domain: sdv info math

text:
Aroa Suárez-Vega

Beatriz Gutiérrez-Gil

Julio Benavides

Valentín Pérez

Gwenola Tosser-Klopp

Christophe C Klopp

Stephen J Keennel

Juan José Arranz

Valentín Perez

Combining GWAS and RNA-Seq Approaches for Detection of the Causal Mutation for Hereditary Junctional Epidermolysis Bullosa in Sheep

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

Introduction

Epidermolysis bullosa (EB) is a clinically and genetically heterogeneous group of inherited mechanobullous disorders caused by mutations in several structural skin proteins. EB is characterized by structural and mechanical fragility of the skin, leading to recurrent blistering and erosions that impair the life of EB affected individuals [START_REF] Fine | The classification of inherited epidermolysis bullosa (EB): Report of the third international consensus meeting on diagnosis and classification of EB[END_REF]. Following the latest classification [START_REF] Fine | Inherited epidermolysis bullosa: Updated recommendations on diagnosis and classification[END_REF], there are four major types of EB: EB simplex (EBS), junctional EB (JEB), dystrophic EB (DEB) and Kindler. This classification has been developed based on the level of blisters and the location of the aberrant proteins involved.

JEB encompasses all of the subtypes of EB that have mechanical fragility and blistering confined to the lamina lucida of the basement membrane and severe alteration of the hemidesmosomal junctions [START_REF] Fine | The classification of inherited epidermolysis bullosa (EB): Report of the third international consensus meeting on diagnosis and classification of EB[END_REF]. The hemidesmosomes are multi-protein complexes that play a critical role in the stable association of basal epithelial cells to the underlying basement membrane in stratified and pseudostratified epithelia [START_REF] Borradori | Structure and function of hemidesmosomes: More than simple adhesion complexes[END_REF]. Mutations in the genes encoding hemidesmosomal proteins and functionally associated structures have been shown to result in different subtypes of JEB [START_REF] Uitto | Epidermolysis bullosa: A spectrum of clinical phenotypes explained by molecular heterogeneity[END_REF][START_REF] Varki | Epidermolysis bullosa. I. molecular genetics of the junctional and hemidesmosomal variants[END_REF]. JEB occurs in various mammalian species [START_REF] Olivry | Absent expression of collagen XVII (BPAG2, BP180) in canine familial localized junctional epidermolysis bullosa[END_REF][START_REF] Spirito | Animal models for skin blistering conditions: Absence of laminin 5 causes hereditary junctional mechanobullous disease in the belgian horse[END_REF][START_REF] Milenkovic | A mutation in the LAMC2 gene causes the herlitz junctional epidermolysis bullosa (H-JEB) in two french draft horse breeds[END_REF][START_REF] Alhaidari | Junctional epidermolysis bullosa in two domestic shorthair kittens[END_REF][START_REF] Capt | Inherited junctional epidermolysis bullosa in the german pointer: Establishment of a large animal model[END_REF][START_REF] Graves | Partial deletion of the LAMA3 gene is responsible for hereditary junctional epidermolysis bullosa in the American saddlebred horse[END_REF][START_REF] Medeiros | Junctional epidermolysis bullosa in a calf[END_REF][START_REF] Peters | DNA-based diagnosis of rare diseases in veterinary medicine: A 4.4 kb deletion of ITGB4 is associated with epidermolysis bullosa in charolais cattle[END_REF]. In sheep, a Herlitz subtype of JEB has been described in German Black-headed Mutton sheep [START_REF] Kerkmann | Epidermolysis bullosa in german black headed mutton sheep[END_REF][START_REF] Momke | A frameshift mutation within LAMC2 is responsible for herlitz type junctional epidermolysis bullosa (HJEB) in black headed mutton sheep[END_REF][START_REF] Ostmeier | Inherited junctional epidermolysis bullosa (herlitz type) in german black-headed mutton sheep[END_REF].

In Spanish Churra sheep, JEB has been detected in two commercial flocks belonging to the National Churra Sheep Breeders' Association (ANCHE). The affected animals show erosions and ulcers of the skin and mucous membranes (Fig 1). Initial analyses showed that the JEB in Churra sheep is a recessive autosomal inherited defect. Microscopically, JEB with congenital absence of skin have been diagnosed. In humans, the concurrence of JEB and congenital absence of skin is described in a subtype of JEB known as JEB with pyloric atresia (JEB-PA) [START_REF] Fine | Inherited epidermolysis bullosa: Updated recommendations on diagnosis and classification[END_REF]. Pyloric atresia has not been identified in the affected Churra lambs. Because of the clinical and molecular heterogeneity of EB, there are some rare cases of JEB-PA-affected humans who did not show pyloric atresia [START_REF] Carmi | Aplasia cutis congenita in two sibs discordant for pyloric atresia[END_REF]. JEB-PA is related to mutations in the ITGA6 and ITGB4 genes [START_REF] Vidal | Integrin beta 4 mutations associated with junctional epidermolysis bullosa with pyloric atresia[END_REF][START_REF] Ruzzi | A homozygous mutation in the integrin alpha6 gene in junctional epidermolysis bullosa with pyloric atresia[END_REF][START_REF] Inoue | A homozygous missense mutation in the cytoplasmic tail of beta4 integrin, G931D, that disrupts hemidesmosome assembly and underlies non-herlitz junctional epidermolysis bullosa without pyloric atresia?[END_REF][START_REF] Nakano | Epidermolysis bullosa with congenital pyloric atresia: Novel mutations in the beta 4 integrin gene (ITGB4) and genotype/phenotype correlations[END_REF][START_REF] Birnbaum | Deletion of the first pair of fibronectin type III repeats of the integrin beta-4 gene is associated with epidermolysis bullosa, pyloric atresia and aplasia cutis congenita in the original carmi syndrome patients[END_REF].

In recent years, genome-wide mapping strategies have proven to be effective approaches for the identification of defective genes underlying diseases with Mendelian inheritance patterns [START_REF] Momke | A frameshift mutation within LAMC2 is responsible for herlitz type junctional epidermolysis bullosa (HJEB) in black headed mutton sheep[END_REF][START_REF] Shariflou | Brachygnathia, cardiomegaly and renal hypoplasia syndrome (BCRHS) in merino sheep maps to a 1.1-megabase region on ovine chromosome OAR2[END_REF][START_REF] Suarez-Vega | Identification of a 31-bp deletion in the RELN gene causing lissencephaly with cerebellar hypoplasia in sheep[END_REF]. The methods to identify the causal mutation in a Mendelian disorder generally involve the detection of a disease locus followed by candidate gene sequencing. Recently, Next Generation Sequencing (NGS) technologies have enabled high-throughput functional genomic research to be performed [START_REF] Morozova | Applications of next-generation sequencing technologies in functional genomics[END_REF]. High-throughput sequencing of RNA (RNA-seq) has been developed to identify and quantify the gene expression in different tissues [START_REF] Mortazavi | Mapping and quantifying mammalian transcriptomes by RNA-seq[END_REF][START_REF] Wang | RNA-seq: A revolutionary tool for transcriptomics[END_REF]. RNA-seq also offers new opportunities for efficient and cost-effective detection of transcriptome variants (SNPs and short indels) in different tissues and species [START_REF] Canovas | SNP discovery in the bovine milk transcriptome using RNA-seq technology[END_REF][START_REF] Cox | A genome resource to address mechanisms of developmental programming: Determination of the fetal sheep heart transcriptome[END_REF][START_REF] Djari | Gene-based single nucleotide polymorphism discovery in bovine muscle using next-generation transcriptomic sequencing[END_REF]. In humans, recent studies have highlighted the advantages of using NGS in EB diagnostics [START_REF] Poulter | Whole-exome sequencing, without prior linkage, identifies a mutation in LAMB3 as a cause of dominant hypoplastic amelogenesis imperfecta[END_REF][START_REF] Takeichi | Whole-exome sequencing improves mutation detection in a diagnostic epidermolysis bullosa laboratory[END_REF].

In the present study, our aim was to find the causative mutation underlying JEB in Spanish Churra sheep. For this goal, a SNP array was used to map the causative region of JEB, and then, the disease-causing mutation was identified via an RNA-seq variant analysis. This study demonstrates the effectiveness of combining both approaches to map and identify the causal mutations in recessive Mendelian diseases.

Results

Mapping the causative gene for JEB

Approximately 44,800 evenly spaced SNPs were genotyped in 20 JEB-affected lambs and 28 related unaffected and 48 unrelated control sheep. A significant genome-wide association between the SNP genotypes and JEB was shown for sheep chromosome 11 (OAR11) (Fig 2A). The marker rs410387229, located at position OAR11:54,939,690 bp of the OARv3.1 sheep genome assembly, showed a genome-wide corrected p-value of 0.00001. There were three additional SNP markers on OAR11, rs418150485, rs425824336 and rs412661420, located at positions 56,897,973, 54,003,142 and 58,681,220 bp, respectively, with corrected p-values < 0.0001.

