Mathilde Di Filippo
email: filippo@chu-lyon.fr

Hervé Créhalet
email: crehalet_herve@yahoo.fr

Marie Elisabeth Samson-Bouma
email: marie-elisabeth.samson-bouma@inserm.fr

Véronique Bonnet
email: veronique.bonnet@chu-lyon.fr

Lawrence P Aggerbeck

Jean-Pierre Rabès
email: rabèsjean-pierre.rabes@apr.aphp.fr

Frederic Gottrand
email: frederic.gottrand@chru-lille.fr

Gérald Luc

Dominique Bozon
email: dominique.bozon@chu-lyon.fr

Agnès Sassolas
email: agnes.sassolas@chu-lyon.fr

Mathilde Di

Filippo Mathilde Di-

Pcsk9 Proprotein

Molecular and functional analysis of two new MTTP gene mutations in an atypical case of abetalipoproteinemia

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

to determine the relationship between the genotype and this particular phenotype.

The subject exhibited compound heterozygosity for two novel MTTP mutations: one missense mutation (p.Leu435His) and an intronic deletion (c.619-5_619-2del). COS-1 cells expressing the missense mutant protein exhibited negligible levels of MTP activity. In contrast, the minigene splicing reporter assay showed an incomplete splicing defect of the intronic deletion with 26% of the normal splicing being maintained in the transfected HeLa cells.

The small amount of MTP activity resulting from the residual normal splicing in the patient explains the atypical phenotype observed. Our investigation provides an example of a functional analysis of unclassified variations, which is an absolute necessity for the molecular diagnosis of atypical ABL cases.

Supplementary key words: dyslipidemias, familial hypocholesterolemia, chylomicrons, lipoprotein/assembly, hepatosteatosis, genetics, gene expression, genotype-phenotype correlation, functional analysis, splicing Abetalipoproteinemia (ABL; OMIM#200100) is an autosomal recessive hypocholesterolemia usually detected during infancy due to failure to thrive, severe diarrhoea and a lipid malabsorption syndrome. Sixty years ago, Bassen and Kornzweig described this disease for the first time [START_REF] Bassen | Malformation of the erythrocytes in a case of atypical retinitis pigmentosa[END_REF] and forty years later, the role of MTP (microsomal triglyceride transfer protein large subunit) was discovered [START_REF] Wetterau | Absence of microsomal triglyceride transfer protein in individuals with abetalipoproteinemia[END_REF][START_REF] Sharp | Cloning and gene defects in microsomal triglyceride transfer protein associated with abetalipoproteinaemia[END_REF][START_REF] Shoulders | Abetalipoproteinemia is caused by defects of the gene encoding the 97 kDa subunit of a microsomal triglyceride transfer protein[END_REF][START_REF] Berriot-Varoqueaux | The role of the microsomal triglygeride transfer protein in abetalipoproteinemia[END_REF].

MTP forms a heterodimer with protein disulfide isomerase (PDI) also named prolyl 4hydroxylase beta polypeptide (P4HB) which is responsible of the assembly of apolipoprotein B (ApoB) containing lipoproteins in the liver and the intestine.

Following the discovery of the first mutations, many mutations have been identified in the microsomal triglyceride transfer protein large subunit gene (MTTP) in patients [START_REF] Sharp | Cloning and gene defects in microsomal triglyceride transfer protein associated with abetalipoproteinaemia[END_REF][START_REF] Shoulders | Abetalipoproteinemia is caused by defects of the gene encoding the 97 kDa subunit of a microsomal triglyceride transfer protein[END_REF][START_REF] Berriot-Varoqueaux | The role of the microsomal triglygeride transfer protein in abetalipoproteinemia[END_REF][START_REF] Narcisi | Mutations of the microsomal triglyceride-transfer-protein gene in abetalipoproteinemia[END_REF]. The identification of mutations in the MTTP gene in DNA from patients is important for establishing the diagnosis of ABL in the context of two other hereditary hypocholesterolemias (homozygous familial hypobetalipoproteinemia OMIM#107730 and Anderson's disease/Chylomicron retention disease, OMIM#246700) which are due to mutations in the APOB and SAR1B genes, respectively.

To date, mutations in the MTTP gene have been established in about 50 cases of ABL [START_REF] Zamel | Abetalipoproteinemia: two case reports and literature review[END_REF][START_REF] Chardon | Identification of two novel mutations and long-term follow-up in abetalipoproteinemia: a report of four cases[END_REF][START_REF] Najah | Identification of patients with abetalipoproteinemia and homozygous familial hypobetalipoproteinemia in Tunisia[END_REF][START_REF] Uslu | Abetalipoproteinemia in an infant with severe clinical phenotype and a novel mutation[END_REF][START_REF] Pons | A severe form of abetalipoproteinemia caused by new splicing mutations of microsomal triglyceride transfer protein[END_REF](12). In some instances, the prediction of altered MTP function is readily made on the basis of premature stop codons, mutations in canonical splice sites or frameshift mutations. In other cases, the functional consequences have been difficult to predict [START_REF] Ohashi | Novel mutations in the microsomal triglyceride transfer protein gene causing abetalipoproteinemia[END_REF][START_REF] Wang | Microsomal triglyceride transfer protein (MTP) gene mutations in Canadian subjects with abetalipoproteinemia[END_REF][START_REF] Al-Shali | Ileal adenocarcinoma in a mild phenotype of abetalipoproteinemia[END_REF], even if the mutations co-segregate with clinical phenotype.

The measurement of MTP activity [START_REF] Wetterau | Absence of microsomal triglyceride transfer protein in individuals with abetalipoproteinemia[END_REF][START_REF] Sharp | Cloning and gene defects in microsomal triglyceride transfer protein associated with abetalipoproteinaemia[END_REF][START_REF] Narcisi | Mutations of the microsomal triglyceride-transfer-protein gene in abetalipoproteinemia[END_REF][START_REF] Pons | A severe form of abetalipoproteinemia caused by new splicing mutations of microsomal triglyceride transfer protein[END_REF][START_REF] Ohashi | Novel mutations in the microsomal triglyceride transfer protein gene causing abetalipoproteinemia[END_REF][START_REF] Ricci | A 30-amino acid truncation of the microsomal triglyceride transfer protein large subunit disrupts its interaction with protein disulfide-isomerase and causes abetalipoproteinemia[END_REF][START_REF] Rehberg | A novel abetalipoproteinemia genotype. Identification of a missense mutation in the 97-kDa subunit of the microsomal triglyceride transfer protein that prevents complex formation with protein disulfide isomerase[END_REF] and the consequence of intronic mutations [START_REF] Najah | Identification of patients with abetalipoproteinemia and homozygous familial hypobetalipoproteinemia in Tunisia[END_REF][START_REF] Pons | A severe form of abetalipoproteinemia caused by new splicing mutations of microsomal triglyceride transfer protein[END_REF] have been studied in only a few cases due to the necessity of additional intestinal or hepatic biopsies.

ABL patients are usually diagnosed during infancy and they exhibit marked lipid malabsorption, very low levels of cholesterol and triglycerides, the absence of ApoB in the plasma, the absence of chylomicrons after fat loading, and essential fatty acid and fat-soluble vitamin deficiencies (particularly vitamin E). Hepatic steatosis has not been reported uniformly (although it is frequent), and hepatic fibrosis or cirrhosis has been reported in a few cases [START_REF] Black | Intestinal and hepatic apolipoprotein B gene expression in abetalipoproteinemia[END_REF][START_REF] Sakamoto | An atypical case of abetalipoproteinaemia with severe fatty liver in the absence of steatorrhoea or acanthocytosis[END_REF]. We report here an atypical case of ABL presenting in childhood with severe liver injury, hypocholesterolemia associated with a low (but not absent) levels of plasma ApoB and a subnormal level of plasma vitamin E. Furthermore, the patient exhibited normal development into adulthood. ABL was suspected because of the presence, at the ultrastructural level, of large amounts of free lipid droplets accumulated in the cytoplasm of enterocytes and hepatocytes in intestinal and liver biopsies.

The patient was found to be compound heterozygous for two novel mutations in the MTTP gene, one intronic (c.619-5_619-2 del, from her mother) and the other a missense mutation (p.Leu435His, from her father). The possible functional impact of these two mutations was not readily apparent. Since no additional biopsy material was available from the patient, two different assays were established. COS-1 cells were transfected with wild type and several mutant MTTP cDNAs in order to evaluate the impact of the missense mutation on MTP activity and HeLa cells were transfected with wild-type and mutant minigenes to evaluate possible defects in splicing.

Material and methods

Patient (diagnosis and follow-up)

A 4 year-old girl was referred for an anicteric chronic hepatitis fortuitously discovered 10 months previously. The level of blood transaminases were consistently elevated and ranged from 5 to 7 times the normal values associated with bilirubinemia in the absence of inflammation or hepatic insufficiency. Upon clinical examination, the liver was moderately enlarged and firm, but there was no other sign of chronic liver disease. The patient's growth was normal. The patient's parents altered her diet to avoid fatty foods following episodes of diarrhoea in the first months of life. The levels of total cholesterol and ApoB in the plasma were found to be low, whereas the level of triglyceride was normal (Table1). The parents were not related and they had normal levels of blood lipids.

