postnatal coordinated program. As expected, hospital readmission up to 7 days after childbirth decreased the probability of full participation. In terms of factors related to the healthcare system, we found that the accessibility to midwives impacted the probability of full participation in the PRADO program. Using the index of spatial accessibility (ISA), we observed that increased accessibility to a midwife increased the probability of full participation. Accessibility to a gynecologist and accessibility to a general practitioner were not associated with any change in the PRADO program follow-up. Unexpectedly, although the number of children in the household affected full participation in the program before adjustment on other variables, we found that the number of children did not affect full participation in the program after controlling for healthcare accessibility and the prenatal education program. As robustness checks, presented in Table A1 in the Appendix, we also included the hospital characteristics variables and hospital organization variables, and we found that the characteristics and the organization of the hospitals were not associated with the women's full participation in the home-based postnatal coordinated care.

Thus far, we assumed that the enrollment in the PRADO program was a random process. We then considered the self-selection bias in the decision to participate in the PRADO program.

Table 4 shows the results regarding the probability of full participation in the home-based postnatal coordinated care program for all of the eligible women when taking into account this potential self-selection. We thus regressed a probit Heckman selection model. The results are reported as coefficients. Taking into account the initial enrollment of women in the PRADO program, we controlled for the same entire set of observable characteristics as used before. The impact of the accessibility to a midwife on full participation in the home-based postnatal coordinated care program remained statistically significant, that is to say, women with a higher level of accessibility to a midwife were more likely to fully participate in the PRADO program. Moreover, the effect of postnatal readmission to the hospital during the first seven days was still significant. However, the effect of a prenatal education program was no longer significant after taking into account the self-selection bias. The results of the selection equation are reported in Table A2 in the Appendix. The independent significant variables of the selection equation were quite similar to what was found when the probability of PRADO program enrollment was estimated without accounting for self-selection (Table 2).

We did not interpret Rho because its nature is extremely sensitive to model specification.

Thus, these results show that a number of patient-, municipality-and hospital-level factors impacted the use of the home-based postnatal coordinated care program (PRADO).

We also computed the correlation between the woman's level of information and the socioeconomic environment (measured as the median annual income in the municipality). The correlation was significantly positive for both prenatal education and prenatal information regarding postpartum care (coefficient = 0.17, p < 0.001, and coefficient = 0.13, p < 0.001, respectively). Thus, the higher the median annual income in the municipality, the higher the woman's level of information. We also estimated the correlation between the accessibility to a midwife and the socioeconomic environment on the subsample of all of the women's municipalities (n = 279) . The correlation was significantly positive (coefficient = 0.35, p < 0.001). Thus, the higher the median annual income, the greater the accessibility to a midwife. As a sensitivity analysis, we also computed the same correlation at the national level using all French municipalities. The results remained unchanged (Table A3 in the Appendix).

Discussion

Key findings

Of the total pool of eligible low-risk women giving birth in hospitals in the Yvelines district, 59.6% of them enrolled and subsequently fully participated in the home-based postnatal coordinated care program after discharge. Controlling for the full set of independent factors, the household's intrinsic characteristics, the woman's level of information, and the hospital characteristics and organization impacted enrollment in the PRADO program, while accessibility to health professionals impacted full participation in the PRADO program.

Furthermore, the level of information and accessibility to healthcare professionals correlated with a higher socioeconomic environment.

Strengths and limitations

To the best of our knowledge, this study is the first research analysis of access to the PRADO program. On the one hand, our study has many strengths. First, we had access to a large dataset of women eligible for the home-based PRADO program and who had given birth in different hospitals of the Yvelines district. Also, the dataset used for the analysis was checked by the statistics department of the public health insurance of the Yvelines district. Notably, it did not include any missing values for the variables considered in the study. Both the large scale and the high quality of the data allowed us to obtain reliable and accurate results.

Secondly, we used many available variables, which enabled us to consider different characteristics of the women throughout their maternity trajectories at the patient, municipality, and hospital levels. This reduced the risk of confusion bias. Thirdly, we considered the entire population of eligible women with respect to the postnatal coordinated care rather than just the subset of women who chose to participate in the PRADO program. This allowed us to address the possible individual self-selection in enrollment in this program, as we employed a Heckman selection model to manage self-selection bias. Fourthly, in order to estimate the accessibility to primary health professionals, we used the index of spatial accessibility (ISA), which made it possible to simultaneously consider healthcare supply, demand, and access. Indeed, this new indicator is more accurate than the usual indices of healthcare accessibility such as access time, access distance, and density by living area including health professional population ratios.