The location of the JEB locus was refined using the identical-by-descent (IBD) mapping method. The IBD analysis narrowed the defective region to an 868-kb homozygous segment, from 54,632,309-55,500,100 bp on OAR11, in all 20 affected lambs (Fig 2B). The consensus region contains 16 consecutive SNP loci. Regarding the chromosomal location of the genes related to JEB in humans (OMIM #226730: ITGB4,ITGA6), the ITGB4 gene was identified as a promising positional and functional candidate, as it is located at 54,848,050-54,874,496 on OAR11, which is within the identified block of homozygosis common to all of the cases (Fig 2C). The ITGB4 gene codes for integrin β4 protein. In stratified epithelia, integrin α6β4 is located in specialized epidermal cell-basement membrane adhesion structures, called hemidesmosomes [START_REF] Stepp | Alpha 6 beta 4 integrin heterodimer is a component of hemidesmosomes[END_REF].

Ovine ITGB4 gene sequence and annotation

Because of its association with human JEB [START_REF] Carmi | Aplasia cutis congenita in two sibs discordant for pyloric atresia[END_REF][START_REF] Birnbaum | Deletion of the first pair of fibronectin type III repeats of the integrin beta-4 gene is associated with epidermolysis bullosa, pyloric atresia and aplasia cutis congenita in the original carmi syndrome patients[END_REF][START_REF] Chavanas | Splicing modulation of integrin beta4 pre-mRNA carrying a branch point mutation underlies epidermolysis bullosa with pyloric atresia undergoing spontaneous amelioration with ageing[END_REF], the ITGB4 gene was identified as the most promising candidate. Our first aim was to identify the causal mutation via Sanger sequencing of the ITGB4 gene in four JEB cases and four unrelated controls. Through the Sanger approach for genomic sequencing, we were able to obtain 84% (29,936 bp) of the complete genomic sequence of the ITGB4 gene (ENSOARG00000009764), in the four cases and four controls. By comparing the sequences of the unaffected and the JEB-affected lambs, we identified eight single nucleotide polymorphisms (SNPs) that had have different genotypes in the cases (g.54857417T>G. g.54857293C>T, g.54856642A>G, g.54853969C>G, g.5485255C>A, g.54851357C>T, g.54850814C>T and g.54848707G>A). All of the identified SNPs were located in non-coding ITGB4 regions. A search for the SNPs in the International Sheep Genomic Consortium (ISGC) database (https://data.csiro.au/dap/landingpage?execution = e1s2) allowed us to discard these mutations as causal mutations, as all of them were already published on healthy sheep from other breeds.

Because of the difficulties in performing Sanger DNA sequencing of the whole gene using the ITGB4 reference sequence available in Ensembl (ENSOARG00000009764), we isolated the ITGB4 sequence from three ovine BAC clones (CH243-344E15, CH243-306J7 and CH243-504K6) [START_REF] Dalrymple | Using comparative genomics to reorder the human genome sequence into a virtual sheep genome[END_REF]. The BAC clone sequencing allowed us to obtain the complete genomic sequence of ITGB4 (GenBank: KP025765). Pairwise alignment between the sheep ITGB4 reference obtained via BAC clone sequencing and the ITGB4 reference available in Ensembl (ENSOARG00000009764) revealed an identity of 83.1% between the two sequences and a gap percentage of 16.6%. Excluding the 5'-and 3'UTR regions that were not available in the Ensembl sequence, 14 gaps, ranging from 2 to 1119 bp, were detected (S1 Fig).

In parallel to the BAC clone sequencing, an RNA-seq analysis was conducted. Sequencing of the cDNA libraries from the RNA skin samples from one JEB case and one control generated a total of 69.31-million paired-end reads, with an average of 34.65-million paired-end reads for each sample. The aligner STAR [START_REF] Dobin | STAR: Ultrafast universal RNAseq aligner[END_REF] was used to map the paired-end reads from each sample to the ovine reference genome sequence (Oar_v3.1). Approximately 83.54% of the reads (85.69% in the case and 81.39% in the control) were mapped to a unique location in the ovine v3.1 genome. The combination of the ITGB4 sequence obtained from the BAC clone with the RNA-seq data from the control allowed us to predict the complete CDS and the integrin β4 protein sequence. To determine the gene structure of the ITGB4 sequence, Augustus software was used [START_REF] Stanke | Using native and syntenically mapped cDNA alignments to improve de novo gene finding[END_REF][START_REF] Keller | A novel hybrid gene prediction method employing protein multiple sequence alignments[END_REF]. In total, of 5360 reads that were overlapping the ITGB4 region in the control sample were used to generate a hints file for Augustus. A complete CDS of 5256 bp was found. This transcript is distributed into 38 exons. The sheep ITGB4 CDS sequence encodes a predicted protein of 1751 amino acids. The BLAST tool (http://blast.ncbi.nlm.nih.gov/Blast.cgi) was used to compare the predicted amino acid sequence from the ovine ITGB4 gene with several available sequences, including the cow (DAA18168.1), camel (EQB77252.1) human (aai43743.1) and mouse (NP_598424.2) sequences. The predicted ovine integrin β4 protein sequence showed 92% homology with the bovine protein, 92% homology with the camel sequence, 89% homology with the human, and 88% homology with the mouse sequence.

Detection of the genetic variation between the JEB-affected and unaffected skin samples via RNA-Seq GATK [START_REF] Mckenna | The genome analysis toolkit: A MapReduce framework for analyzing next-generation DNA sequencing data[END_REF] was used for variant detection in the RNA-seq data from the JEB-affected and unaffected samples. GATK can identify single base substitutions in addition to small insertions and deletions. In total, 495921 different variants were detected using the RNA-seq reads in both samples. The VEP tool from Ensembl processed a total of 490,196 variants, among which 5.2% (25247) corresponded to already-described variants and 94.8% (464949) corresponded to novel variants. There were 20,828 variants overlapping with known transcripts. Most of the variants detected were SNPs (91.5%), and 8.4% (40956) were indels.

Among the variants, we selected those that overlapped with the ITGB4 gene. In total, 54 variants were found in the ITGB4 region (OAR11: 54,848,050-54,874,496). Following the assumption that JEB in the Churra sheep is caused by a homozygous recessive mutation, the variants were sifted to retain those present at a homozygous stage in the affected case (1/1) and absent in the control animal (0/0). Fourteen variants matched this filter: nine intron variants, three synonymous variants, one missense variant, and one frameshift variant and feature truncation. We discarded the mutations located in introns, which had average DP (coverage) values of 3.2 in the case and 5.6 in the control, and the synonymous variants.

The missense variant detected in the RNA-seq variant analysis is a G>A substitution located at OAR11, which was 54,868,894 bp of the sheep Oar_v3.1 assembly. Regarding the ITGB4 sequence (GenBank: KP025765) the mutation is positioned at 9,381 bp of the ITGB4 genomic sequence. In the predicted CDS, the SNP is located at 1337 bp (c.1337G>A), in exon ten of the ITGB4 gene (Fig 3). The mutation leads to an arginine-to-glutamine residue change at position 446 of the integrin β4 protein (G>A p.Arg446Gln). The genotype quality (GQ) value for c.1337G>A was 99 for the control and 42 for the case. In general, the determined coverage (DP value) for the ITGB4 variants was higher in the control lamb than in the case; for this SNP the DP value was 75 and 14, respectively. This non-synonymous SNP has been observed at a homozygous stage in other sheep breeds (ISGC SNP database) and was predicted to be non-deleterious by SIFT [START_REF] Kumar | Predicting the effects of coding non-synonymous variants on protein function using the SIFT algorithm[END_REF] and PolyPhen-2 [START_REF] Adzhubei | A method and server for predicting damaging missense mutations[END_REF]. Therefore, the c.1337G>A variant was discarded as a putative causal mutation of JEB.

The frameshift variant detected in the RNA-seq variant analysis is a four-bp deletion located at OAR11:54,849,767-54,849,770 bp of sheep Oar_v3.1 assembly. In the ITGB4 sequence (GenBank: KP025765) the mutation is positioned at 30,204-30,207 bp of the ITGB4 genomic sequence. In the predicted CDS, the deletion is located at 4,412-4,415 bp (c.4412_4415del). The GQ value for the deletion was 99 for the control and 35 for the case, and the DP value was 180 and 11, respectively. This mutation is located on sheep ITGB4 predicted exon 33 and leads to a shift in the open reading frame of the ITGB4 coding sequence and a stop codon at position 1,473 (Fig 3). This mutation could lead to a truncated protein of 1,472 amino acids [START_REF] Fine | The classification of inherited epidermolysis bullosa (EB): Report of the third international consensus meeting on diagnosis and classification of EB[END_REF]470 amino acids of normal integrin β4 followed by two missense amino acids and a premature termination codon) in contrast to the 1,751-amino-acid length of the normal integrin β4 protein.