A perendoscopic jejunal biopsy, performed as previously described [START_REF] Bouma | Description of two different patients with abetalipoproteinemia: synthesis of a normal-sized apolipoprotein B-48 in intestinal organ culture[END_REF], showed a white mucosa. Electron microscopic examination of the biopsy showed enterocytes with many small or very large free lipid droplets scatterred throughout the cytoplasm (Figure 1a,1b). Examination by ultrasound showed that the liver was moderately enlarged and slightly hyper-echogenic. A percutaneous liver biopsy showed extensive fibrosis (scored F3 according to the METAVIR scoring system [START_REF]Intraobserver and interobserver variations in liver biopsy interpretation in patients with chronic hepatitis C. The French METAVIR Cooperative Study Group[END_REF]) which was associated with a massive micro-and macro-vesicular steatosis.

Large amounts of free lipid droplets were observed in the cytoplasm by electron microscopy (Figure 1c). Abetalipoproteinemia was suspected although retinitis pigmentosa and acanthocytosis were absent and the blood vitamin E level was only slightly decreased. The fecal fat content, measured over a 3 day period, was also normal (mean 2.8 g/24h, normal < 3g/24h). However, when the child consumed a normal fat diet (33% fat), diarrhoea occurred and steatorrhoea was 24 g/day. After an oral fat load (Table1), the level of serum triglycerides increased slightly and a small amount of chylomicrons appeared in the plasma, suggesting that some intestinal lipid absorption occurs accompanied by low levels of intestinal lipoprotein secretion.

A diet limited in saturated fatty acids (22% of total energy) and enriched with medium chain triglycerides (MCT) was begun and resulted in the elimination of diarrhoea, progressive improvement in liver function (transaminases 2 to 4 X normal at the age of 7 years, 2 to 3 X normal at 14 years, normal values at 18 years of age), the normalization of the levels of essential fatty acids and fat soluble vitamins, and normal growth and puberty. At the age of 14 years, the patient no longer adhered to the MCT diet and 36% of total energy intake was composed of fat. Hepatitis recurred (transaminases increased to 150 to 200 X normal) but resolved when the MCT diet was re-instituted.

The patient is now 22 years of age and is in good health. Fat soluble vitamins are in the normal range without any supplementation. The levels of transaminases are < 1.5 X normal and hepatic steatosis persists (16% of hepatic parenchyma as determined by magnetic resonance imaging). There are no neurological, ophthalmological or retinal abnormalities.

Mutation analysis

Following the extraction of genomic DNA from the blood (Nucleon Bac3, GE Healthcare®, Chalfont St. Giles, UK, http://www.gehealthcare.com), each of the coding exons, and the flanking intronic junctions, of the MTTP, APOB, proprotein convertase subtilisin/kexin type 9 (PCSK9) and angiopoietin-like 3 (ANGPTL3) genes was amplified by PCR. The amplicons were sequenced directly with the BigDye® Terminator v3.1Cycle Sequencing Kit on an ABI PRISM 3130 or 3730 DNA sequencer (Applied Biosystems, Foster City, USA, www.appliedbiosystems.com).

In Silico Analysis

Analysis of the mutations was performed with Alamut v2.0 (Interactive Software), Polyphen (http://genetics.bwh.harvard.edu/pph/) and SIFT (http://sift.jcvi.org/www/SIFT_aligned_seqs_submit.html). Several algorithms were used for computational scoring of 3' splice site based on different concepts using default parameter settings (Neural Network Splice Prediction (NNSplice) [START_REF] Reese | Improved splice site detection in Genie[END_REF], MaxEntScan [START_REF] Yeo | Maximum entropy modeling of short sequence motifs with applications to RNA splicing signals[END_REF], Splice site Finder Like, GeneSplicer [START_REF] Pertea | GeneSplicer: a new computational method for splice site prediction[END_REF], Human Splicing Finder (HSF) ( 25)).

Protein expression

To evaluate the p.Leu435His protein expression, two assays were performed.

Minigene Splicing Reporter Assay

A 371 bp MTTP fragment (the last 147 bases of intron 5, 140 bp of exon 6, and the first 84 bases of intron 6) was amplified from the patient's DNA and inserted (In-Fusion Advantage PCR Cloning Kit) into the Nde I restriction site of the pTB minigene vector [START_REF] Baralle | Splicing in action: assessing disease causing sequence changes[END_REF]. The transfection of normal and mutant minigenes into HeLa cells and RT-PCR procedures were as previously described [START_REF] Crehalet | U1 snRNA mis-binding: a new cause of CMT1B[END_REF].

The fluorescence of the ethidium bromide bands obtained following gel electrophoresis of RT-PCR products was integrated under unsaturated conditions (Quantity One ® 1-D Analysis Software Cat. No170-9600, BIO-RAD, www.biorad.com) to derive band intensities.

Metabolic labelling of intestinal biopsies

Intestinal biopsies from normal individuals and from the patient were placed into organ culture and metabolically labelled with [ 35 S] methionine as described previously [START_REF] Wetterau | Localization of intracellular triacylglycerol and cholesteryl ester transfer activity in rat tissues[END_REF]. After homogenization and solubilization, the labelled intestinal biopsy extracts and the corresponding media were immunoprecipitated with polyclonal antibodies against Apo B (Rabbit polyclonal antibodies to Apo B were the gifts of Dr A. Mazur of Institut National de la Recherche Agronomique, Champanelle, France)

and MTP [START_REF] Wetterau | Localization of intracellular triacylglycerol and cholesteryl ester transfer activity in rat tissues[END_REF]. For semi-quantitative analysis, densitometric analysis was performed using the PC version of NIH Image software (Scion Image) after photography with a computer-assisted camera (GS-800 Calibrated Densitometer, BIO-RAD). The values were normalized with respect to the amount of TCA precipitable incorporated material in the biopsies.

Results

Identification of mutations

Sequencing was performed on the genomic DNA of the patient and her parents. Two novel variants of the MTTP gene were identified in the patient. The first, inherited from her father, is a change from T to A at position c.1304 in exon 10 (Supplemental Data, Figure 1A) (Nucleotide numbering starts at A of the ATG initiating codon and exon 1 is the first coding exon) which changes the amino acid Leu 435 to a His. The second, inherited from her mother, is a 4 bp deletion in intron 5: c.619-5_619-2 del (Supplemental Data, Figure 1B). This deletion of a repeated motif (TTTA) is upstream the acceptor site. These mutations were found neither in our panel of 100 normal alleles from unrelated subjects nor in the 1000 genomes database [START_REF]A map of human genome variation from population-scale sequencing[END_REF].

The patient is also heterozygous for a well described polymorphism p.Gln95His inherited from her father. The frequency of this allele is between 5.4 and 6% in a healthy adult Caucasian population (England) and in a sample of 270 unrelated French Canadian men (6, 33) and 6.2% in a cohort of abetalipoproteinemia [START_REF] Narcisi | Mutations of the microsomal triglyceride-transfer-protein gene in abetalipoproteinemia[END_REF].

The patient had no mutation in the APOB, PCSK9 or ANGTL3 genes.

Analysis of c.1304 T>A, p.Leu435His:

The missense mutation (p.Leu435His) is located in exon 10 and affects a highly conserved residue in the middle 1-helical domain of MTP. The substitution changes a hydrophobic to a hydrophilic, basic residue (Grantham distance: 99 [0-215]). This missense mutation is predicted by Polyphen to be "probably damaging" with a score of 0.99 and by SIFT to "affect protein function".

To determine whether the p.Leu435His missense mutation produces a protein that is functional or not, we expressed, in COS-1 cells, the wild type MTP as well as the p.Leu435His and several other mutants (p.Leu435Glu, p.Leu435Val and p.Cys194Stop). These different mutations were tested in COS-1 cells to evaluate the sensitivity and specificity of our MTP activity assay: p.Leu435Glu is expected to be as severe as p.Leu435His as it changes the hydrophobic Leu to a hydrophilic acidic Glu, whereas p.Leu435Val is expected to be a mild change (Leu and Val are both hydrophobic and closely related amino acids). The introduction of a premature stop codon at the position p.Cys194 is expected to lead to the complete abolition of MTP activity since the ApoB and PDI binding sites are absent from the mutant. Further, the homozygous p.Cys194Stop mutation has been reported in an ABL patient [START_REF] Chardon | Identification of two novel mutations and long-term follow-up in abetalipoproteinemia: a report of four cases[END_REF].

As shown in Figure 2 2A and2B). Fluorescent tagged MTP showed an identical cellular distribution (Supplemental Data, Figure 2C).The p.Leu435His mutant, therefore, is functionally defective.

Analysis of c.619-5_619-2del:

Splicing predictions

All the algorithms predicted an effect on splicing with a decrease in the score for the acceptor site of intron 5 ranging from -10% for SSF-like to a complete abolition for GeneSplicer, (-44% for MaxEnt Scan, -38.9% for NNSplice, -79% for HSF). Given that all the algorithms predicted an effect on splicing, experimental analysis of splicing was performed. HeLa cells transfected with wild type or mutant minigenes were analyzed (size and sequence) by RT-PCR to determine the presence of abnormal transcript processing of the mutant.