On the other hand, our analysis could also have some limitations. The study involved women affiliated with a French local public health insurance system. However, the single-payer structure of the French healthcare system (100% of the population) eliminated the potential for program choice to be dictated by the type of health insurance coverage and thus provided a good setting in which to carry out this study. Moreover, the Yvelines district includes differences in terms of socioeconomic environment and accessibility to health professionals [17]. The diversity of geographic conditions covered by the Yvelines district supports the representativeness of the study and, therefore, the transferability of our results to the French situation. Finally, we used the index of spatial accessibility (ISA) produced by the French Ministry of Health. This indicator is estimated at the municipality level. However, this multidimensional index is assessed using different parameters of healthcare supply and demand that allows disparities in access to healthcare to be captured that a standard density indicator will tend to mask [START_REF] Gao | Assessment of the spatial accessibility to health professionals at French census block level[END_REF].

Interpretation

The first finding of our study was that only a portion of the women eligible for the homebased postnatal coordinated care decided to enroll and fully participated in this program after their hospital discharge. During the hospital stay, similar healthcare services are offered to all patients. However, once they have been discharged from the hospital, the patient is expected to be active and responsible for their health. They should, therefore, understand the medical recommendations and organize their care pathway accordingly. This raises the role of the patient and their environment in the use of ambulatory healthcare services [START_REF] Menichetti | Giving patients a starring role in their own care: a bibliometric analysis of the on-going literature debate[END_REF].

This observation led us to consider why some patients did not use the recommended homebased postnatal coordinated care. We sought to identify the determinant factors that explain both the initial enrollment and full participation in this postpartum care program. We considered a wide range of different patient, area-based, and hospital variables, and we found that the household's intrinsic characteristics including demographic and family-context variables, the woman's level of information including prenatal education and prenatal information regarding postpartum care, and hospital variables including characteristics and organization of maternity units impacted enrollment in the PRADO program, while we found that accessibility to primary healthcare professionals, particularly accessibility to a midwife, impacted full participation in the PRADO program. Notably, pregnancy in middle age, a low number of children, a high level of information, and some hospital characteristics increased enrollment in the home-based postnatal coordinated care program, while greater accessibility to a midwife increased full participation in the program. A number of previous studies have reported the same findings for different community medical care services. A US study has observed substantial differences in diagnostic practices across regions that could be explained by patient characteristics but that could also be related to local conditions [START_REF] Song | Regional variations in diagnostic practices[END_REF]. A German study has found that differences in demography and the supply of services explained part of the variation in the utilization of ambulatory services for general practitioner, specialist, and psychotherapist consultations [START_REF] Kopetsch | Regional variation in the utilisation of ambulatory services in Germany[END_REF]. A Swedish study has indicated that demography could explain a considerable part of the regional variation in visits to outpatient specialists, while basic supply-side factors, including the proportion of providers and physicians per inhabitants, could explain part of the regional variation in primary physician visits [START_REF] Johansson | Regional variation in health care utilization in Sweden -the importance of demand-side factors[END_REF].

Another US study found that patient information regarding integrated care was associated with outpatient utilization but not inpatient utilization [START_REF] Fryer | patient perceptions of integrated care and their relationship to utilization of emergency, inpatient and outpatient services[END_REF]. A German study has reported that cancer-screening services were significantly higher in areas with higher physician density [START_REF] Vogt | Examining regional variation in the use of cancer screening in Germany[END_REF]. Another German study found that supply-side factors accounted for most of the regional inequities in the actual utilization of outpatient health services [START_REF] Ozegowski | Understanding the gap between need and utilization in outpatient care--the effect of supply-side determinants on regional inequities[END_REF].