Based on the deleterious effect on the composition and structure of the resulting protein, this 4-bp deletion was identified as the putative causal mutation for JEB in sheep.

Functional analysis identifies ITGB4 as a causative gene

To quantify the levels of the ITGB4 mRNA in the affected lambs and compare them with the levels in unaffected animals, we performed qRT-PCR using six replicates for each condition. The qRT-PCR confirmed that the ITGB4 transcript levels were significantly lower in the cases than in the controls (relative expression average: 8.3 in the cases vs 100 in the controls; P<0.001). The mRNA expression analysis suggested that the JEB was likely caused by reduced expression of ITGB4 in the skin.

Immunohistochemical labeling of integrin β4 showed the absence of this protein in the skin of lambs with gross JEB lesions, whereas it was widely expressed in the basal cell layer in the control animal (Fig 4). The labeling of the two other epidermal basement membrane proteins that were studied, collagen VII and integrin α6, was similar between the control and the affected lambs. In the lambs showing gross JEB lesions, collagen VII was expressed primarily at the base of the blister, whereas integrin α6 was found at the roof. This labeling pattern confirms the localization of the cleavage between the lamina lucida and the lamina densa of the basement membrane (Fig 4).

Validation of c.4412_4415del at the DNA level

After the putative causal mutation in the RNA-seq variant analysis was identified, the mutation was confirmed via analysis of 46 samples at the genomic DNA level. The association between the c.4412_4415del allelic variant and the JEB phenotype was confirmed by genotyping the deletion in the gDNA sequences of affected, alleged carrier and control animals. From the DNA samples, the amplicon of the putative exon 33 was obtained via PCR for 27 cases, 10 unrelated controls and sires and dams of affected lambs. The resulting PCR product was sequenced and analyzed using SeqScape v2.5 (Applied Biosystems). All of the samples from the JEB-affected animals were homozygous for the identified 4-bp deletion (c.4412_4415del/c.4412_4415del or EB/EB), the sires and dams for later animals were heterozygous (EB/+) and the controls from the different flocks were homozygous (+/+) for the wild-type allele.

Discussion

We herein demonstrated that a 4-bp deletion within exon 33 of ITGB4 gene (c.4412_4415del) is responsible for JEB in Churra sheep, based on medium-resolution mapping and RNA-seq variant analysis. The JEB locus was mapped to Ovis aries chromosome 11 via a GWA analysis using cases, related animals and unrelated controls. The region was fine-mapped to an 868-kb homozygous segment using IBD analysis. The ITGB4 gene, which is located within this region, was the best positional and functional candidate gene. The RNA-seq variant analysis of control and JEB-affected skin RNA samples allowed us to find the causal mutation (c.4412_4415del). In this study, we found that individuals that are homozygous for this mutation lack the integrin β4 polypeptide. The mutation was confirmed at the genomic level, and three genotypes segregated with the disease status: homozygous mutants (EB/EB) were the affected lambs, heterozygous animals (EB/+) corresponded to the phenotypically normal carriers (ewes and rams of the affected lambs), and the wild-type genotype (+/+) was identified in all of the analyzed control individuals. These genotypes completely agreed with the autosomal recessive mode of Mendelian inheritance inferred for JEB in Churra sheep. The discovery of this mutation has allowed screening of affected flocks to identify phenotypically normal individuals carrying the 4-bp deletion to avoid at-risk mattings. Simple and rapid JEB genotyping methods like fragment length analysis in capillary electrophoresis sequencer and restriction fragment length polymorphism analysis are proposed (S2 Fig) . The implementation of this gene-assisted selection approach will solve the problem of JEB in flocks of Churra sheep.

De novo BAC clone sequencing allowed us to obtain the complete ITGB4 gene sequence (GenBank KP025765). The previous ITGB4 ovine gene sequence (ENSOARG00000009764) was missing 16.6% of the bases when compared with KP025765 (S1 Fig) . The RNA-seq approach enables the generation of extensive transcriptome information in the absence of a wellannotated genome. In the present study, the transcriptome information was essential in the annotation of the ITGB4 gene sequence (GenBank: KP025765). The sheep ITGB4 CDS obtained from the KP025765 sequence is distributed into 38 exons and encodes a predicted protein of 1751 amino acids. The transcript available in Ensembl (ENSOART00000010637) has 42 exons and encodes a predicted protein of 1835 residues. The percentage of identity of the ovine integrin β4 protein with that of other species, such as cow (DAA18168.1) or human (AAI43743.1), is higher for the integrin β4 protein obtained from KP025765 (92% and 89%, respectively) than for the sequence obtained from the ENSOART00000010637 transcript (86% and 82%, respectively), which suggests that the ITGB4 sequence obtained in this study is more reliable than the available sequence from the ovine genome assembly (Oar_v3.1).

Integrin β4 associates with integrin α6 to form a transmembrane constituent of hemidesmosomes anchoring complex [START_REF] Borradori | Structure and function of hemidesmosomes: More than simple adhesion complexes[END_REF]. Mutations in α6β4 have been associated with JEB-PA in humans (OMIM: #226730). Although several mutations have been described in these two genes, most mutations were found in the ITGB4 gene (approximately 70), whereas only five cases have been reported in the ITGA6 gene [START_REF] Schumann | Phenotypic spectrum of epidermolysis bullosa associated with alpha6beta4 integrin mutations[END_REF]. In this study we found a causal mutation in the ITGB4 gene which leads into JEB in sheep. Several forms of EB have been associated with congenital absence of skin, primarily with DEB (Bart type (OMIM: #132000)). Churra lambs lack PA; however, previous reports on humans suggest that PA is not necessarily a feature of integrinassociated JEB [START_REF] Schumann | Phenotypic spectrum of epidermolysis bullosa associated with alpha6beta4 integrin mutations[END_REF][START_REF] Lee | ITGB4-associated junctional epidermolysis bullosa without pylori atresia but profound genito-urinary involvement[END_REF]. A recent report about JEB in cattle due to a deletion in ITGB4 also describes the absence of PA [START_REF] Peters | DNA-based diagnosis of rare diseases in veterinary medicine: A 4.4 kb deletion of ITGB4 is associated with epidermolysis bullosa in charolais cattle[END_REF]. Lack of PA could be due to the particular anatomical characteristics of ruminant's pyloric sphincter, less muscular than in monogastric animals. The resemblance between the integrin β4-associated EB lesions described in humans [START_REF] Vidal | Integrin beta 4 mutations associated with junctional epidermolysis bullosa with pyloric atresia[END_REF], cattle [START_REF] Peters | DNA-based diagnosis of rare diseases in veterinary medicine: A 4.4 kb deletion of ITGB4 is associated with epidermolysis bullosa in charolais cattle[END_REF] and ovine JEB supports the causative nature of the 4-bp deletion in the ITGB4 gene that was reported here in relation to the JEB phenotype observed in Churra sheep.

The 4-bp deletion on putative exon 33 of the ITGB4 gene (c.4412_4415del) results in a premature termination codon (PTC) and is predicted to delete the region spanning the last 281 amino acids in the intracellular domain (Fig 3), which has been shown to interact with the 180-kD bullous pemphigoid antigen [START_REF] Aho | Direct interaction between the intracellular domains of bullous pemphigoid antigen 2 (BP180) and beta 4 integrin, hemidesmosomal components of basal keratinocytes[END_REF]. This results in the loss of the C-terminal region, including the third and fourth FNIII repeats. The lack of labeling by the monoclonal antibody (clone 450-10D) used against integrinβ4 is consistent with this prediction, as this antibody binds to the intracellular region of the integrin [START_REF] Kennel | Second generation monoclonal antibodies to the human integrin alpha 6 beta 4[END_REF]. The persistency of the extracellular domain could enable the formation of an α6β4 dimer. Also supporting this possibility, is the wide expression of integrin α6, which was observed via immunohistochemistry at its physiological location, the lamina lucida. However, the highly decreased amount of mRNA in the JEB skin samples compared with the control samples (identified through the qRT-PCR analysis) suggests that the truncated mRNA products are degraded by nonsense-mediated mRNA decay (NMD) machinery. Therefore, the absence of both the extracellular and intracellular domains, of integrin β4 cannot be ruled out, as deficiency of the whole protein would also produce a lack of labeling by the monoclonal antibody specific for the intracellular epitope. In humans, mutations related with PTC in both alleles are predominantly associated with lethal forms of the disease, whereas missense mutations are associated with mid-range phenotypes [START_REF] Pulkkinen | Novel ITGB4 mutations in lethal and nonlethal variants of epidermolysis bullosa with pyloric atresia: Missense versus nonsense[END_REF]. However, lethal/non-lethal consequences are primarily associated with the existence of a functional integrin β4, which, is why the immunological labeling of the protein is also important in the diagnostic procedure [START_REF] Nakano | Epidermolysis bullosa with congenital pyloric atresia: Novel mutations in the beta 4 integrin gene (ITGB4) and genotype/phenotype correlations[END_REF]. Similar to the integrin β4 subunit, when transmission electron microscopy (TEM) is performed in the skin samples of JEB lambs we could see that hemidesmosomes were also absent. Schaapveld et al (1998) [START_REF] Schaapveld | Hemidesmosome formation is initiated by the beta4 integrin subunit, requires complex formation of beta4 and HD1/ plectin, and involves a direct interaction between beta4 and the bullous pemphigoid antigen 180[END_REF] indicated that keratinocytes lacking β4 expression are not capable of forming organized hemidesmosome-like structures. No hemidesmosome structures were found in ITGB4 knock-out mouse [START_REF] Van Der Neut | Epithelial detachment due to absence of hemidesmosomes in integrin beta 4 null mice[END_REF], it was thus proposed that alternative adhesion molecules, such as integrin α3β1, could explain the residual adhesion of the non-detached skin areas.