Minigene splicing reporter assay

Transfection of the wild type minigene into HeLa cells produced, as expected, a 386 bp RT-PCR fragment (Figure 3, lanes 1, 2 3). In contrast, transfection of the mutant c.619-5_619-2del minigene produced 2 different RT-PCR fragments: a small amount of a fragment of normal size (386bp) and a large amount of a 248 bp product (lanes 4, 5, 6). The sequence of the 386 bp PCR product includes exon 6 of MTTP whereas exon 6 is skipped in the 248 bp product (Supplemental Data, Figure 3). This result shows that c.619-5_619-2del mutation produces two differently spliced transcripts: one containing exon 6 and the other without exon 6. Exon 6 skipping would result in a protein containing the first 206 MTP amino acids but followed by 26 aberrant amino acids and a premature stop codon. In vivo, this mutant mRNA might be targeted for nonsense mediated decay and degraded. By densitometry of the agarose gel, the amount of normal splicing for the mutant minigene is estimated to be 26% of that of the wild type (Figure 3). These data indicate that the c.619-5_619-2del is a splice site mutation which can lead to the skipping of exon 6. However, the effect of this mutation on splicing is not complete and about 26% of the transcript is correctly spliced.

Biosynthesis of ApoB and MTP in intestinal organ culture

Immunoprecipitation with polyclonal antibodies to ApoB of the medium and the total homogenate of the organ culture of intestinal biopsies from the patient showed the synthesis of an ApoB48 identical in size to that of normal control subjects (Figure 4). Analysis by densitometry with correction for the amount of incorporated TCA precipitable incorporated radioactivity showed that there is an intracellular retention of ApoB48 (1.6 fold more) and a decreased ApoB48 secretion (1.3 fold less).

Immunoprecipitation with polyclonal antibodies to MTP of the total homogenate of the organ culture of intestinal biopsies from the patient showed the synthesis of a large subunit of identical in size to that of normal control subjects.

However, by densitometric analysis, there was 2.49 fold less MTP synthesized in the patient's biopsy as compared to that of the normal control subject after correction. A protein band corresponding to the molecular mass of PDI co-immunoprecipitated with the MTP in the patient as was observed for the normal control subject (identical in amount as compared to that of the normal subject after correction).

Discussion

In this paper, we describe two new mutations in the MTTP gene: p.Leu435His and a 4 bp deletion in intron 5: c.619-5_619-2 del. Since the impact of these mutations was unclear and the phenotype was atypical, cellular functional tests were used to further understand this new case of ABL.

The diagnosis of ABL is made readily if the following criteria are present during the first months of life: severe diarrhoea, failure to thrive, undetectable vitamin E, severe hypocholesterolemia and hypotriglyceridemia with undetectable amounts of plasma ApoB, and the absence, post-prandially, of chylomicrons in the serum. Our patient presented with a mild phenotype and lacked the major criteria for the diagnosis of ABL. Surprisingly, the patient was referred only for anicteric chronic hepatitis with major liver steatosis at 4 years of age. The other causes of hypobetalipoproteinemias were ruled out because of the patient's lipids profile and normal lipid values of the parents [START_REF] Cohen | Low LDL cholesterol in individuals of African descent resulting from frequent nonsense mutations in PCSK9[END_REF][START_REF] Kotowski | A spectrum of PCSK9 alleles contributes to plasma levels of low-density lipoprotein cholesterol[END_REF][START_REF] Tarugi | Molecular diagnosis of hypobetalipoproteinemia: an ENID review[END_REF][START_REF] Abifadel | A PCSK9 variant and familial combined hyperlipidaemia[END_REF][START_REF] Musunuru | Exome sequencing, ANGPTL3 mutations, and familial combined hypolipidemia[END_REF][START_REF] Martin-Campos | Identification of a novel mutation in the ANGPTL3 gene in two families diagnosed of familial hypobetalipoproteinemia without APOB mutation[END_REF][START_REF] Pisciotta | Characterization of Three Kindred with Familial Combined Hypolipidemia Due to Loss of Function Mutations of ANGPTL3[END_REF]. Furthermore, no mutation was found in the PCSK9, ANGPTL3, APOB genes. The diagnosis of ABL was finally suspected because of hypocholesterolemia and the typical aspect of intestinal and liver biopsies suggesting a defect in the assembly of ApoB-containing lipoproteins.

Seven atypical cases of ABL have been reported (13-15, 19, 41, 42). For five of these patients (Table 2) a molecular diagnosis of the mutation in MTTP was established. However, a functional assay of the mutation was performed in only one case [START_REF] Ohashi | Novel mutations in the microsomal triglyceride transfer protein gene causing abetalipoproteinemia[END_REF]. Two of the cases (cases 1 and 2 in Table 2) are very surprising. Both the patients have deleterious mutations, however, the first patient exhibits a mild phenotype [START_REF] Al-Mahdili | A mild case of abetalipoproteinaemia in association with subclinical hypothyroidism[END_REF] and the other is almost asymptomatic [START_REF] Ohashi | Novel mutations in the microsomal triglyceride transfer protein gene causing abetalipoproteinemia[END_REF]. Two other patients (cases 4 and 5 in Table 2) are homozygous for the same p.Ser590Ile mutation [START_REF] Wang | Microsomal triglyceride transfer protein (MTP) gene mutations in Canadian subjects with abetalipoproteinemia[END_REF][START_REF] Al-Shali | Ileal adenocarcinoma in a mild phenotype of abetalipoproteinemia[END_REF].

Unfortunately the functional impact of this mutation has not been established. Only the third case (19) (Table 2) resembles our case, clinically and biologically. This patient exhibits compound heterozygosity for a splice-site and a missense mutation, along with liver dysfunction and slightly decreased level of vitamin E.

The p.Leu435His mutation affects an amino-acid in helix 8 of the predicted 1helical domain of MTP [START_REF] Bradbury | A common binding site on the microsomal triglyceride transfer protein for apolipoprotein B and protein disulfide isomerase[END_REF]. Although this helix is not included in the major binding site for PDI and ApoB [START_REF] Bradbury | A common binding site on the microsomal triglyceride transfer protein for apolipoprotein B and protein disulfide isomerase[END_REF][START_REF] Shoulders | The abetalipoproteinemia gene is a member of the vitellogenin family and encodes an alpha-helical domain[END_REF][START_REF] Mann | The structure of vitellogenin provides a molecular model for the assembly and secretion of atherogenic lipoproteins[END_REF], the MTP activity of the mutant protein is very low and similar to that of "severe" mutations. The p.Leu435His mutation leads to a nonfunctional protein probably by affecting the MTP folding. Misfolded MTP may be targeted for degradation in the intestine or the liver; however, this recognition of misfolded proteins cannot be observed in heterologous expression systems (transfected COS-1 cells).

The c.619-5_619-2del is probably responsible for the mild phenotype as it produces two transcripts. One is normal-sized and contains exon 6 of the MTTP. The other transcript is aberrantly spliced and lacks exon 6. The phenotype may result from the correctly spliced RNA which is able to produce an active MTP. Although there may be some variability in the amounts of correctly spliced RNA between HeLa cells and the liver or the intestine, the marked effect of the intronic deletion observed in HeLa cells most likely reflects what might be expected in other cell types. A possible molecular mechanism for this splicing anomaly, predicted by the in silico analysis, is the shortening of the polypyrimidine tract by the deletion of one TTTA repeat. Short polypyrimidine tracts have been showed to be associated with variable levels of correctly spliced transcripts in the CFTR gene [START_REF] Rave-Harel | The molecular basis of partial penetrance of splicing mutations in cystic fibrosis[END_REF] thus providing evidence for incomplete penetrance of some splicing mutations in disease.

In our patient, the lipid transfer activity of the MTP produced from the normally In conclusion, our study of this atypical case of abetalipoproteinemia shows that the combination of molecular diagnosis and functional analysis resulted in a Supplemental Data Figure 3

  Blood samples, intestinal and liver biopsies were obtained from the patient using the procedures and the experimental methods approved by INSERM (RBM 0256) and by a bioethics committee (Comité Consultatif de Protection des Personnes dans la Recherche Biomédicale de Paris Bichat-Claude Bernard, Paris, France, CCPPRB Bichat-C. Bernard-2003/05). Informed, written consent was obtained from the patient's parents.