Finally, we observed that the woman's level of information and the area-based accessibility to professionals correlated with the socioeconomic environment. That is to say, a higher level of information and accessibility to a midwife correlated with a higher socioeconomic environment. Past analyses have shown that socioeconomic factors impact women's participation in prenatal education during their maternity pathway [START_REF] Milcent | Prenatal care and socioeconomic status: effect on cesarean delivery[END_REF]. Furthermore, a number of recent studies have reported a relationship between local socioeconomic conditions, the distribution of healthcare professionals, and the efficiency of healthcare provision [START_REF] Schley | Health care service provision in Europe and regional diversity: a stochastic metafrontier approach[END_REF][START_REF] Herwartz | Improving health care service provision by adapting to regional diversity: An efficiency analysis for the case of Germany[END_REF]. The French healthcare system, although it has a high level of favorable health outcomes and patient satisfaction, still suffers from socioeconomic, geographic, and cultural health inequalities, including iniquity in terms of access to relevant health information and to healthcare professionals, which could both be major barriers to health equity [START_REF] Chevreul | France: Health System Review[END_REF]. This is also the current situation in many other developed countries [START_REF] Pietro | Switzerland: Health System Review[END_REF][START_REF] Kroneman | Netherlands: Health System Review[END_REF]. Moreover, the issue of socioeconomic disparities is the focus of much attention in reproductive health and still needs to be addressed more thoroughly [START_REF] Milcent | Prenatal care and socioeconomic status: effect on cesarean delivery[END_REF].

Policy implication and prospects

In recent years, ambulatory care has been promoted as an effective healthcare service that could replace unnecessary hospital stay care, which should only be reserved for patients requiring continuous medical surveillance. This ambulatory shift has accentuated the need for adequate investment in patient empowerment and community infrastructure to ensure continuity of care.

The results of our study may have a number of public policy implications. A straightforward implication would be that health policies promoting the level of information of patients or accessibility to primary health professionals, which are possibly editable components of the healthcare system, could lead to better follow-up of community care, especially after hospital discharge. Furthermore, since these two factors differ across different socioeconomic environments, patient and spatial needs along with social characteristics should be considered in order to improve the allocation of medical services.

The French public insurance system recently implemented financial incentives to promote the appointment and retention of independent health professionals, including midwives, in areas where the supply of care is insufficient or the access to care is difficult [START_REF] Bowers | Continuity of care in community midwifery[END_REF]. Future research should study the impact of such experimentations on the use of outpatient care. Moreover, other interventions that may enhance access to healthcare professionals should be investigated. For instance, in the National Health Service, one study on home-based postnatal care that involved a series of home visits reported that small increases in travel time of midwives boosted continuity of care and, therefore, access to community midwifery services [START_REF]Convention nationale des sages-femmes et ses avenants[END_REF]. Financial incentives can also be used to promote the degree to which women are informed. As an example, the financial aid usually allocated to future parents to prepare for the arrival of their child, which is more important for the socially disadvantaged population, could be modulated according to the degree of the woman's use of prenatal care.

Conclusion

In conclusion, enrollment in the PRADO home-based postnatal coordinated care program was determined primarily by the household intrinsic characteristics, the woman's level of information, and hospital characteristics and organization; while full participation (adherence) in the program was determined primarily by the accessibility to health professionals. These findings emphasize the importance of improving the level of information and the accessibility to professionals, which are both lower for less affluent women, as potential ways to get more women to use community postnatal care. Future high-quality studies are needed to evaluate the effect of interventions that increase patient empowerment or healthcare access on postnatal care use.

Tables

Table 1 .