In conclusion, we have identified the causal mutation for ovine JEB in Churra sheep. This mutation (c.4412_4415del) is located in putative exon 33 of the ITGB4 gene and leads to the formation of a premature termination codon without protein expression. The ITGB4 gene can be rapidly applied as a selection marker for identification of carriers to avoid at-risk mattings, enabling the eradication of this lethal mutation from the Churra sheep breed. We have also demonstrated the successful combination of medium-density SNP panels with RNA-seq approaches for localization of mutant locus causing a recessive defect, which can serve as a reference for other studies performed in species in which high resolution exome sequencing is not straightforward. Furthermore, given the relatively low cost of maintaining the carriers, sheep with JEB could be used as a valuable large-animal model for the study of this disease and the development of therapeutic approaches.

Material and Methods

Ethics statement

The blood and tissue samples were collected from rams, ewes and lambs by qualified veterinarians following standard procedures and conducted under a license issued in accordance with the European Union legislation (European Community Directive, 86/609/E and Directive 2010/63/EU of the European Parliament and of the Council). All of the animals were managed in accordance with the guidelines for the accommodation and care of animals. The DNA samples used in this study were extracted from leucocytes in 3 ml of blood obtained via jugular venipuncture. The tissues were collected immediately after euthanasia (performed by a qualified veterinarian via intravenous injection of T-61 (Intervet)); animals were euthanized as standard of care. As the samples were from two commercial flocks that underwent veterinary examination, we were in a special situation in veterinary medicine and there was no "animal experiment" according to the legal definitions in Spain (Animal care legislation "Ley 32/2007"). According to the Ethics Commission of the University of León, formal ethical approval is not required under these circumstances.

Animals

Blood and/or tissue samples were collected from a total of 27 JEB-affected lambs from two commercial flocks belonging to the Spanish Churra Sheep Breeders' Association (ANCHE). Additionally, samples from 28 healthy Churra individuals from the same flocks were collected. The samples included the parents, siblings and half-siblings of the JEB-affected offspring. Fifty samples from unaffected Churra sheep from different flocks with unknown relatedness to the affected lambs were collected for use as controls.

Genome-wide association and homozygosity mapping studies

In total, 96 DNA samples were genotyped using the Illumina OvineSNP50 BeadChip. These samples included 48 unrelated, healthy Churra individuals from different flocks of the Churra Selection Nucleus and 48 animals from the affected flock. Twenty of the animals from the affected flock were JEB lambs, and the rest of the animals were related to them (sires, dams, siblings and half-siblings).

The results were analyzed using PLINK, v1.07 [START_REF] Purcell | PLINK: A tool set for wholegenome association and population-based linkage analyses[END_REF]. A quality control procedure was performed to eliminate SNPs with genotyping rates lower than 0.05 and minor allele frequencies lower than 0.01 and to eliminate animals with call rates lower than 0.95. After the QC pruning, 44,785 SNPs were considered in the subsequent analysis. The-assoc option in PLINK was applied to perform a case-control genome-wide association study (GWAS). To correct empirical p-values, a one-million permutation procedure was performed.

After the GWAS was performed, a run of homozygosity or identity-by-descent (IBD) analysis was performed. The run of homozygosity is performed by filtering the cases to identify the allele-sharing regions. The SNPs flanking the consensus region obtained from this analysis were used to trace this region in the Ovine Genome Assembly Oar_v3.1 Browser (http:// gbrowse-ext.bioinformatics.csiro.au/fgb2/gbrowse/oarv3/). The genes identified within the homozygosity block were evaluated as putative positional candidate genes.

Isolation of the ovine ITGB4 gene

ITGB4 was isolated from three sheep CHORI-243 bacterial artificial chromosome (BAC) libraries [START_REF] Ratnakumar | Quality control of the sheep bacterial artificial chromosome library, CHORI-243[END_REF]. The selected BAC clones predicted to span the region in which the ITGB4 gene is located were CH243-344E15, CH243-306J7 and CH243-504K6. The BACMAX DNA Purification Kit (Epicentre) was used for purification of the DNA.

Ion Torrent PGM data were generated from the three BAC libraries constructed from 500 ng of DNA quantitated using the Qubit Fluorometer 2.0 (Life Technologies, Grand Island, NY). The fragment libraries were constructed using the Ion Xpress Plus Fragment Library kit based on Ion Shear chemistry according to the user guide. The barcode and adaptor ligation were performed per the manufacturer's protocol using the Ion Xpress Barcode Adapters 1-16 Kit. The quantitation and size distribution of the fragments were analyzed on an Agilent Bioanalyzer using the High Sensitivity Kit (Agilent). The template preparation and emulsion PCR were performed using the Ion PGM Template OT2 200 Kit (Life Technologies) according to the manufacturer's instructions. The template product then was quantitated using the Ion Sphere Quality Control Kit on at Qubit Fluorometer 2.0. OneTouch ES was used for the enrichment of the Ion Sphere Particles (ISP) template products. Finally, the samples were loaded onto a 316 chip V2 and sequenced using the Ion PGM 200 Sequencing Kit V2 (Life Technologies).

The reads were collected using Ion Torrent Suite v4.0, which also sorts the data according to the barcodes. The Torrent Suite software scores the quality of the reads by assigning Q20 scores according to Ion Torrent's quality scoring computation. MIRA (version 3.4.0) was used for de novo assembly of all of reads from each of the three BACs. The mean coverage was 120X for each BAC. The Torrent Mapping Alignment Program (TMAP) was used for alignment of all of the reads from each of the three BACs against the reference (Chr 19 Cow sequence; chromosome: UMD3.1:19:1:64057457:1). Both, the BAC DNA sequencing and the de novo assembly were performed at AC-gen Reading Life (Valladolid, Spain).

RNA-seq analysis

For the RNA-seq analysis, skin samples from a control lamb and a JEB-affected lamb were collected for RNA extraction. The skin samples were harvested immediately after euthanasia and preserved in RNA Stabilization Reagent (RNAlater, Ambion). Slices of up to 500 mg of skin tissue were processed using the RNeasy Fibrous Tissue Midi Kit (Qiagen) to extract the total RNA. RNA integrity and concentration were evaluated using an Agilent Technologies 2100 Bioanalyzer. Both samples had RNA Integrity Number (RIN) values higher than 7.3. The True-Seq RNA Sample Prep Kit v2 (Illumina) was used to prepare paired-end libraries with fragments of 300 bp. Two paired-end cDNA libraries were constructed, one for each of the two samples (JEB-affected and unaffected skin samples). The cDNA libraries were loaded onto the flow cell channels of an Illumina Hi-Seq 2000 platform to perform massive parallel sequencing at the CNAG (Centro Nacional de Análisis Genómico, Barcelona), generating 75-bp pairedend reads.

The Ovis aries genome, the annotation file and the reference variant file were downloaded from the Ensembl database (http://www.ensembl.org/index.html). The quality of the reads generated was assessed using the program FastQC (http://www.bioinformatics.babraham. ac.uk/projects/fastqc/). The paired ends for each sample were aligned to Oar v3.1 using STAR [START_REF] Dobin | STAR: Ultrafast universal RNAseq aligner[END_REF].