First, to measure

  wild type and mutant MTP activities, we transfected COS-1 cells with the MTTP and P4HB cDNA. Second, to compare the production of wild type and p.Leu435His, we transfected COS-1 cells with wild type and mutated MTTP cDNA Cter tagged with GFP. MTP activity A 2699 bp fragment containing the entire MTTP coding sequence, extending from c.-5 to c.*9 (NM_000253.2) and a 1582 bp fragment containing the entire P4HB coding sequence (from c.-14 to c.1527, NM_000918.3) were obtained by RT-PCR from 1µg of human liver total RNA (Cat.No.636531, Clontech, Mountain View, U.S.A, www.clontech.com) with the Transcriptor High Fidelity cDNA Synthesis Sample Kit (Cat.No.05081963001 Roche Applied Science, Indianapolis, USA, www.rocheapplied-science.com). The MTTP cDNA was inserted, with the In-Fusion Advantage PCR Cloning Kit (Cat.No.639616, Clontech, Mountain View, U.S.A, www.clontech.com), into the Kpn I site of the pBudCE4.1 expression vector (Cat.No.V532-20 Invitrogen, Carlsbad, U.S.A, www.invitrogen.com) downstream of the human elongation factor 11-subunit promoter. The P4HB cDNA was inserted into the Hind III site of the same vector, downstream of the human cytomegalovirus immediate-early promoter, allowing the production of the two proteins from the same plasmid. The p.Leu435His (c.1304T>A), p.Leu435Glu (c.1303_1305delinsGAA), p.Leu435Val (c.1303C>G) and p.Cys194Stop (c.582C>A) mutants were constructed from the wild type sequence with the QuickChange II XL Site-Directed Mutagenesis Kit (Cat.No.200521 Stratagene, Agilent Technologies, Cedar Creek, USA, www.agilent.com) according to the manufacturer's instructions. Transient expression of MTP and P4HB in COS-1 cells (Cat. No. CRL-1650 ATCC) was carried out by transfecting 6 µg of plasmid per T25 flask in the presence of 9 µL of FuGENE® HD (Cat.No.4709691001 Roche Applied Science, Indianapolis, USA, www.roche-applied-science.com), according to the manufacturer's instructions.COS-1 cells were harvested by trypsinization 48 hours post-transfection and disrupted by sonication. Triglyceride transfer from donor to acceptor vesicles was measured by a fluorescent-labelled method using a commercial kit (R100 MTP activity, Chylos Inc, USA, www.chylos.com) according to the manufacturer's instructions and previously described fluorescent methods[START_REF] Athar | A simple, rapid, and sensitive fluorescence assay for microsomal triglyceride transfer protein[END_REF][START_REF] Rava | Transfer of cholesteryl esters and phospholipids as well as net deposition by microsomal triglyceride transfer protein[END_REF][START_REF] Mirandola | Liver microsomal triglyceride transfer protein is involved in hepatitis C liver steatosis[END_REF]. The results are expressed as % transfer/mg total proteins/h. The method was evaluated with intestinal and hepatic biopsies and the MTP activities were in agreement with previous published results (data not shown).Visualisation of MTP in transfected cellsA fragment containing the MTTP cDNA, extending from c.1 to c.2682 (NM_000253.2) of each sequence (wild type, p.Cys194Stop, p.Leu435His) was amplified from the previously obtained pBudCE4.1 vector. The MTTP cDNA was inserted, with the In-Fusion Advantage PCR Cloning Kit into the KpnI site of pAcGFP1-N1 expression vector (Cat.No.632469 Clontech, Mountain View, U.S.A, www.clontech.com) between the immediate early promoter of the CMV (PCMV IE) and the AcGFP1 cDNA allowing the fusion of the MTP and the GFP proteins. COS-1 cells were electroporated with 0.5 µg of MTP-AcGFP1 plasmid per 80 000 cells (MicroPorator, DigitalBio Technology and Neon Transfection System 10µL Kit, Cat.No. MPK1096, Invitrogen Carlsbad, U.S.A, www.invitrogen.com) according to the manufacturer's instructions (1050V, 30ms, 2 pulses). Intracellular fluorescence was observed with a Nikon Eclipse TE 2000-U microscope 48h after transfection.

  spliced transcript may be sufficient to allow the assembly and secretion of a relatively limited amount of ApoB-containing lipoproteins by liver and intestine and could explain the presence of the lipid absorption (as shown by metabolic labelling biosynthesis data) observed in the intestine of the patient. Both the intracellular accumulation and the decreased secretion of ApoB48 from the intestine are consistent with a partial defect in the assembly of ApoB which leads to a partial defect in secretion of ApoB48 (unusual in ABL). In addition, the data also suggest that a low amount of intestinal MTP was present with a normal molecular weight and with an intact PDI binding domain. Finally, the presence of a limited amount of fat absorption and lipoprotein secretion could explain the normal level of vitamin E and the normal development with only a low fat diet as treatment. However, the residual MTP activity is not sufficient to prevent the accumulation of lipids in the enterocytes and the hepatocytes.

Figure 1 :

 1 Figure 1: Ultrastructure of intestinal and liver biopsies after a 12-hour fast.

Figure 2 :

 2 Figure 2: Triglyceride transfer activity of normal and mutant MTTP in COS-1 cells over a period of 48h.

Figure 3 :Figure 4 :

 34 Figure 3: Size separation of RT-PCR products as determined by 2% agarose gel electrophoresis:

  The enterocytes (a,b) and hepatocytes (c) were engorged with numerous small or very large lipid droplets (L) free in the cytoplasm. The Golgi apparatus (G) is empty (b). Hepatic intercellular spaces were sometimes enlarged with fibrosis (c). The cell nucleus is labelled N.

Figure 1 Figure 2

 12 Figure 1

Figure 3 M

 3 Figure 3

Supplemental Data 1 :5 2 C

 12 Figure 4

Table 1 :

 1 Biological data for the patient and the parents.

		TC	TG	HDLC	LDLC	ApoB	ApoA1	VitE	VitA	VitD	ALT	AST	ALB	BMI
		mmol/L	mmol/L	mmol/L	mmol/L	g/L	g/L	µmol/L	µmol/L	nmol/L	UI/L	UI/L	g/L	kg/m	2
	Reference interval*	2.58-4.39 0.34-1.25	>1.03	1.81-3.10 0.5-1.2	1.1-2.0	11-35	1.4-3.0 34-130	<30	<30	35-50
	Patient
	(at diagnosis, 4yrs of	1.68	0.58	0.85	0.57	< 0.18	ND	9.9	1.7	159	287	272	32	14
	age)
	Oral fat load (5yrs)
		2.27	1.15	2.17		0.20	0.93
	T0
	Oral fat load
		2.06	1.50	1.86		0.20	0.99
	T 90 min
	Patient
	(22yrs old)	2.24	0.64	1.70	0.26	ND	ND	18.9	3	96	49	41	45	17.8
	(reduced fat diet)
	Father	5.42	0.97	1.42	3.57
	Mother	4.64	1.84	1.52	2.77	1.01	1.19				18	14
	TC total cholesterol, TG triglyceride, HDLC HDL cholesterol, LDLC LDL cholesterol, Vit vitamin, ALT Alanine aminotransferase, AST Aspartate
	aminotransferase, ALB albumin, BMI body mass index, ND : not determined
	* normal values for 4 a year old child.
	Oral fat load with 15g of fat.

Table 2 :

 2 Atypical mild cases of ABL reported in the literature

		case 1	case 2	case 3	case 4	case 5
	ref.	(42)	(13)	(19)	(15)	(14)
		[c. 61+2T>C]+	homozygous	[c. 61+1G>C]+	homozygous	homozygous
	mutation
		[c.419-420insA]	p.Asn780Tyr	[p.Ile564Thr]	p.Ser590Ile	p.Ser590Ile
	age at diagnosis
		13	27	1.25	52	24
	(year)
	age at present
		32	27	5	52	44
	(year)
			during a routine		during a routine
	context of			hepatomegaly +
		NA	medical		medical	NA
	diagnosis			liver dysfunction
			examination		examination
	malabsorption	+	0	NA	+	+
	liver	ALT, AST, GGT		hepatomegaly,
			mild fatty liver		NA	NA
	impairements	increased		steatosis, fibrosis
	neurological
		reduced reflexes	0	0	0	0
	impairements
		minor
		xerophtalmia,
	ophtalmological				atypical retinitis
		abnormal dryness	0	0		retinopathy
	impairements				pigmentosa
		of membrane of
		the eyes
		14 (RI: 12-36) on
	Vit E (µmol/L)	replacement	<2.4	10 (RI: 18-34)	23 (RI: 12-46)	NA
		therapy
	TC (mmol/L)	0.90	0.87	1.25 to 2.36	0.85	NA
	TG (mmol/L)	0.50	0.03	0.11 to 1.14	0.06	NA
	HDLC (mmol/L)	0.50	0.59	NA	0.68	0.56
	LDLC (mmol/L)	0.07	NA	NA	0.16	NA
	ApoB (g/L)	0.06	0.006	< 0.007	NA	NA
	MTTP activity	NA	negligible	NA	NA	NA

TC total cholesterol, TG triglyceride, HDLC HDL cholesterol, LDLC LDL cholesterol, Vit + present, 0 absent, NA not available, RI reference interval

Acknowledgments

We thanks S. Dumont (Service de Biochimie, CBE, HCL, Lyon), S. Faïna (Service de Biochimie et Génétique Moléculaire, CHU A Paré, AP-HP et PIFO-UVSQ, Boulogne) and M. Lannoy (INSERM U 698, Paris) for technical assistance; N Verthier for her expertise in electron microscopy methods and JP Laigneau for his expertise in illustration (INSERM, IFR 94 IRNM, Hôpital Necker-Enfants Malades, Paris).

definitive diagnosis of MTP deficiency in a patient when insufficient biopsy material was available for the analysis of the MTP protein or RNA. Second, the functional studies of the substitution of Leu435 to a charged amino acid (acid Glu or basic His) highlight the requirement for this hydrophobic un-charged residue for MTP activity.

Third, in cases of unusual phenotype, the functional characterization of the MTTP mutants allows a better understanding of the milder ABL phenotype.


