Annelies Rotthier

Anke Penno

Bernd Rautenstrauss

Michaela Auer-Grumbach

Georg M Stettner

Bob Asselbergh

Kim Van Hoof

Heinrich Sticht

Nicolas Lévy

Dr Vincent Timmerman
email: vincent.timmerman@molgen.vib-ua.

Thorsten Hornemann

Katrien Janssens

Characterization of two mutations in the SPTLC1 subunit of serine palmitoyltransferase associated with hereditary sensory and autonomic neuropathy type I

Keywords: HSAN-I, SPTLC1, serine palmitoyltransferase, 1-deoxy-sphinganine

Hereditary sensory and autonomic neuropathy type I (HSAN-I) is an axonal peripheral neuropathy leading to progressive distal sensory loss and severe ulcerations. Mutations in SPTLC1 and SPTLC2, encoding the two subunits of serine palmitoyltransferase (SPT), the enzyme catalyzing the first and rate-limiting step in the de novo synthesis of sphingolipids, have been reported to cause HSAN-I. Here, we demonstrate that the SPTLC1 mutations p.S331F and p.A352V result in a reduction of SPT activity in vitro and are associated with increased levels of the deoxysphingoid bases 1-deoxy-sphinganine and 1-deoxymethyl-sphinganine in patients' plasma samples. Stably expressing p.S331F-SPTLC1 HEK293T cell lines likewise show accumulation of deoxysphingoid bases, but this accumulation is not observed in HEK293T cells overexpressing p.A352V-SPTLC1. These results confirm that the increased formation of deoxysphingoid bases is a key feature for HSAN-I as it is associated with all pathogenic SPTLC1 and SPTLC2 mutations reported so far, but also warrant for caution in the interpretation of in vitro data.

INTRODUCTION

Inherited peripheral neuropathies are common neurodegenerative disorders of the peripheral nervous system. They are genetically and clinically heterogeneous, but based on the predominant involvement of motor or sensory neurons, they fall into three main subclasses: hereditary motor and sensory neuropathies (HMSN), hereditary motor neuropathies (HMN) and hereditary sensory and autonomic neuropathies (HSAN). The clinical spectrum of HSAN is wide and led to the further subdivision into six subtypes [START_REF] Dyck | Hereditary motor and sensory neuropathies[END_REF]. Patients with hereditary sensory and autonomic neuropathy type I (HSAN-I; MIM 162400) present with progressive loss of pain and temperature sensation in the limbs, variable distal muscle weakness and limited autonomic dysfunction. Typically, first symptoms appear between the second and third decade of life. The loss of sensation leads to painless injuries complicated by ulcerations and osteomyelitis, often necessitating amputation [START_REF] Auer-Grumbach | Autosomal dominant inherited neuropathies with prominent sensory loss and mutilations: a review[END_REF]. HSAN-I has a dominant mode of inheritance and is associated with mutations in SPTLC1 (MIM 605712) [START_REF] Bejaoui | SPTLC1 is mutated in hereditary sensory neuropathy, type 1[END_REF][START_REF] Dawkins | Mutations in SPTLC1, encoding serine palmitoyltransferase, long chain base subunit-1, cause hereditary sensory neuropathy type I[END_REF][START_REF] Rotthier | Genes for hereditary sensory and autonomic neuropathies: a genotype-phenotype correlation[END_REF] and SPTLC2 (MIM 605713) [START_REF] Rotthier | Mutations in the SPTLC2 subunit of serine palmitoyltransferase cause hereditary sensory and autonomic neuropathy type I[END_REF], two subunits of the enzyme serine palmitoyltransferase (SPT). SPT is located at the outer membrane of the endoplasmic reticulum (ER), where it catalyzes the pyridoxal-5'-phosphate (PLP) dependent condensation of Lserine with palmitoyl-CoA. This is the first and rate-limiting step in the de novo biosynthesis of sphingolipids [START_REF] Hanada | Serine palmitoyltransferase, a key enzyme of sphingolipid metabolism[END_REF]. Sphingolipids, having both a structural and a signaling function, are essential for all eukaryotic cells. Mutations in various enzymes of the sphingolipid metabolism are associated with neurodegenerative diseases [START_REF] Kolter | Sphingolipid metabolism diseases[END_REF], highlighting the importance of these ubiquitous components in neuronal functioning.