For the ITGB4 gene prediction, Augustus software was used [START_REF] Stanke | Using native and syntenically mapped cDNA alignments to improve de novo gene finding[END_REF][START_REF] Keller | A novel hybrid gene prediction method employing protein multiple sequence alignments[END_REF]. Once the alignment to the reference genome was performed, Samtools [START_REF] Li | The sequence alignment/map format and SAMtools[END_REF] was used to sort, index and extract the IDs of the reads aligned within the ITGB4 region from the bam files (OAR11: 54,830,000-54,880,000). Seqtk tool (https://github.com/lh3/seqtk) was used with the ID list from the bam file and the initial fastq file to create a new fastq file that contained only the reads that align within the ITGB4 region. The new fastq files were mapped against the ITGB4 sequence obtained from the BAC clones using STAR. Cufflinks [START_REF] Roberts | Identification of novel transcripts in annotated genomes using RNA-seq[END_REF] was used to assemble the bam files and to create an exonpart hints file for Augustus. The putative coding transcript for the ITGB4 BAC clone sequence was identified with Augustus by providing the ITGB4 sequence, exon boundaries and an integrin β4 protein profile and allowing the program to predict exactly one gene.

For variant detection, GATK was used [START_REF] Mckenna | The genome analysis toolkit: A MapReduce framework for analyzing next-generation DNA sequencing data[END_REF]. The GATK analysis was performed on the STAR alignments following the RNA-seq workflow recommended in the best-practices section of the webpage for the GATK software [START_REF] Depristo | A framework for variation discovery and genotyping using next-generation DNA sequencing data[END_REF][START_REF] Van Der Auwera | From fastq data to high-confidence variant calls: the Genome Analysis Toolkit best practices pipeline[END_REF]. Prior to the variant analysis, the PCR duplicates from the bam files were removed using Picard tools (http://picard.sourceforge.net/). Then, an indel realignment and a base quality score recalibration were performed using the Ovis aries vcf file downloaded from Ensembl as a reference. These steps were followed by the variant calling analysis performed using the "-T HaplotypeCaller" option of GATK. The biological impact predictions for detected variants were obtained using Ensembl Variant Effect Predictor (VEP: http://www.ensembl.org/tools.html). Once the variant calling analysis was performed, the variants located in the ITGB4 region were selected. These variants were filtered to keep only those located in exons homozygous for the reference sequence in the control and homozygous and distinct from the reference in the case.

Quantification of the ITGB4 mRNA via qRT-PCR

Total RNA was isolated from the skin using the RNeasy Fibrous Tissue Midi Kit (Qiagen) according to the manufacturer's instructions. The-high-capacity cDNA Reverse Transcription Kit (Applied Biosystems) was used to synthesize the DNA according to the manufacturer's instructions. The primers were designed using Primer3 [START_REF] Untergasser | Primer3-new capabilities and interfaces[END_REF][START_REF] Koressaar | Enhancements and modifications of primer design program Primer3[END_REF] resulting in two primer sets (S1 Table ) spanning the region containing the 4-bp-deletion. One primer set produced a 334-bp product and the second produced a 182-bp product. The expression profiling of ITGB4 was performed on a StepOne Real-Time PCR System (Applied Biosystems) with the Power SYBR Green PCR Master Mix according to the manufacturer's instructions. ITGB4 transcriptspecific expression was evaluated via relative quantification against the sheep glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression level (ΔCt) using one of the control homozygous replicates samples as a calibrator.

Immunohistochemical studies

Samples of grossly normal skin (metacarpal region) were taken from three lambs showing gross JEB lesions. Control samples, taken from the same area, were also obtained from one lamb from the same flock that was euthanized because of diarrhoea and not affected by JEB.

The samples were snap-frozen immediately after euthanasia in 2-methylbutane cooled in liquid nitrogen and stored at -70°C until used. Frozen sections (5 μm) were cut with a cryostat, collected on slides and fixed in acetone for 10 min. The samples underwent immunohistochemical examination with monoclonal antibodies against collagen VII, clone LH7.2, (100 dilution; Neomarkers), integrin α6, clone GoH3 (100 dilution; Abcam) and integrin β4, clone 450-10D (2000 dilution; [START_REF] Kennel | Second generation monoclonal antibodies to the human integrin alpha 6 beta 4[END_REF]) was carried out, using Dako EnVision System-HRP (Dako North America Inc.) with 3,3'diaminobenzidine (Dako North America Inc.) as chromogen substrate.

Confirmation and genotyping of the causal mutation DNA was extracted from the blood samples collected from the 27 cases, the 6 mothers and 3 half-brothers of the affected animals, and the 10 unrelated controls. Primers (ITGB4ovi-ne_ex33fw: 5'-CCAGCAGTCAGGGAGGTG and ITGB4ovine_ex33rv: 5'-CTCACCGCCGTT CAGCAG) were designed using Primer3 [START_REF] Untergasser | Primer3-new capabilities and interfaces[END_REF][START_REF] Koressaar | Enhancements and modifications of primer design program Primer3[END_REF]. The primers ITGB4ovine_ex33fw and ITG-B4ovine_ex33rv amplify a 243-bp fragment of the gene sequence that contains the 4-bp deletion that was identified has the putative causal mutation. The products were amplified via PCR in a 30 μL reaction volume containing 100 ng of gDNA, 480 μM dNTPs, 1.5 μM MgCl2, buffer 10X diluted 1X (Applied Biosystems), the specific primers at 0.5 μM, 0.25 M betaine and 0.75 μl of AmpliTaq Gold polymerase (5 U/ml) (Applied Biosystems). The PCR conditions were 5 min at 96°C; 40 cycles of 30 sec at 96°C, 45 sec at 58°C, and 40 sec at 72°C; and then a final extension of 10 min at 72°C. The amplicons were purified using Illustra Exonuclease I and alkaline phosphatase (Illustra ExoProStar 1-Step, GE Healthcare Life Sciences) treatment and were dideoxy-sequenced in both directions using the Big Dye Terminator Cycle Sequencing Kit, v3.1 (Applied Biosystems) using the same primers used for the fragment amplification. After sequencing on an ABI Prism 3130 automatic sequencer (Applied Biosystems), the sequence data were analyzed using SeqScape v2.5 (Applied Biosystems).

Fig 1 .

 1 Fig 1. Clinical features of JEB in Churra sheep. (A) Affected lamb showing skin lesions at the ears and limbs. (B) Erosions in the tongue. (C) Affected lamb showing severe skin lesions that involve the ears, limbs and caudal part of the body. doi:10.1371/journal.pone.0126416.g001

Fig 2 .

 2 Fig 2. Workflow chart for the discovery of the mutant locus of inherited JEB in Churra sheep. (A) Manhattan plot that shows the results of the casecontrol GWA analysis performed. The X-axis shows the positions of the SNPs analyzed across the 26 ovine autosomes; different colors represent each different chromosome. The Y-axis represents the -log10P values obtained after performing 1 million permutations using PLINK. The most significant associated SNP is located at position 54,939,690 bp on OAR11. (B) IBD analysis on OAR11. The analysis of SNPs genotypes from affected lambs shows an extended homozygous region on chromosome 11. All twenty affected lambs shared overlapping homozygous blocks (black and yellow blocks, which represent the genes within the homozygous block of each animal). The consensus haplotype block (indicated by a solid red box) that contains the putative causal mutation expands a region of 868-kb, from 54,632,309-55,500,100 bp on OAR11. (C) Gene content of the 868-kb interval shared by the JEB affected. doi:10.1371/journal.pone.0126416.g002

Fig 3 .

 3 Fig 3. ITGB4 mutation analysis. (A) Genomic structure of ITGB4 sequence based in the information obtained by BAC clone sequencing and RNA-seq data. Grey boxes represent exons. Grey lines crossing the exons represent the two mutations with protein effects found in this study (c.1337G>A and c.4412_4415del). The red box highlights the exon 33 in which c.4412_4415del is located. (B) The first sequence corresponds to exon 33 and its translation in a wild type protein, the second sequence corresponds to mutant exon 33 and the resulting translation. The affected codons at exon 33 are highlighted in red. This 4-bp deletion leads to a truncated protein of 1,472 amino acids (1,470 amino acids of normal integrin β4 followed by two missense amino acids and a premature termination codon). (C) Putative resulting protein of ITGB4 gene (GenBank KP025765). The mutation is predicted to delete the region spanning the last 281 amino acids in the intracellular domain, including the third and fourth FNIII repeats. doi:10.1371/journal.pone.0126416.g003

Fig 4 .