MD, PhD Cédric Lemogne
email: cedric.lemogne@egp.aphp.fr

MD, PhD Silla M Consoli

MD, PhD e , Béatrice Geoffroy-Perez

MD, MPH Mireille Coeuret-Pellicer

PhD Hermann Nabi

PhD Maria Melchior

MD, PhD Frédéric Limosin

MD Marie Zins

MD, PhD Pierre Ducimetière

MD Marcel Goldberg

PhD Sylvaine Cordier

Personality and the Risk of Cancer: A 16-Year Follow-Up Study of the GAZEL Cohort

Keywords: Emotion, Epidemiology, Incidence, Personality, Risk, Cancer. Acronyms BMI: Body Mass Index, CI: Confidence Interval, Personality Stress Inventory, SD: Standard Deviation

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

Introduction

Personality has long been hypothesized to predispose individuals to cancer initiation or progression. The early observation by Kissen and Eysenck (1) that patients with lung cancer may show high levels of extroversion and low levels of neuroticism led to posit a "cancerprone" personality. However, well-designed large-scale prospective studies have convincingly dismissed this initial hypothesis [START_REF] Schapiro | Extroversion and neuroticism and the associated risk of cancer: a Danish cohort study[END_REF][START_REF] Lillberg | Personality characteristics and the risk of breast cancer: a prospective cohort study[END_REF][START_REF] Nakaya | Personality and the risk of cancer[END_REF][START_REF] Hansen | Personality traits, health behavior, and risk for cancer: a prospective study of Swedish twin court[END_REF][START_REF] Nakaya | Personality traits and cancer risk and survival based on Finnish and Swedish registry data[END_REF]. According to the International Agency for Research on Cancer [START_REF]Preamble to the IARC Monographs on the Evaluation of Carcinogenic Risks to Humans[END_REF], excluding a risk factor as carcinogenic warrants 1) several methodologically sound studies that 2) are mutually consistent in not showing a positive association. Whereas these two criteria may be considered as fulfilled as regards extroversion and neuroticism [START_REF] Ranchor | Invited commentary: personality as a causal factor in cancer risk and mortality--time to retire a hypothesis?[END_REF], most of studies that addressed other personality constructs had serious methodological limitations: a retrospective or cross-sectional design, a focus on cancer mortality rather than cancer incidence, a lack of comprehensive adjustment on confounding variables, a short follow-up period or a relatively small sample size, resulting in insufficient statistical power to analyze specific cancer sites. Overall, these studies have mostly produced negative results or mixed results at best [START_REF] Butow | Epidemiological evidence for a relationship between life events, coping style, and personality factors in the development of breast cancer[END_REF][START_REF] Dalton | Mind and cancer: do psychological factors cause cancer?[END_REF][START_REF] Garssen | Psychological factors and cancer development: Evidence after 30 years of research[END_REF][START_REF] Vries | Alexithymia in Cancer Patients: Review of the Literature[END_REF]. The present study took advantage of the large-scale prospective French GAZEL cohort [START_REF] Goldberg | Cohort profile: the GAZEL Cohort Study[END_REF] to examine the association between cancer incidence and four measures of personality.

Type 1 personality, characterized by suppressed emotional expression in the context of interpersonal relationships, and Type 5 personality, characterized by rational/anti-emotional tendencies, were proposed by Grossarth-Maticek and Eysenck [START_REF] Grossarth-Maticek | Personality, stress and disease: description and validation of a new inventory[END_REF] to account for the early observation of a negative association between neuroticism and cancer [START_REF] Kissen | Personality in male lung cancer patients[END_REF]. These two personality types were claimed to be associated with poor stress coping strategies, resulting in high levels of cortisol and related immune deficiencies [START_REF] Grossarth-Maticek | Personality, stress and disease: description and validation of a new inventory[END_REF]. Owing to serious methodological and ethical issues, it was suggested that the early reports by these authors, supporting a strong link with cancer, should be withdrawn from the literature [START_REF] Ranchor | Invited commentary: personality as a causal factor in cancer risk and mortality--time to retire a hypothesis?[END_REF][START_REF] Van Der Ploeg | What a wonderful world it would be: a reanalysis of some work of Grossarth-Maticek[END_REF][START_REF] Coyne | Meta-analysis of stress-related factors in cancer[END_REF], making these personality constructs unfashionable for psychosomatic research. As a consequence, the hypothesis of their association with cancer incidence remains to be properly tested, whereas these early, questionable results still influence the results of recent impactful meta-analyses [START_REF] Coyne | Meta-analysis of stress-related factors in cancer[END_REF]. The primary aim of the present study was to challenge these results focusing on cancer incidence rather than mortality. Type 5 personality was found to predict poor survival among lung cancer patients [START_REF] Nagano | A prospective Japanese study of the association between personality and the progression of lung cancer[END_REF] but some prospective studies failed to find significant associations between other measures of rational/anti-emotional tendencies and cancer incidence [START_REF] Persky | Personality and risk of cancer: 20-year follow-up of the Western Electric Study[END_REF][START_REF] Hahn | Minnesota Multiphasic Personality Inventory-rated depression and the incidence of breast cancer[END_REF][START_REF] Tijhuis | Prospective investigation of emotional control and cancer risk in men (the Zutphen Elderly Study) (The Netherlands)[END_REF][START_REF] Bleiker | Personality factors and breast cancer risk: a 13-year follow-up[END_REF] or mortality [START_REF] Hirokawa | The relationships of a rationality/antiemotionality personality scale to mortalities of cancer and cardiovascular disease in a community population in Japan[END_REF]. However, none specifically examined Type 5 personality and cancer incidence and most well-designed studies considered only breast cancer [START_REF] Hahn | Minnesota Multiphasic Personality Inventory-rated depression and the incidence of breast cancer[END_REF][START_REF] Bleiker | Personality factors and breast cancer risk: a 13-year follow-up[END_REF] or were underpowered to properly analyze specific cancer sites [START_REF] Persky | Personality and risk of cancer: 20-year follow-up of the Western Electric Study[END_REF][START_REF] Tijhuis | Prospective investigation of emotional control and cancer risk in men (the Zutphen Elderly Study) (The Netherlands)[END_REF]. Finally, there is evidence linking related constructs such as alexithymia with deficiencies in cell-mediated immunity [START_REF] Guilbaud | Is there a psychoneuroimmunological pathway between alexithymia and immunity? Immune and physiological correlates of alexithymia[END_REF], suggesting possible causal pathways between Type 5 and cancer onset.

Here, we examined the associations between cancer incidence and Type 1 and Type 5 personality measures in the GAZEL cohort study, considering several main sites of cancer separately. In addition, we report exploratory analyses regarding two other personality constructs more widely accepted in psychosomatic research: Type A behavior pattern and hostility. Statistical analyses specifically addressed the potential role of several health behaviors, such as smoking habits, in mediating any association between personality and the risk of cancer [START_REF] Garssen | Psychological factors and cancer development: Evidence after 30 years of research[END_REF][START_REF] Vries | Alexithymia in Cancer Patients: Review of the Literature[END_REF].

Material and Methods

Participants

Details of the GAZEL cohort study are available elsewhere [START_REF] Goldberg | Cohort profile: the GAZEL Cohort Study[END_REF]. The target population consisted of 44,992 employees of the French national gas and electricity company "Electricité de France-Gaz de France" (EDF-GDF): 31,411 men aged 40-50 and 13,511 women aged 35-50. The study protocol was approved by the French authority for data confidentiality ("Commission Nationale Informatique et Liberté") and by the Ethics Evaluation Committee of the "Institut National de la Santé et de la Recherche Médicale" (INSERM) (IRB0000388, FWA00005831). In 1989, 20,625 employees (45.8%) (15,011 men and 5,614 women) gave written informed consent to participate in the GAZEL cohort study. Since then, participants were followed by means of an annual mailed questionnaire, as well as through administrative databases. The 1993 questionnaire, which was mailed to the 20,488 alive cohort members, included the Personality-Stress Inventory (PSI), the Buss and Durkee Hostility Inventory (BDHI) and the Bortner Type A Rating Scale (BTARS).