A common feature of the HSAN-I associated mutations in SPTLC1 and SPTLC2 is a reduction of the canonical enzymatic activity, but the effect on total sphingolipid levels remains controversial [START_REF] Bejaoui | SPTLC1 is mutated in hereditary sensory neuropathy, type 1[END_REF][START_REF] Dawkins | Mutations in SPTLC1, encoding serine palmitoyltransferase, long chain base subunit-1, cause hereditary sensory neuropathy type I[END_REF][START_REF] Dedov | Activity of partially inhibited serine palmitoyltransferase is sufficient for normal sphingolipid metabolism and viability of HSN1 patient cells[END_REF][START_REF] Hornemann | Is the mammalian serine palmitoyltransferase a high-molecular-mass complex?[END_REF]. Furthermore, the mutations cause a shift in the substrate specificity of SPT, enabling the mutant enzyme to metabolize, besides serine, also alanine and glycine. This results in the formation of the two atypical deoxysphingoid bases (DSB) 1-deoxy-sphinganine (1-deoxy-SA) and 1deoxymethyl-sphinganine (1-deoxymethyl-SA) [START_REF] Zitomer | Ceramide synthase inhibition by fumonisin B1 causes accumulation of 1deoxysphinganine: a novel category of bioactive 1-deoxysphingoid bases and 1-deoxydihydroceramides biosynthesized by mammalian cell lines and animals[END_REF][START_REF] Gable | A disease-causing mutation in the active site of serine palmitoyltransferase causes catalytic promiscuity[END_REF][START_REF] Penno | Hereditary sensory neuropathy type 1 is caused by the accumulation of two neurotoxic sphingolipids[END_REF]. Both metabolites can be converted to 1-deoxy(methyl)-ceramide and 1-deoxy(methyl)-sphingosine (1-deoxy(methyl)-SO) but because they lack an essential hydroxyl group, conversion to more complex sphingolipids like glyco-or phosphosphingolipids and phosphorylation are hampered, resulting in the accumulation of these intermediate metabolites in the cell. Importantly, it was previously shown that DSBs have pronounced neurotoxic effects on neurite formation in cultured sensory neurons [START_REF] Penno | Hereditary sensory neuropathy type 1 is caused by the accumulation of two neurotoxic sphingolipids[END_REF].

Recently, we performed a systematic screening of the known HSAN genes in a large cohort of HSAN patients and identified two novel SPTLC1 mutations, p.S331F and p.A352V [START_REF] Rotthier | Genes for hereditary sensory and autonomic neuropathies: a genotype-phenotype correlation[END_REF], bringing the total number of SPTLC1 mutations to six (the others being p.C133W, p.C133Y, p.C133R and p.V144D) [START_REF] Bejaoui | SPTLC1 is mutated in hereditary sensory neuropathy, type 1[END_REF][START_REF] Dawkins | Mutations in SPTLC1, encoding serine palmitoyltransferase, long chain base subunit-1, cause hereditary sensory neuropathy type I[END_REF][START_REF] Rautenstrauss | Late onset hereditary sensory neuropathy type 1 (HSN1) is caused by a novel p.C133R missense mutation in SPTLC1[END_REF]. The p.S331F mutation occurred de novo in a patient with a severe congenital phenotype, the p.A352V mutation was detected in an isolated patient with a typical HSAN-I phenotype. Further evaluation of the pathogenicity of this latter variant is warranted because of the absence of DNA of family members for segregation analysis, the limited evolutionary conservation of the targeted amino acid and the similar chemical properties of alanine and valine.

In the present study, we report the identification of the p.S331F mutation in a patient with severe and earlyonset HSAN-I. Furthermore, functional characterization of both the p.S331F and p.A352V mutations was undertaken to a) identify differences between the p.S331F and previously described SPTLC1 mutant proteins that could explain the severe phenotype associated with the p.S331F mutation and b) assess the pathogenicity of the p.A352V mutation.

MATERIALS AND METHODS

Subjects

Patient II.2 (MGZ-48522; Fig. 1a) has been described in Huehne et al., 2008. Patients CMT-791.01 and CMT-186.05 have been described in [START_REF] Rotthier | Genes for hereditary sensory and autonomic neuropathies: a genotype-phenotype correlation[END_REF].

Mutation analysis

After DNA extraction from EDTA blood using the Flexigene kit (Qiagen, Hilden, Germany), standard PCR and direct sequencing with ABI PRISM 3100 Avant (Big Dye v1.1, Applied Biosystems, Foster City, USA) covering all 15 coding exons and the flanking regions of the SPTLC1 gene (GenBank: NM_178324.1) was performed. Primer sequences are available upon request. Nucleotide numbering reflects cDNA numbering with +1 corresponding to the A of the ATG translation initiation codon in the reference sequence. The initiation codon is codon 1.

Cloning

The SPTLC1 cDNA was amplified and cloned into the Gateway® entry vector pDONR221 (Invitrogen, Carlsbad, USA) using the primers SPTLC1_attb1 and SPTLC1_attb2. The SPTLC1 mutations p.C133W, p.S331F and p.A352V were introduced by site-directed mutagenesis, using the primers listed in supplementary Table 1. The constructs were recombined in the destination vector pEF5/FRT/V5-DEST (Invitrogen), fusing the cDNA with a C-terminal V5-tag. Additionally, C-terminally EGFP-tagged constructs were constructed and recombined in the destination vector pLenti6/V5-DEST (Invitrogen). All constructs were validated by sequencing.

Stable expression of SPTLC1 constructs in HEK293T cells

Stable cell lines expressing wild type (wt) or mutant SPTLC1 were generated via calcium phosphate based transfection of the Flp-in host cell line HEK293T following manufacturer's instructions (Invitrogen). The Flp-in system ensures the stable insertion of a single copy of the transgene at a specific location. In this way, moderate and equal expression of the different transgenes is obtained.

Lymphoblastoid cell lines

Lymphoblastoid cell lines were generated as described previously in [START_REF] Rotthier | Mutations in the SPTLC2 subunit of serine palmitoyltransferase cause hereditary sensory and autonomic neuropathy type I[END_REF].