 4 Fig 4. Immunohistochemical labeling of collagen VII and integrins α6 and β4. The three proteins are labeled (brown signal) in the skin samples of the control lamb, at the basal cell layer while in the samples from the affected lamb integrin β4. The signal of the other two proteins is normal. Note the localization of collagen VII at the base of the blister in the sample from the affected lamb while the integrin α6 remains attached to the basal keratinocytes, at the roof of the blister. doi:10.1371/journal.pone.0126416.g004

PLOS ONE | DOI:10.1371/journal.pone.0126416May 8, 2015

Funded by Spanish Ministry of Economy and Competitiveness (grant AGL2012-34437 to JJA); Aroa Suarez-Vega is funded by an FPU contract from Spanish Ministry of Education. Beatriz Guterrez-Gil is in the species in which high resolution exome-sequencing is not straightforward.

funded by an Ramon y Cajal contract from the Spanish Ministry of Economy and Competitiveness. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

The transcriptomic data (RNA-Seq) are available from the European Nucleotide Archive database (http://www.ebi.ac.uk/ ena) with the accession numbers: ERS631692 and ERS631693. The association data, genotypes and phenotypes have been submitted to the Dryad repository (doi:10.5061/dryad.4j7jh).

Supporting Information


Remi Maniak
email: remi.maniak@

Christophe Midler
email: christophe.midler@polytechnique.fr

Sylvain Lenfle
email: sylvain.lenfle@polytechnique.fr

TRACKING THE ROUTES OF INNOVATION MANAGEMENT ACROSS PROJECTS: A CROSS-CASES PERSPECTIVE

Although innovation management has been extensively studied over the past decades, current literature hardly screens the current challenge for large project-based firms. For these firms, innovation management no longer deals with launching breakthrough products, but more likely with integrating innovative value propositions within a regular stream of platforms. This paper provides an analytical framework to understand this new form of challenge. We applied this framework to analyze four cases of innovation routes at two different OEMs. The article specifies the process of the innovation route and identifies key moments that stand as manageable turning points and variables.

INTRODUCTION

Time-paced competition led companies to put in place rationalized design organizations that guarantee a constant stream of new products. As a consequence, the challenge of innovation management stands as the pattern of making the stream of products evolve in a value-creating way. In this perspective, the evolution of product lines is a balanced game between regular platforms of products based on incrementally evolving knowledge, and a constellation of value propositions that challenge the dominant design of the product lines and the core competencies of the organization.

Companies adopt different strategies regarding this features/products game. In the automotive industry, Toyota dedicated a new line of products to a value proposition (hybrid engine) and then to deploy this value proposition on regular product lines. In the software industry, Microsoft makes its software platforms evolve by versioning different packages of features. Facing this renewed competitive landscape, we are still at the early stages of definition of a satisfying theoretical framework that can highlight the key challenges and provide decision making canvas for such innovation management issues.

The purpose of this paper is to provide an analytical framework that can address the main rules of the featuresproducts interplay. To tackle this issue, we chose to study several value propositions' routes across several generations of products, and to analyze their evolution. The goal is to highlight key moments and mechanisms paving the route that goes from a brilliant idea of value to a proven and profitable innovation, deployed on several ranges of platforms, and part of the core competencies of the firm.

In the first part we base on the product development literature to delimitate the new challenges for innovation management in large project-oriented firms. We then settle the innovation route framework and explain how we use it as an analytical artefact to track innovative features across products. In the following part we review four cases of innovation routes: ACCESS and CROSS at two different global automotive OEMs. We finally underline the major findings of this experimental implementation of the analytical grid both from an academic and managerial view.

FROM PRODUCT DEVELOPMENT TO INNOVATION MANAGEMENT

The empowerment and routinization of development activities

In the 80s, the increasing competitive pressure put emphasise on the ability of industrial firms to improve the quality level, to lower the cost, to increase the pace of products launches, and, last but not least, to manage the increasing complexity of several kinds of products. This shift was addressed to numerous industries: automotive, medical devices, consumer goods, electronics, computers... A lot of attention has been paid by practitioners and researchers on the way manufacturers could meet this challenge, mainly by studying the Japanese firms [START_REF] Imai | Managing the New production Development[END_REF]. Pioneer researches defined concepts and organizational frameworks for effective product development: heavyweight project management teams, concurrent engineering and early supplier involvement [START_REF] Clark | Product Development in the World Auto Industry[END_REF][START_REF] Clark | Product Development Performance: Strategy, Organization and Management in the World Auto Industry[END_REF][START_REF] Midler | L'Auto qui n'existait pas: Management des projets et transformation de l'entreprise[END_REF][START_REF] Midler | Projectification"of the firm: The Renault case[END_REF]. These frameworks and methodologies spread rapidly among industrial firms along the 90s.

The "fat-design" problem is a collateral effect of this overwhelming success [START_REF] Fujimoto | The Evolution of a Manufacturing Systems at Toyota[END_REF]. The focus on the cost-quality-timeline (CQT) indicators of a single product tends to prefer one-shot solutions that favoured the project disregarding the firm global performance. Platform strategies aim at promoting a global parts approach, by sharing components and subsystems among different products [START_REF] Cusumano | Thinking beyond lean[END_REF][START_REF] Meyer | The Power of Product Platforms[END_REF]. The platform approach maximizes the commonality of components among several projects and reduces the coordination burden between projects and functional departments. The platform approach manages diversity among the different products in order to save costs and improve lead-time.

Given the pressure on development performance, it is more and more difficult for firms to take risks in the context of development routines [START_REF] Aggeri | Innovation and project development: an impossible equation?[END_REF]. Moisdon & Weil (1998) thus show that the pressure upon project managers leads them to consider innovation as a potential danger with regards to cost, quality and delivery time. This leads to "frontload" all the problems to the pre-project phase (the "front-end" of the project). As a consequence, the pre-project phase was increasingly considered both as a product definition process (Smith & Reinertsen's 1991;Ulrich and Eppinger 2003) but also as a risk-elimination process aimed at reducing the problem-solving effort of the development phase [START_REF] Ciavaldini | Des projets aux avant-projets : l'incessante quête de réactivité[END_REF][START_REF] Cooper | An investigation into the new product process: steps, deficiencies and impact[END_REF][START_REF] Gerwin | An Evaluation of Research on Integrated Product Development[END_REF][START_REF] Thomke | The Effect of "Front-Loading" Problem-Solving on Product Development Performance[END_REF].

Innovation management in the projectified firm

Firms are now well armed to develop rapidly new products, and to lead a global parts strategy in order to manage the diversity implied by this evolution. But new product development did not explicitly refer to innovation management [START_REF] Brown | Product development : past research, present findings, and future directions[END_REF]. Researchers paid a lot of attention on the CQT factors, partly because innovation has been regarded as a micro-economical event. For several years, this shaping of the innovation topic is balanced by numerous researches in marketing, organization, and operations and management sciences. They take for common basis that the CQT criteria are only a part of the product attractiveness. Innovation management implies to complete the CQT criteria by questioning the firm's capability to embed within the stream of products enough customer value to provide product and brand attractiveness.

Building such value propositions appear to be a major challenge for project-based firms, whose organization clearly mirrors the traditional knowledge base of the firm (market and technical). This setting is likely to be reluctant to integrate value propositions that are disruptive towards this organizational structure [START_REF] Galunic | Architectural Innovation and Modular Corporate Forms[END_REF][START_REF] Henderson | Architectural innovation: The reconfiguration of existing product technologies and the failure of established firms[END_REF]. The projectified organizations [START_REF] Midler | Projectification"of the firm: The Renault case[END_REF] instituted core capabilities that maximize the CQT indicators. The core capabilities developed in NPD tend to turn into core rigidities that model potential products through a stable architecture [START_REF] Leonard-Barton | Core Capabilities and Core Rigidities: A Paradox in Managing New Product Development[END_REF].

Innovation as an interplay between knowledge and development activities

As the development activities became empowered and autonomous, innovation management has been increasingly considered as a matter of interplay between the empowered development activities and knowledge activities. Several researches highlighted specific mechanisms of this interplay.

Iansiti's work (1998) permits to better understand the linkage between technological knowledge activities and product development activities. He shows that development projects that "create a match between technological options and application context" (p.21) perform better than others. In other words, knowledge creation should be oriented through the future context of integration. If Iansiti's work permits to better understand the integration of a technology within a product under development, his work focuses on the technical improvement of well defined value propositions, and leaves aside deeper reshuffling of the product hierarchy. Furthermore, the multi-product deployment of the technology remains at the background of his work.

Cusumano & Selby (1995) describe the products / features interplay at Microsoft as a "syncand-synchronize" process. The development teams build an increasingly reliable product by testing the integration of the different features at a day-to-day basis. Features remain relatively independent so they can change along the project from more than 30% depending on updated knowledge. On the same way as Iansiti, since the authors focus on product performance, they do not really question the deployment and capitalization issue.

Marsh and Stock settle a model of "intertemporal integration" that address this multiproducts issue [START_REF] Marsh | Building Dynamic Capabilities in New Product Development through Intertemporal Integration[END_REF]. By modelling the product learning cycle they aim at identifying key mechanisms of dynamic capabilities in the interplay between development activities and knowledge activities. This framework looks promising, but remains at an emerging phase and still misses some empirical insights.