Personality measures

The PSI is a 70-item questionnaire with "true-false" answers that aims to identify 6 personality types, each claimed to be specifically associated with increased or decreased morbid risks [START_REF] Grossarth-Maticek | Personality, stress and disease: description and validation of a new inventory[END_REF]. Five of the personality scales are measured by 10 items each (sum of the "true" responses), and one (Type 4) is measured by 20 items (sum of the "true" responses, divided by 2). Six personality scores ranging from 0 to 10 are thus calculated. A pilot study in retest reliability of the French version of the PSI [START_REF] Consoli | Validation of a personality questionnaire designed for defining subgroups at risk for ischemic cardiopathy or cancer in the GAZEL cohort[END_REF]. The 6 subscales of the PSI assess the following constructs: Type 1 = dependence on withdrawing objects (i.e. either persons or situations), leading to suppressed emotional expression in the context of interpersonal relationships; Type 2 = dependence on disturbing objects, leading to hostile thoughts and feelings; Type 3 = dependence on objects that are both withdrawing and disturbing, leading to ambivalent behaviors oscillating between the positive and negative aspects of the objects; Type 4 = autonomy and self-regulation; Type 5 = rational/anti-emotional tendencies ; Type 6 = antisocial tendencies. Here, we focused on Type 1 and Type 5 subscales for three main reasons: first, we aimed to limit multiple comparisons as much as possible; second, we nonetheless aimed to challenge previous results linking Type 1 and Type 5 with cancer; third, a principal component analysis suggested that Types 1, 2, 3 and, inversely, Type 4 may indeed relate to the same latent construct, whereas Types 5 and 6 may represent independent constructs [START_REF] Amelang | Using personality variables to predict cancer and heart disease[END_REF]. Type 1 personality (Cronbach"s α = 0.61 in the GAZEL cohort, 3-month retest r coefficient = 0.65, p<.001), is close to the type C personality proposed by Temoshok [START_REF] Temoshok | Personality, coping style, emotion and cancer: toward an integrative model[END_REF]. It is characterized by a tendency to suppress negative emotions, especially anger, and to be unassertive in order to seek harmony with others (e.g. "I often feel inhibited when it comes to openly showing negative feelings such as hatred, aggression, or anger"). Type 1 correlates positively with emotion-oriented coping strategies, neuroticism and alexithymia, and negatively with extraversion [START_REF] Schmitz | Personality, stress-reactions and disease[END_REF]. Type 5 personality (Cronbach"s α = 0.57 in the GAZEL cohort, 3-month retest r coefficient = 0.63, p<.001), once referred to as "rational/anti-emotional", shares a tendency to suppress emotion with Type 1 personality but also features non-emotional and rational tendencies such as inhibited emotional reactions and lack of confidence in one's own feelings (e.g. "I can only express feelings when they have a rational basis"). Type 5 correlates positively with task-oriented coping strategies and alexithymia, and negatively with psychoticism. It does not correlate with neuroticism or extraversion [START_REF] Schmitz | Personality, stress-reactions and disease[END_REF].

Hostility was assessed with the French version of Buss and Durkee Hostility Inventory (BDHI) [START_REF] Consoli | Validation of a personality questionnaire designed for defining subgroups at risk for ischemic cardiopathy or cancer in the GAZEL cohort[END_REF][START_REF] Buss | An inventory for assessing different kinds of hostility[END_REF]. The BDHI is composed of 75 items with "true-false" answers. It has eight subscales, seven of which are designed to measure different components of hostility: assault, verbal aggression, indirect hostility, irritability, negativism, resentment, and suspicion. The sum of these seven sub-scales leads to a "total hostility" score with a high 3-month test-retest reliability (r = 0.87, p<.001) [START_REF] Consoli | Validation of a personality questionnaire designed for defining subgroups at risk for ischemic cardiopathy or cancer in the GAZEL cohort[END_REF]. In order to limit multiple comparisons, we focused on the total hostility score (Cronbach"s α = 0.88 in the GAZEL cohort).

Although the 1991 pilot study [START_REF] Consoli | Validation of a personality questionnaire designed for defining subgroups at risk for ischemic cardiopathy or cancer in the GAZEL cohort[END_REF] considered the Jenkins Activity Survey as index of Type A personality, the 1993 questionnaire included the Bortner Type A Rating Scale (BTARS) [START_REF] Bortner | A short rating scale as a potential measure of pattern A behavior[END_REF]. It consists of 14 items each comprising two statements with a graded scale between the two statements (24-point scale in the original version, 6-point scale in the version adapted for the GAZEL cohort). Examples of statements include "never late" versus "casual about appointments". Importantly, the BTARS captures time urgency, job involvement, hard driving, need for achievement, ambition and competitiveness, but not hostility. The sum of the 14 items yields a global score ranging from 14 to 84 (Cronbach"s α = 0.64 in the GAZEL cohort). This scale was translated and validated for the French population against the Friedman and Rosenman (30) structured interview for assessing Type A, agreement observed 71.5% [START_REF] Belgian-French | Assessment of type A behavior by the Bortner Scale and ischaemic heart disease[END_REF].

Cancer cases

All participants were followed-up for diagnoses of primary cancers from January 1, during the period of employment came from a registry kept by the medical departments at EDF-GDF and that has been validated for accuracy and completeness [START_REF] Chevalier | Cancer incidence among active male workers at Electricite de France-Gaz de France[END_REF]. Diagnoses after retirement came from the systematic validation of each self-reported primary cancer through a diagnosis validation survey that began in 2009. Each annual questionnaire asked participants to report whether or not they were hospitalized or diagnosed with several diseases; including cancer. All participants who self-reported a cancer at least once during the follow-up were contacted (if alive) to give consent for a detailed diagnostic investigation with their physician.

In a first set of analyses, we considered as cases all participants with a validated diagnosis as well as participants who reported a diagnosis of primary cancer but who died from a cancer before the onset of the diagnosis validation survey. Living status and the date of death were obtained annually for all participants from EDF-GDF itself as it pays out retirement benefits. Causes of death were available from baseline (i.e. January 1, 1994) to December 31, 2009 and were coded by the French national cause-of-death registry (CépiDc, INSERM) using the ICD 9 th and 10 th Revision.

We planned to examine the four most frequent types of cancer in France, separately: prostate cancer in men, breast cancer in women, smoking-related cancers (i.e. cancer of the oral cavity and pharynx, esophagus, larynx, trachea, bronchi and lungs, and bladder) and colorectal cancer [START_REF]La situation du cancer en France en 2011. Collection Rapports & synthèses[END_REF]. A fifth category encompassing all other sites was also examined. Nonmelanoma skin cancers and in situ neoplasms were not considered as cancer cases.

Covariates

Age, sex, and occupation grade (blue-collar workers or clerks, first-line supervisors or sales representatives, management) were obtained from employer"s human resources files at baseline. Alcohol consumption, smoking, fruits and vegetables consumption (<1, 1-2, >2 times per week), height, weight, physical activity (at least one time per week, occasionally, none) and perceived health status were self-reported at baseline. Alcohol consumption, as drinks per week, was categorized as non-drinkers, occasional and moderate drinkers (1-27 for men, 1-20 for women) or heavy drinkers (≥28 for men, ≥21 for women). Smoking in the same period was categorized into 5 classes: never-smokers, ex-smokers of fewer than 20 pack-years, current smokers of fewer than 20 pack-years, ex-smokers of more than 20 packyears and current smokers of more than 20 pack-years. Body mass index (BMI) was calculated by dividing weight in kilograms by height in meters squared and categorized as < 18.5, 18.5-24.9, 25-29.9 or ≥30 kg/m 2 . Perceived health status was reported with an 8-point Likert scale ranging from 1 ("very bad") to 8 ("very good").

Statistical analyses

All statistical analyses were computed with PASW 18.0.0 software (SPSS Inc.).

Personality scores had a normal distribution. Coefficients of correlation and ANOVAs were computed to examine the relation of personality scores with continuous and discrete covariates, respectively. The association of personality scores and covariates with cancer incidence was estimated with Hazard Ratios (HR) and 95% confidence intervals (CI) computed in Cox regressions. The follow-up ran from January 1, 1994 to the date of cancer diagnosis, death, refusal to receive further questionnaire, or December 31, 2009, whichever occurred first. For participants who reported a diagnosis of cancer after retirement, but who died from a cancer before the onset of the diagnosis validation survey, the estimated date of diagnosis was the date of the first self-report minus 180 days (i.e. the mean interval between two annual questionnaires). Discrete covariates were considered as nominal variables. Each personality score was considered as a continuous variable as we did not have a priori hypotheses to posit a more sophisticated relationship than a linear one.

To test the hypothesis that some personality features might increase the risk of cancer through their influence on health behaviors (e.g. smoking habits), unadjusted analyses and two multivariate models were computed. Model 1 was adjusted for all covariates except for health behaviors, whereas model 2 was further adjusted for health behaviors (i.e. alcohol consumption, smoking, fruits and vegetables consumption and physical activity). Whenever a personality measure was significantly associated with cancer in model 1, the contribution of health behaviors to this association was appreciated by the percentage of change in the HR from model 1 to model 2, calculated with the following formula: 100 × (HR model 2 -HR model 1 ) / (HR model 1 -1).

Model 2a included all the cancer cases for which we had a date of diagnosis (see above). In sensitivity analyses, we also considered two additional models. In model 2b, which aimed at optimizing the diagnosis sensitivity, all the participants who died from a cancer were considered as cases, including those who did not report a diagnosis of cancer during the follow-up. The estimated date of diagnosis was the date of death minus 886 days (i.e. the mean survival time among participants with an ascertained date of diagnosis and who subsequently died from a cancer). In model 2c, which aimed at optimizing the diagnosis specificity, only participants with a validated diagnosis of cancer were considered as cases, excluding participants who reported a cancer during the follow-up but died from a cancer before the diagnosis validation survey.

Results

All personality scores (i.e. Type 1, Type 5, hostility and Type A) were available for 14,522 GAZEL cohort members (70.8%). Compared with non responders, responders were more likely to be male, older, to have a higher occupational grades, to eat fruits and vegetables more than twice a week and to have physical activity at least once a week, and were less likely to be heavy or non drinkers or current smokers and to have extreme BMI (all p<.05).