Cell culture material and conditions

HEK293T Flp-in cells were cultivated in DMEM supplemented with 10% fetal bovine serum, 2 mM Lglutamine and 100 µg/ml penicillin/streptomycin. SH-SY5Y neuronal cells were cultured in MEM supplemented with 10% fetal bovine serum, 2 mM glutamine, 1% non-essential amino acids and 100 µg/ml penicillin/streptomycin. Lymphoblastoid cell lines were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 1mM sodium pyruvate and 200 units/ml penicillin/streptomycin. All cells were cultivated at 37°C and 5% CO 2 . All cell culture media and supplements were from Invitrogen.

In vitro SPT activity

SPT activity was measured using the radioactivity-based assay described by Rütti et al., 2009.

Fumonisin B1 block assay, lipid extraction and hydrolysis and LC-MS

These assays were performed as described in [START_REF] Penno | Hereditary sensory neuropathy type 1 is caused by the accumulation of two neurotoxic sphingolipids[END_REF].

Structure modelling

The structure of human SPT was modelled using the high-resolution crystal structure of the Sphingomonas paucimobilis SPT homodimer (PDB code: 2JG2; [START_REF] Yard | The structure of serine palmitoyltransferase; gateway to sphingolipid biosynthesis[END_REF] as a template. The two subunits of heterodimeric human SPT exhibit a sequence similarity of respectively 47% and 50% to the bacterial template. Model building was performed using Modeller 6.2 (Sanchez and Sali, 2000) and included also the PLP cofactor covalently bound as an aldimine to K379 of SPTLC2. The resulting model was refined by energy minimization [START_REF] Hooft | Errors in protein structures[END_REF] revealed a correct local geometry and no steric clashes. Rasmol [START_REF] Sayle | RASMOL: biomolecular graphics for all[END_REF] was used for structure analysis and visualization.

Statistics

The two-tailed unpaired Student's t-test and the ANOVA with post hoc correction were used for statistical analysis. Error bars (standard deviation) and p-values (Student's t-test) were calculated based on three independent experiments.

RESULTS

Identification of the SPTLC1 mutation p.S331F in a HSAN-I patient

Mutation analysis of the complete coding region of the SPTLC1 gene in patient MGZ-48522 (II.2 in Fig. 1a), with a severe and early-onset HSAN with additional juvenile cataract and retinal detachment, led to the identification of the heterozygous missense mutation c.992C>T (p.S331F). This mutation is absent in the parents (Fig 1a) as well as in 300 control individuals. Mutations in PMP22, MPZ, GJB1, EGR2, MFN2, NTRK1 and NGF had been previously excluded [START_REF] Huehne | Novel missense, insertion and deletion mutations in the neurotrophic tyrosine kinase receptor type 1 gene (NTRK1) associated with congenital insensitivity to pain with anhidrosis[END_REF]. The identification of the p.S331F mutation in this patient provides additional genetic evidence for the pathogenicity of this variant, that was previously identified in patient CMT-791.01 with an atypically severe HSAN-I phenotype [START_REF] Rotthier | Genes for hereditary sensory and autonomic neuropathies: a genotype-phenotype correlation[END_REF]. The c.1055C>T (p.A352V) mutation was previously described in patient CMT-186.05 with a typical HSAN-I phenotype [START_REF] Rotthier | Genes for hereditary sensory and autonomic neuropathies: a genotype-phenotype correlation[END_REF].

S331F and p.A352V mutant proteins have a reduced SPT activity

Complementary to genetic evidence, proof of the pathogenicity of a sequence variant can be inferred from its effect on protein function. Therefore, functional characterization of the p.S331F and p.A352V mutant proteins in stably transfected HEK293T cells was undertaken. Equal expression of the mutant proteins in HEK293 cells was confirmed by Western Blot analysis (Suppl Fig 1). First, we investigated the effect of the p.S331F and p.A352V mutations on the canonical SPT activity using two different methods. In a first assay, in vitro SPT activity was determined in cell lysates by measuring the incorporation of 14 C labeled L-serine into lipids. SPT activity in wt SPTLC1 expressing cells was slightly different from control cells, however this difference was not observed in an alternative assay (see below). The previously characterized p.C133W mutant caused a more than 50% reduction in SPT activity which is in agreement with earlier reports [START_REF] Bejaoui | Hereditary sensory neuropathy type 1 mutations confer dominant negative effects on serine palmitoyltransferase, critical for sphingolipid synthesis[END_REF][START_REF] Hornemann | Is the mammalian serine palmitoyltransferase a high-molecular-mass complex?[END_REF][START_REF] Hornemann | A systematic comparison of all mutations in hereditary sensory neuropathy type I (HSAN I) reveals that the G387A mutation is not disease associated[END_REF]. SPT activity was reduced to a similar extent in cells expressing the mutant proteins p.S331F (35-55% reduction) and p.A352V (60-65% reduction) (Fig 2a).

In an alternative assay, growing cells were treated with Fuminosin B1 to block the sphingolipid pathway downstream of SPT. This results in a time dependent accumulation of SA. Since SPT catalyzes the rate-limiting step in the sphingolipid biosynthesis pathway, the amount of SA accumulating over time reflects canonical SPT activity. No difference in SPT activity was observed between wt SPTLC1 and control cells whereas the stable expression of the mutants resulted in a significant reduction of SA accumulation (Fig 2b). This confirms that, like the p.C133W mutant, the p.S331F and p.A352V mutations in SPTLC1 lead to a reduced SPT activity.

Like wt SPTLC1 protein, the two mutant forms of SPTLC1 colocalized with the ER marker calreticulin in SH-SY5Y neuroblastoma cells (Suppl Fig 2 ), rendering it unlikely that mislocalization of the mutant protein causes the loss in SPT activity.