FRAMEWORK & METHODOLOGY Figure 1 -The Interplay among Products Lines and Innovative Value Propositions

Products / Plateforms

Value Propositions / Features

Products / features interplay

This paper completes and enlarges the existing literature by providing a framework that can address the trajectory of an innovative feature across products. This framework is used to analyze empirical data on innovative features that are not only technological components, but more radical features that question both the regular architecture of the product, and the regular customer value.

Defining a value proposition and its evolution

We define an innovative feature as a technical solution aimed at providing a supplement of customer value that is not included in the definition of the traditional products. A value proposition tends to present a set of valuable functionalities (1) that match with the specific embedment constraints of several products contexts (2). This solution should be technically reliable -product and process (3) and bring with it a recognized advantage for the firm (4). We thus characterize a value proposition through the four following ranked criteria.

Level 1

Level 5

Customer Value

Basic idea expected to increase the customer value

Proven strong customer value associated with a product/service.

Integrability

Context related constraints are not taken into account by the feature.

Multi-product integrability -A generative model to deploy it on a range of products.

Maturity

Underlying technology is unclear, no system test, no process.

Underlying bodies of knowledge are explicitly known.

Profit

Fuzzy assessment on profitability for the firm.

Defined business model, positive margin, recognized benefit.

Framing the products/features interplay: the innovation route

We distinguish between knowledge related activities and development related activities. Development activities are defined as the set of investigations aimed at renewing existing lines of products based on firm's core capability. These activities encompass the investigations focused on CQT criteria and kaizen improvement of the products. We define the knowledge activities as the set of investigations aimed at exploring innovative value propositions, preparing them for the embedment in specific contexts, and capitalizing the related knowledge in the regular product-process organization. We finally define four phases in the route of an innovative feature. These phases are delimited by the targeted contexts and by the nature of the investigations. The exploration phase consists in exploring and preparing an innovative feature for upcoming development projects. The contextualization phase consists in preparing a specific proposal to embed the feature within an ongoing pre-project. As soon as the non-return point is passed, the development phase consists in effectively developing the feature within the product development process. Once the first feature is marketed, the deployment phase consists in capitalizing on this first experience to deploy the feature on a coherent range of products. This framework will be used to analyze empirical data following a cross-cases methodology.

Methodology

We aim at better understanding the process and organizational setting that support the emergence, integration and deployment of value propositions. We are thus looking for a process theory [START_REF] Mohr | Approaches to explanation: Variance theory and process theory[END_REF]) that includes several development and knowledge-based activities. Therefore, we chose to shift our analytical lenses from a product to a value proposition that is to be deployed on a broad range of products.

Trying to formalize emerging practices in companies, we relied on multiple exploratory case studies [START_REF] Yin | Case Study Research: design and methods[END_REF] which provide empirical insights of the above-described illstructured process. The two cases ACCESS and CROSS at OEM A were used to generate the initial framework. The same cases at OEM B were added to the sample as theoretical replications and extensions to challenge and refine the framework [START_REF] Leonard-Barton | A Dual Methodology for Case Studies: Synergistic Use of a Longitudinal Single Site with Replicated Multiple Sites[END_REF][START_REF] Yin | Case Study Research: design and methods[END_REF].

We aim at characterizing the route of the 4 features from their early beginnings to their multi-product deployment. On each case, we tracked back the 4 above described criteria in order to identify the main shifts. This tracking process permitted to identify key milestones of the route, and to characterize the corresponding management challenges.

Data collection

We chose to investigate the automotive industry. Although it was a privileged source of learning on project management, it has been relatively neglected as a research field for innovation management. The choice of this industry was also guided by the opportunity we had to collect rich data. Top Management from two global automotive manufacturers promoted the study through financial support and time consuming involvement.

The two cases ACCESS and CROSS were selected in order to cover a contrasted scope of value propositions. The ACCESS feature is a keyless system which is very innovative towards customer use (new set of functionalities). The CROSS value proposition is a compact cockpit module which stands as an integrated system putting into question the traditional architecture of the dashboard perimeter.

On each case, we had access to key managers and in-house documentation of car manufacturers and tier-1 suppliers that were involved in the innovation process. We conducted 42 interviews of diverse manager's profiles (project, research, purchasing, technical…) that were involved in the management of the features.

TRACKING THE ROUTES OF 4 VALUE PROPOSITIONS Case ACCESS at OEM A

VP Preparation -In the 80s, the OEM A's Exterior Equipment Department and their supplier's counterparts had lived an exciting period: in less than ten years they passed from mechanical key controlled locks to electrical radio-controlled centralized locking system. Both OEM and suppliers thought they can do more, and studied how they could make this opening system fully automatic. In the early 90s, no one showed enough interest in the topic to continue such efforts in the Technical Departments. The topic remained active from 1992 to 1994 at the OEM's Research Centre, which was responsible to make a first draft of an ACCESS system. It finally delivered a patented functioning scheme mainly based on electronic specifications.

VP Contextualization -In June 1996, a Development Program suddenly broadcasted its need to include in the definition of the product several value propositions that could make a difference for the customer. In order to abide by the constraints of the product (parts layout, market segment, selling price…), the Vehicle Program Manager merged the pilot in-house studies and the studies of consulted suppliers, and finally proposed to the Board a mixed version of ACCESS.

VP Development -In December 1997, the Board decided that ACCESS should be included as original equipment. Another important decision was to nominate an Innovation Project Manager who was responsible to coordinate the two main contracted suppliers and the development teams impacted by the system (12 out of 26). What he put in place was an actual "development project within the development program", since he created ad hoc validation patterns and contractual engagements among people that were already contracted within the vehicle project. The ACCESS project involved numerous surprises caused by vehicle embedment, or related with customer functional misanticipations. The system was finally right on-time for vehicle market launch.

VP Deployment -The first version of ACCESS did not fully meet the initial Marketing Department demand. Indeed the technical incertitude took the lead and lowered the functional ambition. The Program Manager decided not only to deploy the feature on the two upcoming models, but also to enrich the functionalities in order to meet the initial customer value. Today, the ACCESS system includes all the initially wished functionalities, and is deployed on 8 models from 3 platforms. ACCESS is placed under the responsibility of a joint electronicmechanic team, which was recently co-located.

Case ACCESS at OEM B

VP Preparation -In the early 80s, the Mechanical Division wondered how it could benefit from the generalization of electric power in cars. It launched a two year study focus on the electrification of the steering column lock. At the same time, the Exterior Equipment Division triggered a parallel study aimed at making the door locking and unlocking procedure automatic. At the end of the day, both studies were unable to show enough benefit to justify more resources, even if the auto-lock project finally sold a pilot feature as optional equipment on a luxury car.

VP Contextualization -The topic got silent until 1995, when the Board voted a customer value based strategy, which implied to add attractive features to upcoming vehicles. The dedicated steering committee nominated a taskforce responsible for introducing the ACCESS feature on the market. The members of this taskforce were the former pilots of the 2 above described studies, who had reached heavyweight positions in their respective Departments. They rapidly merged their experience to propose a coherent ACCESS system. They initially targeted the directly upcoming car project, which was a luxury one. After 6 months of study, they realized that no solution could match the cost and functional expectations. Taking advantage from this initial study, they targeted another upcoming vehicle project -a low end urban carand managed to build a scenario that met the cost (scale effect), technical functional requirements. This was the kick-off of the ACCESS project within the traditional vehicle project.

VP Development -The early collaboration engaged among the different technical departments was identified as dramatic for the development of the ACCESS system, at least at two levels. First, this system spread among more than ten regular car perimeters, and required a tight coordination among project teams. Secondly, they had to agree on the specific requirements of the ACCESS feature, which had sometimes to take the lead in front of institutionalized vehicle validation procedures. The OEM finally launched the car on time, and widely based the marketing plan on the promotion of the ACCESS system (TV ads, show-cars). The car sales and ACCESS equipment rate reached the previsions, and allowed meeting the economic balance.

VP Deployment -Based on this successful experience, the OEM decided to deploy the feature on other from other platforms. The ACCESS topic has been promoted by the same steering committee as at the beginning of the story, which supported its development within more than a dozen of different vehicles in 5 years. They triggered ACCESS-focused cross-functional investigations that clearly stand out of the regular scope of vehicle-focused cross-functional investigations. By doing so, they were able to guarantee a persuasive balance between the customer value and the pattern of associated costs. In order to enhance the customer value, it demanded to refine functionalities in seek for the best fit with the market segment specificities.

Case CROSS at OEM A

VP Preparation -During the 1990s, the Technical Department in charge of the cockpit had great troubles in preparing innovative solutions within such an integrated perimeter. The technical teams conducted several advanced studies in order to improve the performance of their parts, but once the time to develop a vehicle came, these advanced studies did not match together in a coherent manner. That's why the traditional parts layout survived despite the increasing importance of the "life-on-board" concept. In 2002, the Director of the Inner Design Engineering tried to bridge this paradox by triggering a new form of advanced study. He requested several suppliers to investigate an integrated cockpit solution in the context of a real ongoing vehicle program.