Among the responders, 754 (5.2%) were excluded from this study: 18 died and one asked to receive no further questionnaire before the beginning of the follow-up (i.e. January 1, 1994); 203 had previously had a cancer diagnosis at baseline; 408 self-reported either a cancer or an hospitalization for cancer but these cases were not confirmed owing to the following reasons: lack of written consent to participate in the diagnosis validation survey (N=296 volunteers including 17 who died before the onset of the survey), death of the volunteer (N=7), refusal to respond to the survey when contacted (N=2) or failure to contact the volunteer and his or her physician (N=103); 124 died from a cancer without having reported a diagnosis of cancer or an hospitalization for cancer during the follow-up. These 124 individuals were excluded from subsequent analyses to increase the specificity of cancer diagnoses, except in model 2b, which aimed at optimizing the diagnosis sensitivity (see above). The study population included 13,768 participants whose characteristics are displayed in Table 1.

Associations between personality scores and discrete and continuous variables are displayed in Table 2 and3 participants (881 men, 258 women) received at least one diagnosis of a primary cancer (other than nonmelanoma skin cancer), including 121 participants (103 men, 18 women) who selfreported a cancer or a hospitalization for cancer during follow-up but died from a cancer before the diagnosis validation survey. Among participants who received at least one diagnosis of a primary cancer, the mean duration of follow-up prior to the first cancer diagnosis was 9.3 years. There were 413 prostate cancer cases among men, 146 breast cancer cases among women, 124 colorectal cancer cases (106 men, 18 women), 137 smoking-related cancer cases (132 men, 5 women), and 352 cases of other cancers (256 men, 96 women).

Among this residual category, the most frequent cancers were lymphoid and hematopoietic cancers (N=95), cancers of the urinary organs (N=49), non colorectal digestive cancers (N=45) and melanomas (N=44). Associations between covariates and cancer incidence in multivariate analyses are displayed in Table 4.

Associations between personality scores and cancer incidence are displayed in Table 5.

Type 1 personality was not associated with an increased risk of cancer, regardless of cancer site, and was even associated with a decreased risk of breast cancer in women. Type 5 personality score was associated with an increased risk of other cancers. Hostility was associated with an increased risk of smoking-related cancers in model 1, but no longer in model 2a (HR change: -56%). Indeed, this association was no longer significant when further adjusting model 1 with smoking only (HR for one standard deviation: 1.14, 95% CI: 0.96-1.35, p=.13, HR change: -38%). Type A was not associated with the risk of cancer, regardless of cancer site. Including participants who died from a cancer without having reported a cancer during the follow-up (model 2b) or excluding those who reported a cancer during the followup but died from a cancer before the diagnosis validation survey (model 2c) yielded similar results. There was no interaction between gender and Type 5 personality as regards its association with other cancers (Wald"s statistic = 0.717, degree of freedom = 1, p=.40). Some post hoc analyses were also performed.

To examine a potential dose-response relationship between Type 1 and the risk of breast cancer, and between Type 5 and the risk of other cancers, we divided these personality scores into quartiles. As regards Type 5 personality, we found that the highest quartile (versus the lowest) was significantly associated with an increased risk of other cancers, with intermediate HR for the second and third quartiles (Table 6). As regards Type 1 personality, we did not find evidence for a clear dose-response relationship, the lowest HR being associated with the third quartile, with intermediate HR for the second and the fourth quartiles (Table 6).

To examine whether Type 1 and Type 5 were associated with biases in reporting diseases, we examined the correlations between the total number of diseases reported by each participant in the 1993 questionnaire and these personality scores. It was positively associated with Type 1 personality (r = 0.129, p<.001) and negatively associated with Type 5 personality (r = -0.055, p<.001), suggesting that Type 1 was not associated with under-reporting diseases and that Type 5 personality was not associated with over-reporting diseases.

To examine the validity and the independence of Type 1 and Type 5 measures, we performed a principal component analysis over the 20 items of the two personality subscales.

The Kaiser-Meyer-Olkin measure of sampling adequacy was 0.78 and the Bartlett's test of sphericity was significant (χ 2 =28124.742, df=190, p<.001). As expected, the scree test was clearly consistent with a two-factor solution. After varimax rotation, all except two items of the Type 1 subscale were selectively loading on the first factor and all except two items of the Type 5 subscale were selectively loading on the second factor. Computing the two personality scores without these items yielded slightly higher alpha coefficients for both Type 1 (Cronbach"s α = 0.63) and Type 5 (Cronbach"s α = 0.58). Type 1 was still associated with a decreased risk of breast cancer (HR for one standard deviation: 0.83, 95% CI: 0.69-1.00, p=.047) and Type 5 was still associated with an increased risk of other cancers (HR for one standard deviation: 1.19, 95% CI: 1.06-1.34, p=.004).

Discussion

To our knowledge, this is the first large-scale prospective study to examine the association between the PSI personality types and the incidence of cancer. Adjusting for an extensive set of covariates, we found no evidence for an association between Type 1 personality and an increased risk of cancer, regardless of cancer site. Type 1 personality was indeed associated with a decreased risk of breast cancer. This association remained significant in sensitivity analyses but there was no evidence for a clear dose-response relationship. Type 5 personality, which is characterized by rational/anti-emotional tendencies, was associated with an increased risk of other cancers. Post hoc analyses with a quartile approach suggested a dose-response relationship. Type 5 personality, however, was not associated with the risk of breast, prostate, colorectal or smoking-related cancers. In addition, there was a positive association between hostility and the risk of smoking-related cancers, which was explained by smoking habits. Finally, Type A personality was not associated with the risk of cancer, regardless of cancer site.

Strengths of the present study include its large sample size, the long duration of followup (i.e. 15.2 years on average), the wide set of covariates, the study of different cancer sites and the study of cancer incidence rather than mortality. Cancer diagnoses were carefully ascertained and validated and sensitivity analyses yielded similar results. Our data are consistent with known associations for established risk factors such as age, alcohol consumption and smoking, as well as for social and demographic variables and protective factors [START_REF]La situation du cancer en France en 2011. Collection Rapports & synthèses[END_REF]. They are also consistent with the lack of association between rational/antiemotional tendencies and the incidence of breast cancer [START_REF] Hahn | Minnesota Multiphasic Personality Inventory-rated depression and the incidence of breast cancer[END_REF][START_REF] Bleiker | Personality factors and breast cancer risk: a 13-year follow-up[END_REF]. In comparison with previous studies that failed to find significant associations between other measures of rational/antiemotional tendencies and cancer incidence [START_REF] Persky | Personality and risk of cancer: 20-year follow-up of the Western Electric Study[END_REF][START_REF] Tijhuis | Prospective investigation of emotional control and cancer risk in men (the Zutphen Elderly Study) (The Netherlands)[END_REF], the present study had more statistical power owing to a five to ten-fold greater number of events. Indeed, one of these studies found a positive association between control of depressive feelings and cancer mortality, with a similar trend for cancer incidence [START_REF] Tijhuis | Prospective investigation of emotional control and cancer risk in men (the Zutphen Elderly Study) (The Netherlands)[END_REF].

Several limitations should nevertheless be acknowledged. Although most of the cancer cases were thoroughly ascertained and validated, thus making false positives unlikely, there may have been false negatives cancer cases that were diagnosed after retirement. Indeed, 124 individuals died from a cancer without having self-reported a diagnosis of cancer or a hospitalization for cancer during the follow-up. Several of them died from a cancer with poor prognosis (e.g. lung cancer) and the lack of self-reported cancer incidence data may be due to the short time period between diagnosis and death. However, including these participants in the analysis yielded similar results. Owing to the possibility of false negatives, our results may partially result from reporting biases. For instance, the tendency to report cancer or to engage in screening procedures could have been lower in Type 1 and higher in Type 5.

Theses hypotheses are, however, unlikely. First, the number of self-reported diseases tended to be positively associated with Type 1 and negatively with Type 5. Second, Type 1 and Type 5 were not associated with an increased risk of prostate cancer diagnosis, which may capture both real incidence and excessive screening based on the prostate specific antigen, or with colorectal cancer, for which routine screening is recommended for everyone over 50 years in France [START_REF]La situation du cancer en France en 2011. Collection Rapports & synthèses[END_REF].

Since our study is observational, our findings should be interpreted with caution. For instance, our results might have been partially confounded by some unmeasured variables, such as genetic factors or early socioeconomic adversity, or mediated by unmeasured health behaviors, such as adherence to medical recommendations or preventive measures. For instance, the inverse association between Type 1 and the risk of breast cancer may have been partially explained by some specific risk factors such as exposure to estrogen, history of full-term pregnancies or breastfeeding. As regards potential mediators, personality factors have been found to be associated with behavioral risk factors such as smoking status or alcohol consumption. In the present study, the association of hostility with the incidence of smokingrelated cancers vanished after adjustment for health behaviors, as did similar trends regarding Type 1 and Type 5. Such findings could be of some relevance for cancer prevention. For instance, smoking cessation programs may benefit from taking into account the potential role of smoking in coping with hostile thoughts and feelings. Given the considerable role of smoking as a risk factor, one may argue that residual confounding due to inadequate adjustment may account for previous findings linking personality with smoking-related cancers [START_REF] Dalton | Mind and cancer: do psychological factors cause cancer?[END_REF]. It is thus noteworthy that smoking-related cancers were not included in the other sites category, which was significantly associated with Type 5 personality. Likewise, although Type 5 personality appeared to be connected with alcohol consumption, the risk of cancers at other sites was not. However, the measure of some health behaviors, such as diet or physical exercise, may have been too crude to rule out residual confounding.