Accumulation of 1-deoxysphingoid bases in p.S331F but not in p.A352V expressing HEK cells

To assess the effect of the observed reduction in SPT activity on total sphingolipid levels, we subsequently compared the sphingoid base profile of HEK293T cells expressing wt or mutant SPTLC1. We subjected the extracted total lipid fraction to a combined acid-base hydrolysis reaction; the resulting free sphingoid bases, which reflect the total amount of extracted sphingolipids, were analyzed by LC-MS. For both the HEK293T cell lines expressing the transgenic proteins and the lymphoblast cell lines of patients and controls, no significant differences in total SO levels between mutant and control cell lines could be observed (Fig 3a and3b), indicating that despite of a reduced canonical SPT activity, total sphingolipid levels are not altered. We furthermore analyzed the DSBs 1-deoxy-SA/1-deoxy-SO and 1-deoxymethyl-SA/1-deoxymethyl-SO, which have been shown to accumulate in cells expressing the previously characterized HSAN-I mutants and reflect the ability of the mutant proteins to use respectively alanine and glycine instead of serine as a substrate for the enzymatic reaction [START_REF] Penno | Hereditary sensory neuropathy type 1 is caused by the accumulation of two neurotoxic sphingolipids[END_REF][START_REF] Rotthier | Mutations in the SPTLC2 subunit of serine palmitoyltransferase cause hereditary sensory and autonomic neuropathy type I[END_REF]. In the HEK293T cells stably expressing wt SPTLC1, the amount of 1-deoxy-SA was similar to control cells; 1-deoxy-SO was below the detection limit in these cell lines (Fig 3c). Expression of the p.C133W mutant resulted in highly elevated 1-deoxy-SA and 1-deoxy-SO formation compared to wt SPTLC1 expressing cells, in accordance with previous observations [START_REF] Penno | Hereditary sensory neuropathy type 1 is caused by the accumulation of two neurotoxic sphingolipids[END_REF]. The p.S331F mutant also showed significantly higher levels of 1-deoxy-SA and 1-deoxy-SO compared to wt SPTLC1 expressing cells (p-value < 0.001). However, there was no statistical difference between the levels in the p.S331F expressing cells and in the p.C133W expressing cells. In the p.A352V expressing cells, the level of 1deoxy-SA was similar to the level measured in wt cells; 1-deoxy-SO was above the detection limit, but low.

1-deoxysphingoid bases are elevated in biosamples of p.S331F and p.A352V patients

Interestingly, analysis of plasma samples from all three patients described in this study (the two p.S331F patients and the p.A352V patient) showed significantly elevated plasma levels of 1-deoxy-SA and 1-deoxy-SO when compared to unaffected family members of or unrelated healthy controls (Fig 3e). In addition, levels of 1-deoxymethyl-SA and 1-deoxymethyl-SO were increased in the p.S331F patients' plasma samples, but were not detected in the plasma of the p.A352V patient. This indicates that the p.S331F mutant protein, but not the p.A352V mutant protein, is able to metabolise not only alanine, but also glycine as an alternative substrate.

Highly elevated DSB levels were also observed in lymphoblast cells of the p.S331F patient CMT-791.01 (Fig 3d). Unfortunately, no lymphoblast cell lines were available from the patient carrying the p.A352V mutation or from the p.S331F patient MGZ-48522. Levels of 1-deoxymethyl-SA and 1-deoxymethyl-SO were below the detection limit in HEK293T cells and in lymphoblast cell lines (data not shown).

Taken together, our data show that p.S331F and p.A352V mutant SPTLC1 are associated with a reduction of SPT activity in vitro without affecting total sphingolipid levels. Furthermore, both mutations are associated with elevated DSB levels in patients' plasma samples. For the p.S331F mutation, this finding corroborates the accumulation of DSBs found in two in vitro assays. For the p.A352V mutation, the result challenges the unaltered DSB formation observed in vitro.

DISCUSSION

Recently, we identified two novel HSAN associated sequence variants in SPTLC1, namely p.S331F and p.A352V [START_REF] Rotthier | Genes for hereditary sensory and autonomic neuropathies: a genotype-phenotype correlation[END_REF]. These variants are located far downstream of the 12 amino acid region clustering all other previously reported SPTLC1 mutations (p.C133W, p.C133Y, p.C133R and p.V144D). Structural information based on a model for human SPT (Fig 1b) indicates that the C133 and V144 residues are located close to the SPTLC1:SPTLC2 dimer interface, while the S331 and the A352 amino acids are located more distally. This could imply that the pathomechanism is different for these two groups of mutations. Moreover, both the p.S331F and the p.A352V mutation were detected in a single patient (de novo or isolated case), providing limited genetic evidence for their pathogenicity and calling for a more in-depth analysis of their effect on protein function.