VP Contextualization -Several suppliers had already investigated the theme. Their proposals were promising, and the OEM decided to make them compete on the real vehicle architecture. The OEM firstly indicated a niche vehicle that would be produced by 2010. Suppliers' proposals fitted relatively well with the specific constraints of this product, since product-process constraints were quite loose in that scale of production. After 6 months of interaction with the suppliers, the technical teams responsible for the investigations realized that they would only trigger the required in-house commitment by targeting a current platform program. In January 2004, suppliers were asked to prepare for direct interplay with the development teams of a mass selling car which start of production was programmed for 2008.

The context was evolving on a week-to-week basis, and suppliers had to keep their proposals fitted to this evolving layout. The interlocutors of the suppliers were the regular product-process technical teams of the pre-development project. Each team was evaluated on traditional kaizen performance targets. The suppliers were de facto asked to satisfy not only the global target of compactness, but also the local targets of performance on each part of the module. Six months after the beginning of this experiment, and even if their initial proposals were promising (technically validated, economically feasible…) none of them passed through the final decision. Each solution did lost appeal by adapting to vehicle related constraints (changes in the manufacturing process, need of new components that took more space…).

VP Deployment -Most surprising were the reactions of the involved teams: despite both suppliers and OEM had spent resources to develop a solution that was finally not developed or sold; all of them claimed to have benefited from the experience. The OEM benefited from this experience by promoting the CROSS topic within in-house top-tech meetings. This experience triggered technical studies focused on questions and value axis that have emerged at this occasion, both at the OEM and at the suppliers.

Case CROSS at OEM B

VP Exploration -The research department of OEM B officially considered the interior volume as a valuable innovation domain from the year 2000. In 2001, the research centre allocated resources to a study focused on cockpit module compactness.

The dedicated research team first considered a way to measure the compactness of a cockpit. It defined rough variables of performance and targets to reach. These targets included the traditional conditions of maturity of the different parts composing the cockpit module, and also specific targets of volume and broad architectural orientations. After having validated these elements with the Research Committee, it requested from a parts' supplier to tackle the issue. From 2003 to 2004, the OEM's and the supplier's research team interacted at a month-to-month basis, reviewing the results of this study.

VP Contextualization -After a 12-month period of broad technical feasibility testing and performance criteria refinement, the cooperation shifted to a more contextualized investigation. The OEM gave more precise elements of context, defining a targeted range of vehicle, and several elements of architectural context based on an existing vehicle model that had to be replaced in the next decade. From 2005 to 2006 the supplier had to reach a certain volume target abiding by the previously acquired technical validations.

In July 2006, even if the results of these investigations were promising in term of habitability and cockpit compactness, there was still no guarantee that the targeted development project would be favourable to embed this solution, or that the architectural decisions towards the architecture of this product will fit with the hypothesis made during this investigation.

RESULTS & IMPLICATIONS

The experimental implementation of the innovation route grid makes visible dramatic mechanisms of the process that allow a brilliant idea of feature to be deployed on a range of products. We regarded the features' related sequence of events as a cumulative process aiming at this target. We observe that the route of a value proposition reveals important variations both in the 4 indicators under evaluation and in the organizational settings that support this evolution.

The forms of early explorations

The exploration phase appears as dramatic and particularly difficult to manage. Whereas it is common language for development projects to call for "proven technologies", the cases show that there is an unavoidable space between what the development projects are likely to embed directly, and what the research activities are able to deliver even with consequent resources. On the four cases, no value proposition could be embedded without demanding tremendous further investigations. On the ACCESS A case, even two years of investigations on the keyless entry topic were not sufficient to provide a functioning system.

The case provide evidence that the organization of exploration activities (following technical domains of expertise) and advanced activities (following regular product architecture) act as a major canvas for feature preparation. The ACCESS A research team was specialized in electronics, and disregarded industrialization constraints or functional requirements. We can also observe this phenomenon when the investigations are led by technical product-process departments: the compactness had been studied by different product-process departments in the past, unsuccessfully. The upfront organization orients the early studies towards mono-expertise roadmaps instead of an integrated effort pulled by the seeking for customer value. On the same way, CROSS shows the difficulty to study and develop an architectural value proposition within the regular development organization. Advanced studies were led by different technical departments which failed to coordinate on a valuable parts' setting.

The limits of "off-the-shelves" solutions

The second main insight provided here follows Iansiti's conclusions and directly questions the notion of "proven technology": the exploratory investigations are context dependant. What has been validated out of context can be put into question just by modifying a tight element of the value proposition. On each case, the sudden appearance of specific context related constraints interrogated several hypotheses that have been guiding the investigations so far.

On the CROSS A case, the suppliers made early investigations based on a generic context of embedment that fitted relatively well with a specific niche product. As the targeted product changed, numerous interferences appeared. The adaptation process (scheme 3) putted into question the previously envisaged technical solutions (maturity 3->1), the economic balance because the new technical solutions were much more expensive (profit 2->1). The customer value of the cockpit module was also questioned (3->1) since the new layout demanded volume consuming parts in order to keep abiding by the regular specifications.

The innovative solution is "shelved", "validated" or "proven" in a given functional / technical configuration that is very dependent on the context of embedment. So we can only underline the critical role of the embedment process. The two CROSS cases provide insights from two different choices of contextualization process. Let us have a closer look at the positive and negative aspects of these two settings.

Various track of value proposition contextualization

Whereas OEM A envisaged the embedment process of the CROSS solution within the product development process, OEM B chose to investigate the embedment out of the product development cycle. The table 2 shows a brief synthesis of the characteristics of these two processes.

CROSS A CROSS B Major advantages

The proximity to the product sales provides great incentives for OEM and suppliers

Step-by-step investigations that account a progressively representative context. Major disadvantages VP product and process validations are placed under the same validation regimes than robust traditional parts

Uncertainty towards the context hypothesis and the upcoming valorisation

Table 2 -Advantages and disadvantages observed of both patterns of contextualization

One could imagine a hybrid form of embedment between these two specific settings. We currently carry out further research in order to define the settings of a contextualization process that could maximize namely the context matching, the maturation pace and the incentive effect.

Shaping the innovation routes

We cannot understand the decisions made during case if we limit our scope of evaluation to the performance of the current development project. CROSS was not developed yet but the CROSS's route benefited from the created knowledge for both OEM and suppliers. Several decisions aim not only at increasing the value of the upcoming product, but also to nurture and solidify value proposition related knowledge fields (Hatchuel and al. 2004) in order to make the future features embedment more likely and profitable [START_REF] Lenfle | Innovation-based competition and the dynamics of design in upstream suppliers[END_REF].

In this "rebound" perspective, the cases provide evidence that firms can choose different patterns of learning. These are summarized in Figure 4. OEM A privileged a more customer-value-oriented innovation route. In spite of poor exploratory technical studies, it defined quite rapidly valuable functionalities and brought the feature on the market through a mass selling product. This shift triggered an incentive to learn the missing technical knowledge. The OEM B led a more iterative and technical-oriented innovation route that used several development projects as learning support. The successive trials led to define a relevant set of functionalities and a profitable feature.

The ACCESS case shows extensively how innovative value propositions need different coordination process than the product development routines: they tend to fade the coherence of the innovative value proposition behind the regular validation patterns. At OEM A the lead came from the project management, and needed heavyweight management in order to coordinate the technical teams that were not strongly committed. After the first development, the technical departments formalized written design rules and dedicated a technical team. At OEM B, the lead came from technical departments that had already worked together on the topic. They acted as heavyweight coordinators for inhouse technical teams and suppliers. These roles are still unchanged today.

The multi-products learning perspective enables to regard the improvement of the four criteria on the scale of different products. The cases highlight the need to consider the four criteria at a multi-products scale. Project-oriented organizations (like OEM A) tend to focus on the cost related to the first embedment and disregard the consequences of these early choices towards upcoming embedment efforts. We face here another fatdesign problem: a decision could be rationally justified at the level of project performance, but could represent an over-design cost when considering the integration problem at a multi-context scale.

Further researches

The multi-case approach we adopted here was relevant to build accurate analytical lenses but we still miss a more systematic approach to isolate key levers for the management of this kind of innovation routes. We currently define a quantitative approach to correlate management practices of the innovation routes with differences in the performance of these routes. This framework will be part of the first round of an international benchmark on innovation of the International Motor Vehicle Program.

Figure 2 -

 2 Figure 2 -The Innovation Route model of an innovative feature across products

  Scheme 3 -"CROSS A" indicators before and after contextualization (source: our research)

Table 1 -Ranking of a value proposition on the four criteria

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