Our results may also have been confounded by shared biological processes. For instance, immune and inflammatory processes that are involved in cancer onset and progression may also influence psychological processes that are eventually captured by personality measures [START_REF] Antoni | The influence of bio-behavioural factors on tumour biology: pathways and mechanisms[END_REF][START_REF] Cc | Molecular epidemiology, cancer-related symptoms, and cytokines pathway[END_REF][START_REF] Honkalampi | Alexithymia and tissue inflammation[END_REF]. Direct evidence linking personality with cancer risk through immune mechanisms is sparse in humans [START_REF] Todarello | Alexithymia, immunity and cervical intraepithelial neoplasia: a pilot study[END_REF][START_REF] Todarello | Alexithymia, immunity and cervical intraepithelial neoplasia: replication[END_REF]. However, the activity of some brain regions involved in the neural underpinnings of personality, such as the medial prefrontal cortex [START_REF] Lemogne | Negative affectivity, self-referential processing, and the cortical midline structures[END_REF], has been found to mediate the bi-directional relationships between immune and psychological processes among healthy subjects [START_REF] Harrison | Inflammation Causes Mood Changes Through Alterations in Subgenual Cingulate Activity and Mesolimbic Connectivity[END_REF][START_REF] Slavich | Neural sensitivity to social rejection is associated with inflammatory responses to social stress[END_REF]. Immune processes may therefore shape both cancer vulnerability and some aspects of personality, potentially accounting for the present results without any causal link between personality and cancer. Since combined analyses across cancer sites may blur associations, site-specific analyses are needed. Further studies may look for a specific association between Type 5 personality and cancer sites for which immune mechanisms are central, such as those associated with viral infection (e.g. Bcell lymphomas or cervical cancers) [START_REF] Antoni | The influence of bio-behavioural factors on tumour biology: pathways and mechanisms[END_REF] or those in which immune therapy have proven efficacy (e.g. malignant melanomas, kidney cancers) [START_REF] Kaufman | Vaccines for melanoma and renal cell carcinoma[END_REF]. Interestingly, most of theses cancers were included into the "other sites" category, suggesting promising research avenues for future studies with a greater number of incident cases of these cancers. Our study was indeed underpowered to further test this hypothesis, as a result of the rather young age of study participants at the end of the follow-up.

Other limitations should be considered. First, although the GAZEL cohort covers all regions of France, various areas ranging from small villages to large cities and a wide range of socioeconomic status and occupations, it is not representative of the general population as it includes only middle-aged working individuals with employment security and excluded certain categories of the population (e.g. agricultural workers, self-employed, foreigners) [START_REF] Goldberg | Cohort profile: the GAZEL Cohort Study[END_REF].

Furthermore, cancer incidence among the employees of the French national gas and electricity company was found to be lower in those who participated in the GAZEL Cohort Study than in those who did not [START_REF] Goldberg | Socioeconomic, Demographic, Occupational, and Health Factors Associated with Participation in a Long-term Epidemiologic Survey: A Prospective Study of the French GAZEL Cohort and Its Target Population[END_REF]. Although we believe that this selection is unlikely to have systematically biased our results, we cannot formally rule out this hypothesis. Third, Type 1 and Type 5 personality measures had low internal consistency and 3-month retest coefficients and Type A personality measure also had a low internal consistency. Poor measurement of these personality constructs might have contributed to the generally null pattern of results observed. Fourth, owing to the number of statistical tests performed, we cannot rule out a role of chance in our results. This might especially apply to the unexpected association between Type 1 and a reduced risk of breast cancer, as it did not demonstrate a clear dose-response relationship.

Although there is strong evidence to dismiss the relationships between general personality constructs and cancer risk [START_REF] Ranchor | Invited commentary: personality as a causal factor in cancer risk and mortality--time to retire a hypothesis?[END_REF], several personality constructs have not been

properly tested yet. To our knowledge, our study is the first that examined the association between cancer incidence and the personality types posited to be "cancer-prone" by Grossarth-Maticek and Eysenck [START_REF] Grossarth-Maticek | Personality, stress and disease: description and validation of a new inventory[END_REF]. Although one might have expected negative results, especially given the serious concerns surrounding the early reports by these authors [START_REF] Van Der Ploeg | What a wonderful world it would be: a reanalysis of some work of Grossarth-Maticek[END_REF][START_REF] Coyne | Meta-analysis of stress-related factors in cancer[END_REF], we found some support for a link between rational/anti-emotional tendencies and the risk of at least some cancers. Should these findings be replicated, further studies would be needed to explore the underlying mechanisms of this association, as potential prevention strategies should address the processes through which psychological processes may be associated with cancer, rather than these processes per se [START_REF] Boesen | Impact of psychotherapy on cancer survival: time to move on?[END_REF].

  , respectively. During a median follow-up of 16.0 years [range: 9 days-16 years], 1139 (8.3%)

Table 1 . Characteristics of the participants.

 1

		MEN (N=10,136	WOMEN (N=3,632)
	CONTINUOUS VARIABLES	Mean (SD) Missing: N (%) Mean (SD)	Missing: N (%)
	Age (years)	48.5 (2.9)	0 (0.0)	45.7 (4.2)	0 (0.0)
	Perceived health status	5.7 (1.3)	0 (0.0)	5.5 (1.3)	1 (0.0)
	CATEGORICAL VARIABLES	N (%)	Missing: N (%)	N (%)	Missing: N (%)
	Occupational grade		5 (0.0)		5 (0.1)
	Blue-collar workers, clerks	1092 (10.8)		787 (21.7)
	First-line supervisors, sales	5221 (51.5)		2452 (67.6)
	representatives
	Management	3818 (37.7)		388 (10.7)
	Alcohol consumption		295 (2.9)		88 (2.4)
	Non-drinkers	818 (8.3)		806 (22.7)
	Occasional and moderate drinkers	7472 (75.9)		2596 (73.3)
	Heavy drinkers	1551 (15.8)		142 (4.0)
	Smoking		64 (0.6)		12 (0.3)
	Never-smokers	3729 (37.0)		2450 (67.7)
	Ex-smokers < 20 pack-years	2792 (27.7)		527 (14.6)
	Ex-smokers ≥ 20 pack-years	976 (9.7)		362 (10.0)
	Current smokers < 20 pack-years	1399 (13.9)		97 (2.7)
	Current smokers ≥ 20 pack-years	1176 (11.7)		184 (5.1)
	Fruits consumption		516 (5.1)		252 (6.9)
	< 1 × / week	733 (7.6)		197 (5.8)
	1-2 × / week	1367 (14.2)		316 (9.3)
	> 2 × / week	7520 (78.2)		2867 (84.8)
	Vegetables consumption		586 (5.8)		269 (7.4)
	< 1 × / week	242 (2.5)		56 (1.7)
	1-2 × / week	3400 (35.6)		949 (28.2)
	> 2 × / week	5908 (61.9)		2358 (70.1)
	BMI		38 (0.4)		25 (0.7)
	<18.5	30 (0.3)		139 (3.9)
	18.5-24.99	4451 (44.1)		2700 (74.9)
	25-29.99	4895 (48.5)		594 (16.5)
	≥30	722 (7.1)		174 (4.8)
	Physical activity		592 (5.8)		308 (8.5)
	≥ 1 × / week	3493 (36.6)		1129 (34.0)
	Occasionally	3234 (33.9)		884 (26.6)
	None	2817 (29.5)		1311 (39.4)
	BMI: Body Mass Index; SD: Standard Deviation.

Table 2 . Associations between personality variables and discrete covariates.

 2 Body Mass Index; SD: Standard Deviation. ANOVAs were computed to examine the relation of personality scores with continuous and discrete covariates. a p<.05 for linearity and p≥.10 for deviation from linearity.

	Type 1		Type 5		Hostility		Type A
	Mean SD	p	Mean SD	p	Mean SD	p	Mean SD	p

Acknowledgments

The authors thank the French national gas and electricity company "Electricité de France-Gaz de France" (EDF-GDF), especially the "Service Général de Médecine de

Contrôle", and the "Caisse centrale d"action sociale du personnel des industries électrique et gazière". The GAZEL Cohort Study was funded by EDF-GDF and INSERM, and received grants from the "Cohortes Santé TGIR Program", "Agence nationale de la recherché" (ANR) and "Agence française de sécurité sanitaire de l"environnement et du travail" (AFSSET). The personality data collection was funded by the "Caisse Nationale d"Assurance Maladie" and by the "Ligue Nationale contre le Cancer". The present study was funded by the IReSP, the "Fondation d"entreprise du Groupe Pasteur Mutualité" and the "Société Mutualiste des Médecins du département de Paris". We also wish to acknowledge the "Cohortes" team of the Unit INSERM 1018-Versailles St-Quentin University responsible for the GAZEL data base management.

None of the authors have conflicts of interest to report. CL has accepted paid speaking engagements in industry-sponsored symposia from Astra Zeneca, Euthérapie, Lundbeck, Pierre Fabre and Pfizer. SMC has accepted paid speaking engagements in industry-sponsored symposia from Astra Zeneca, Euthérapie, Lilly, Lundbek, Merck Sharp Dohme and Pfizer.