We now were able to identify a second unrelated HSAN-I patient with the p.S331F mutation (MGZ-48552; II.2 in Fig 1a), which strengthens the genetic evidence that this variant is causative. Both patients carrying the p.S331F mutation show an unusually severe phenotype with early onset insensitivity to pain and pronounced motor impairment (Table 1) and non-typical HSAN-I features. In addition to the classical symptoms, the new patient described in this study presented with juvenile cataract and retinal detachment whereas the previously reported p.S331F patient showed severe growth and mental retardation, vocal cord paralysis and gastro-oesophageal reflux. Whether these features are related to the p.S331F mutation is uncertain since most of them are atypical for HSAN-I, although some have been associated with other inherited peripheral neuropathies [START_REF] Indo | Mutations in the TRKA/NGF receptor gene in patients with congenital insensitivity to pain with anhidrosis[END_REF][START_REF] Kok | A locus for hereditary sensory neuropathy with cough and gastroesophageal reflux on chromosome 3p22-p24[END_REF][START_REF] Aboussouan | Disorders of pulmonary function, sleep, and the upper airway in Charcot-Marie-Tooth disease[END_REF][START_REF] Claeys | Phenotypic spectrum of dynamin 2 mutations in Charcot-Marie-Tooth neuropathy[END_REF]. To explain the more severe phenotype in p.S331F patients, we compared the effect of the p.S331F mutation and the most intensively studied mutation associated with a typical HSAN-I phenotype, p.C133W, on SPT enzyme properties. Our results show that the p.S331F mutation lowers canonical SPT activity without reducing total sphingolipid levels in HEK293T cells or patient lymphoblast cells. In addition, the DSB levels were highly elevated compared to wt cells, but not significantly different from the level observed in p.C133W expressing cells. In this context, it is important to mention that we were likewise unable to observe a correlation between DSB levels and the clinical phenotype in our previous study on HSAN-I causing SPTLC2 mutations [START_REF] Rotthier | Mutations in the SPTLC2 subunit of serine palmitoyltransferase cause hereditary sensory and autonomic neuropathy type I[END_REF]. This might imply that DSB levels are not correlated to the severity of the disease, but that other factors, such as genetic modifiers, modulate the clinical phenotype. Alternatively, the S331 amino acid might be involved in other, yet unidentified functions of the SPT complex that are changed upon mutation. As is apparent from the model of the human SPT structure (Fig. 1b), the S331 amino acid residue is located on the surface of the protein, rendering it possible that this residue is involved in the interaction with other proteins, such as the recently identified small stimulatory subunits of SPT (ssSPT) [START_REF] Han | Identification of small subunits of mammalian serine palmitoyltransferase that confer distinct acyl-CoA substrate specificities[END_REF] and ORM-like proteins [START_REF] Breslow | Orm family proteins mediate sphingolipid homeostasis[END_REF][START_REF] Han | Orm1 and Orm2 are conserved endoplasmic reticulum membrane proteins regulating lipid homeostasis and protein quality control[END_REF]. However, information on functional domains of the mammalian SPTLC1 subunit is still lacking, limiting the conclusions that can be drawn from the model.

The biochemical evidence reported so far indicates that all pathogenic mutants in SPTLC1 and SPTLC2 are consistently associated with a reduction in the canonical SPT activity and an increased formation of DSB [START_REF] Eichler | Overexpression of the wild-type SPT1 subunit lowers desoxysphingolipid levels and rescues the phenotype of HSAN1[END_REF][START_REF] Penno | Hereditary sensory neuropathy type 1 is caused by the accumulation of two neurotoxic sphingolipids[END_REF][START_REF] Rotthier | Mutations in the SPTLC2 subunit of serine palmitoyltransferase cause hereditary sensory and autonomic neuropathy type I[END_REF] (for overview: see Table 1). This is reinforced by the case of the p.G387A variant in SPTLC1. This variant was initially thought to cause HSAN-I in twin sisters [START_REF] Verhoeven | SPTLC1 mutation in twin sisters with hereditary sensory neuropathy type I[END_REF], but does not decrease SPT activity [START_REF] Hornemann | A systematic comparison of all mutations in hereditary sensory neuropathy type I (HSAN I) reveals that the G387A mutation is not disease associated[END_REF] nor increase DSB generation (unpublished data). It turned out to be a rare, benign SNP as it was found in homozygous state in a healthy individual [START_REF] Hornemann | A systematic comparison of all mutations in hereditary sensory neuropathy type I (HSAN I) reveals that the G387A mutation is not disease associated[END_REF]. We used the information above to assess the impact of the p.A352V variant. Although the patient presents with a typical HSAN-I phenotype and despite the absence of the mutation from 300 control individuals, segregation analysis of the p.A352V mutation is lacking, the evolutionary conservation of the targeted amino acid is limited and the chemical properties of alanine and valine are similar, justifying caution in the interpretation of the genetic data. The biochemical experiments performed in this study strengthen the pathogenicity of this variant: it reduces SPT activity in vitro and is associated with accumulation of DSBs in plasma of the patient, but yet does not cause DSB accumulation in stably expressing HEK cells. A similar effect is seen for the p.V144D variant: it shows a clearly reduced SPT activity in vitro and significantly elevated DSB levels in plasma [START_REF] Penno | Hereditary sensory neuropathy type 1 is caused by the accumulation of two neurotoxic sphingolipids[END_REF], but only slightly elevated DSB formation in stably expressing HEK cells (unpublished data). The reason for this discrepancy in DSB accumulation between stably transfected cells and plasma samples is unclear. One possibility is that the effect of the p.A352V and p.V144D mutations on the biochemical properties of SPT is more subtle than that of the other SPTLC1 and SPTLC2 mutations, leading to a slower rate of DSB formation. In HEK cells, that are allowed to produce DSBs over approximately 72 hours, DSB levels might not have reached the limit of detection, while the DSB levels in plasma reflect the long term accumulation in patients. However, since the plasma samples are physiologically more relevant, we conclude that the p.A352V mutation is indeed disease associated.