 1 Descriptive characteristics of the study population.

	Women opting to participate	All of the women eligible for
	in the PRADO program	the PRADO program
		(n = 2,859)	(n = 4,189)
	Household characteristics
	Woman's age at pregnancy (years), n (%)
	18 -23	176 (6.16)	312 (7.45)
	24 -29	978 (34.21)	1,413 (33.73)
	30 -35	1,196 (41.83)	1,711 (40.85)
	36 -41	473 (16.54)	685 (16.35)
	≥ 42	36 (1.26)	68 (1.62)
	Number of children, n (%)
	1	1,720 (60.16)	2,348 (56.05)
	2	713 (24.94)	1,056 (25.21)
	3	286 (10.00)	518 (12.37)
	≥ 4	140 (4.90)	267 (6.37)
	Woman's healthcare coverage, n (%)
	Policyholder	2,625 (91.82)	3,751 (89.54)
	Beneficiary	234 (8.18)	438 (10.46)
	Prenatal care
	Antenatal visits, n (%)
	0 -5	769 (26.90)	1,205 (28.77)
	6 -7	666 (23.29)	974 (23.25)
	≥ 8	1,424 (49.81)	2,010 (47.98)
	Follow-up by a gynecologist a , n (%)
	No	765 (26.76)	1,156 (27.60)
	Yes	2,094 (73.2)	3,033 (72.40)
	Follow-up by a general practitioner a , n (%)
	No	835 (29.21)	1,256 (29.98)
	Yes	2,024 (70.79)	2,933 (70.02)
	Follow-up by a midwife a , n (%)
	No	1,257 (43.97)	1,907 (45.52)
	Yes	1,602 (56.03)	2,282 (54.48)
	Hospital follow-up a , n (%)
	No	898 (31.41)	1,309 (31.25)
	Yes	1,961 (68.59)	2,880 (68.75)
	Community follow-up a , n (%)
	No	388 (13.57)	605 (14.44)
	Yes	2,471 (86.43)	3,584 (85.56)
	Obstetric ultrasound, n (%)
	0 -1	761 (26.62)	1,213 (28.96)
	2 -3	1,346 (47.08)	1,913 (45.67)
	≥ 4	752 (26.30)	1,063 (25.38)
	Prenatal education, n (%)
	No	1,583 (55.37)	2,631 (62.81)
	Yes	1,276 (44.63)	1,558 (37.19)
	Prenatal information regarding postpartum, n (%)
	No	2,416 (84.51)	3,709 (88.54)
	Yes	443 (15.49)	480 (11.46)
	Postnatal care
	Hospital readmission, n (%)
	No	2,811 (98.32)	4,123 (98.42)
	Yes	48 (1.68)	66 (1.58)
	Municipality characteristics
	Location, n (%)
	Urban	2,647 (92.58)	3,890 (92.86)

a At least one antenatal visit b Based on the median annual income c Based on the index of spatial accessibility (ISA) d FTEs (full-time equivalents) per 100 deliveries

Table 2 .

 2 Factors associated with initial enrollment in the home-based postnatal coordinated care (PRADO) for all of the women eligible for this program. Probit regression model (n = 4,189). : p < 0.05; ** : p < 0.01; *** : p < 0.001 a At least one antenatal visit b Based on the median annual income c Based on the index of spatial accessibility (ISA) d FTEs (full-time equivalents) per 100 deliveries

	More deprived	0.09	[-0.24;0.42]
	Most deprived	-0.13	[-0.50;0.24]
	Accessibility to a gynecologist c
	Lowest	reference
	Low	-0.21	[-0.45;0.03]
	High	-0.29	[-0.66;0.09]
	Highest	0.04	[-0.51;0.60]
	Accessibility to a general practitioner c
	Lowest	reference
	Low	0.26	[-0.15;0.66]
	High	0.18	[-0.26;0.61]
	Highest	0.38	[-0.12;0.88]
	Accessibility to a midwife c
	Lowest	reference
	Low	0.58 ***	[0.27;0.90]
	High	0.62 ***	[0.25;0.98]
	Highest	0.72 **	[0.21;1.24]
	CI, confidence interval.

*

Table A3 .

 A3 Correlation between accessibility to health professionals and the socioeconomic environment. All French municipalities (n = 32,948). : p < 0.05; ** : p < 0.01; *** : p < 0.001 a Based on the index of spatial accessibility (ISA)

		Accessibility to	Accessibility to	Accessibility to
		a gynecologist a	a general practitioner a a midwife a
	Socioeconomic environment
	Household median annual income	0.39 ***	0.02 ***	0.12 ***

*

Funding:

The authors have no support or funding to report.

Data Availability Statement:

The data were not generated by the authors but are compiled, maintained, and distributed by the public health insurance agency of the Yvelines district. The authors confirm that access to the data is subject to certain restrictions, thus rendering some of the data not publicly available.

Author Contributions:

PR provided the data. CM and SZ analyzed the data. CM and SZ interpreted the results and wrote up the manuscript. CM, PR, and SZ read and reviewed the final manuscript.

Competing Interests:

The authors declare that there are no competing interests.

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