These results demonstrate the importance of analyzing patient material in addition to performing experiments on cell lines overexpressing the mutant protein. Furthermore, the consistent presence of DSBs in plasma shows that this could be a valuable biomarker for HSAN-I.

Analysis of plasma samples shows that the p.S331F mutation additionally increases the affinity of the SPT enzyme for glycine, an effect that was previously observed for the p.C133W mutation. The absence of increased levels of 1-deoxymethyl-SA in the plasma of patients with the mutation p.C133Y, p.V144D [START_REF] Penno | Hereditary sensory neuropathy type 1 is caused by the accumulation of two neurotoxic sphingolipids[END_REF] or p.A352V (this study) indicates however that 1-deoxymethyl-SA is not essential for the pathogenesis of HSAN-I. Furthermore, the neurotoxic effect on primary sensory neurons was less pronounced with 1-deoxymethyl-SA than with 1-deoxy-SA [START_REF] Penno | Hereditary sensory neuropathy type 1 is caused by the accumulation of two neurotoxic sphingolipids[END_REF]. Nevertheless, a contributory role for 1-deoxymethyl-SA in the pathogenesis cannot be excluded at this point. The question of the causality in HSAN-I is subject of debate. The impact of reduced SPT activity on total sphingolipid levels remains controversial. Reports on decreased ceramide and sphingomyelin synthesis were published [START_REF] Bejaoui | Hereditary sensory neuropathy type 1 mutations confer dominant negative effects on serine palmitoyltransferase, critical for sphingolipid synthesis[END_REF][START_REF] Gable | Mutations in the yeast LCB1 and LCB2 genes, including those corresponding to the hereditary sensory neuropathy type I mutations, dominantly inactivate serine palmitoyltransferase[END_REF], other studies reported increased levels of glucosylceramide [START_REF] Mccampbell | Mutant SPTLC1 dominantly inhibits serine palmitoyltransferase activity in vivo and confers an age-dependent neuropathy[END_REF], while yet other reports did not observe any changes in lipid composition [START_REF] Dedov | Activity of partially inhibited serine palmitoyltransferase is sufficient for normal sphingolipid metabolism and viability of HSN1 patient cells[END_REF][START_REF] Hornemann | Is the mammalian serine palmitoyltransferase a high-molecular-mass complex?[END_REF]. In this study, we showed that total sphingolipid levels are not significantly altered by the reduction in canonical SPT activity (Fig 3a,b). In addition, heterozygous SPTLC1 and SPTLC2 knockout mice, despite showing significantly reduced SPT activity [START_REF] Hojjati | Serine palmitoyl-CoA transferase (SPT) deficiency and sphingolipid levels in mice[END_REF], do not develop a neuropathy even at older age, rendering it unlikely that haploinsufficiency itself is sufficient to cause HSAN-I. Toxicity inherent to the mutant protein is also unlikely to be the causative factor, since double transgenic mice overexpressing both wt SPTLC1 and the p.C133W variant do not develop a neuropathy [START_REF] Eichler | Overexpression of the wild-type SPT1 subunit lowers desoxysphingolipid levels and rescues the phenotype of HSAN1[END_REF]. These observations strengthen the hypothesis that the accumulation of neurotoxic DSBs underlies the pathology in HSAN-I (Eichler et al., 2009;[START_REF] Penno | Hereditary sensory neuropathy type 1 is caused by the accumulation of two neurotoxic sphingolipids[END_REF]. This is further supported by the observation that the exogenous addition of 1-deoxy-SA but not of SA to cultured primary dorsal root ganglia is neurotoxic by impairing neurite outgrowth, probably secondary to disturbing cytoskeletal integrity [START_REF] Penno | Hereditary sensory neuropathy type 1 is caused by the accumulation of two neurotoxic sphingolipids[END_REF]. In this context, it is interesting to note that exogenous addition of 1-deoxy-SA was reported earlier to impair stress fiber formation by interfering with the Rho GTPase signaling cascade [START_REF] Cuadros | The marine compound spisulosine, an inhibitor of cell proliferation, promotes the disassembly of actin stress fibers[END_REF]. Members of the Rho GTPase family regulate various processes involving actin and neurofilament dynamics, cell polarization, axonal stabilization and growth cone formation [START_REF] Heasman | Mammalian Rho GTPases: new insights into their functions from in vivo studies[END_REF]. Destabilization of axonal structures could be envisaged to cause the progressive axonal degeneration seen in HSAN-I patients. However, whether the endogenous generation of 1-deoxy-SA in neuronal cells has the same effect has not been investigated yet. In conclusion, our results demonstrate that the SPTLC1 mutation p.S331F, which is associated with a severe HSAN-I phenotype, has the same effect on SPT activity and DSB formation as the previously characterized SPTLC1 mutations, suggesting that additional factors influence the disease course. Furthermore, we show that the p.A352V mutation, for which genetic evidence for pathogenicity is poor, is truly disease causing, as it is associated with a reduced SPT activity and elevated DSB levels in patient plasma. at room temperature. The primary antibody was detected using a secondary donkey anti-mouse IgG conjugated to Alexa Fluor 594 (1:200; Molecular Probes, Paisley, UK) for 1 h at room temperature, combined with Hoechst 33342 (Invitrogen) for staining of the nuclei. After washing with PBS, cells were mounted in Vectashield medium (Vector Labs, Burlingame, USA). Cells were microscopically examined and images were acquired on a Zeiss Axiovert 200 microscope with a microlens-enhanced dual spinning disc confocal system (UltraVIEW ERS; PerkinElmer, Seer Green, UK), equipped with a 60X 1.4 NA-objective.

  Tripos Inc., St-Louis, USA). Model validation with WHATCHECK

Figure 1 .

 1 Figure 1. De novo p.S331F mutation in a HSAN-I patient and structural model of human SPT. (A) Pedigree of the HSAN-I patient MGZ-48522 carrying the de novo c.992C>T mutation (p.S331F) in SPTLC1, with sequence trace files of the mutation. Nucleotide numbering reflects cDNA numbering with +1 corresponding to the A of the ATG translation initiation codon in the reference sequence. The initiation codon is codon 1. (B) Structure model of the human SPT heterodimer (PDB ID: 2JGT) with the subunits represented in red and blue. The amino acids highlighted in yellow, p.S331 and p.A352, are subject of study in this report. The amino acids highlighted in green, p.C133 and p.V144, are also mutated in HSAN-I. The cofactor PLP is indicated in orange.

Figure 2 .

 2 Figure 2. SPT activity in cells stably expressing SPTLC1 mutant proteins. (A) Radioactive SPT activity assay. Total cell lysate was incubated with L-serine, Palmitoyl-CoA, PLP and L-[U-14 C] serine. After 60 min, lipids were extracted and the incorporation of radioactive serine was determined as a measure for SPT activity. (B) Fumonisin B1 block assay. SPT activity in HEK293T cells stably expressing wt or mutant SPTLC1 was analyzed by measuring SA accumulation after treatment with Fumonisin B1. For both experiments, EGFP expressing cells served as control. Data is represented as a mean with error bars representing standard deviations. Standard deviation was calculated based on six (A) and three (B) independent experiments. CPM: counts per minute; SA: sphinganine; *: p-value < 0.01; ***: p-value < 0.005.

Figure 3 .

 3 Figure 3. Levels of sphingosine and DSBs. (A,B) Levels of SO are measured after an acid and base hydrolysis assay on the extracted lipids in A. HEK293T cells and B. lymphoblast cells. (B) SO levels in a lymphoblast cell line of patient CMT-791.01 carrying the p.S331F mutation are compared to the levels in the unaffected parents (Cnt1 and Cnt2) and unrelated healthy controls (Cnt3 and Cnt4). (C,D) Levels of 1-deoxy-SA and 1-deoxy-SO are measured after an acid and base hydrolysis of the extracted lipids in C. HEK293T cells and D. lymphoblast cells. The levels in lymphoblast cell lines of patient CMT-791.01 with the p.S331F mutation are compared to the levels in the unaffected parents (Cnt1 and Cnt2) and unrelated healthy controls (Cnt3 and Cnt4). (E) DSB levels in plasma samples of p.S331F patient CMT-791.01, p.S331F patient MGZ-48522 and his unaffected family members (father, mother and brother), p.A352V patient CMT-186.05 and of three unrelated and healthy control individuals (Cnt5, Cnt6 and Cnt7). For each assay, a representative experiment is shown. Data is represented as a mean with error bars representing standard deviations. Standard deviation was calculated based on three independent experiments. SA: sphinganine; SO: sphingosine.

  Overview of all currently known SPTLC1 and SPTLC2 mutations and polymorphisms, the associated clinical features and biochemical characteristics. Y: years; Adolesc: adolescent; Cong: congenital; SN: sensory neuropathy; DSB: deoxysphingoid bases; ↓: reduced; ↑: moderately elevated; ↑↑: highly elevated; -: unchanged

John Wiley & Sons, Inc.Human Mutation

ACKNOWLEDGEMENTS

We are grateful to the patients for their contribution to this study. We thank N. Bonnelo for assistance in patient's management and J.-P. Timmermans for access to the microscopy facility at the University of Antwerp. This project was in part funded by a Methusalem grant of the University of Antwerp, the Fund for Scientific Research (FWO-Flanders), the Medical Foundation Queen Elisabeth (GSKE), the 'Association Belge contre les Maladies Neuromusculaires' (ABMM) and the Interuniversity Attraction Poles P6/43 program of the Belgian Federal Science Policy Office (BELSPO). A.R. is supported by a PhD fellowship of the Institute for Science and Technology (IWT). A.R. received an EMBO Short Term Fellowship. K.J. holds a postdoctoral fellowship from BELSPO. Support for H.T. was provided by the German Society for Clinical Chemistry and Laboratory Medicine (DGKL), the Gebert Rüf Foundation and the European Commission (LSHM-CT-2006-037631).

SUPPLEMENTARY MATERIALS AND METHODS

Lentiviral transduction of SH-SY5Y cells

Lentivirus encoding wt or mutant SPTLC1-EGFP fusion constructs was produced in HEK293T cells by cotransfection of 10 µg pLenti-SPTLC1 expression construct together with 3 µg of pMD2-VSV and 6.5 µg of pCMV-R8.91 viral packaging vectors for lentivirus. The calcium transfection medium was replaced 8 h posttransfection with medium containing 10 mM sodium butyrate to enhance promoter activity. The following day the medium was replaced with complete MEM medium and lentiviral production was allowed to proceed for 24 h. The produced lentivirus was harvested by filtering the supernatant.

Stable neuronal cell lines (SH-SY5Y) were obtained by lentiviral transduction, according to the method described by [START_REF] Salmon | Lentiviral vectors for the gene therapy of lympho-hematological disorders[END_REF]. After infection, the cells were selected in complete MEM containing the selection agent blasticidin (3 µg/ml) for 2-3 weeks. The whole population of selected cells was pooled for further experiments.

Western blot

Cell lysates from stably expressing HEK293T cells were prepared in E1A lysis buffer (1% NP-40, 20 mM HEPES (pH 7.9), 250 mM NaCl, 20 mM β-glycerophosphate, 10 mM NaF, 1 mM sodium orthovanadate, 2 mM dithiothreitol, 1 mM EDTA and a protease inhibitor cocktail). Protein concentration was determined using the Bradford method (Biorad, Hercules, USA). Equal amounts of protein extracts were separated on 4-12% Bis-Tris NuPAGE gels (Invitrogen) and transferred to nitrocellulose membranes (Hybond TM -P, GE Healthcare, Uppsala, Sweden). After blocking in 5% milk powder in PBS-Tween (1x PBS supplemented with 0.1% Tween 20), blots were incubated with a primary polyclonal SPTLC1 antibody (1:2500; BD Biosciences, Franklin Lakes, USA) overnight at 4°C, washed with PBS-Tween and incubated for one hour with a secondary horseradish peroxidase conjugated anti-mouse antibody (1:10000; GE healthcare). After washing, blots were developed using the enhanced chemiluminescence Plus TM detection system (GE healthcare).

Immunocytochemistry and microscopy

SH-SY5Y cells stably expressing EGFP-tagged wt or mutant SPTLC1 were fixed in 4% paraformaldehyde in PBS for 20 min, followed by a permeabilization step in 0.1% Triton X-100 in PBS for 3 min. After blocking with 5% FBS and 0.5% BSA in PBS-Triton (1x PBS supplemented with 0.02% Triton) for 1 h, cells were incubated with primary monoclonal antibody recognizing the ER marker calreticulin (1:500; Abcam, Cambridge, UK) for 1 h

SUPPLEMENTARY FIGURES

Supplementary Figure S1. Expression of wt and mutant SPTLC1 in HEK293T cells. Western blot shows equal expression levels of endogenous SPTLC1 (lower band, 53 kDa) and V5-tagged SPTLC1 (upper band). Given the comparable intensity of the wt and mutant transgenic bands, it can be concluded that the mutations do not interfere with the expression of the protein. Moreover, this blot shows that the transgenes are expressed at a moderate level with an approximate 1 to 1 ratio of transgenic and endogenous SPTLC1, mimicking the situation in HSAN-I patients.

Supplementary


Jing Li
email: jing.li2@univ-nantes.fr

Marcus Barkowsky
email: marcus.barkowsky@univ-nantes.fr

Junle Wang
email: junle.wang@univ-nantes.fr

Patrick Le Callet
email: patrick.lecallet@univ-nantes.fr

STUDY ON VISUAL DISCOMFORT INDUCED BY STIMULUS MOVEMENT AT FIXED DEPTH ON STEREOSCOPIC DISPLAYS USING SHUTTER GLASSES

Keywords: Stereoscopic motion images, velocity, disparity, visual discomfort, subjective experiment

Stereoscopic motion images are able to provide observers with realistic and immersive viewing experience. However, observers often experience visual discomfort during the viewing process. In this paper, we investigated the relationship between visual discomfort and the planar motion at different depth levels. The Paired Comparison method was used in the subjective experiments to allow for a precise measurement. The experimental results indicated that the relative angular disparity between foreground object and background played a more important role in determining the visual discomfort than the vergence-accommodation conflict. Furthermore, the results showed that with the increase of planar motion velocity, viewers might experience more visual discomfort. To quantify the effects of relative angular disparity and velocity on visual discomfort, two visual discomfort models were constructed. The preferred model was chosen based on the performance as well as the algorithmic complexity. This model can be used as an index for other related researches.

INTRODUCTION

Stereoscopic images have drawn more and more attention recently as they can give viewers a totally different and enhanced viewing experience. The three-dimensional (3-D) technology can be applied on numerous areas, e.g. television broadcast system, video games, telecommunications, telemedicine, education, etc. Nevertheless, viewers often experience visual discomfort during the 3-D viewing process which may mangle the viewer's enthusiasm. And it seems to be one of the critical factors that impede the development of 3-D on some applications.

To solve this problem, a number of researches on the relationship between visual discomfort and 3-D technology have been conducted [START_REF] Lambooij | Visual Discomfort and Visual Fatigue of Stereoscopic Displays: A Review[END_REF]. One of the widely accepted main reasons that may cause visual discomfort is the vergence-accommodation conflict [START_REF] Hoffman | Vergence-accommodation conflicts hinder visual performance and cause visual fatigue[END_REF]. When viewing an object by means of a 3-D screen, the eyes will converge to the virtual object which is in front of or behind the screen plane. However, the accommodation has to be performed at the screen depth level. This discrepancy may induce the declination of visual function and cause visual discomfort. Additionally, some studies about the effect of distortions on visual discomfort have been conducted [3][4]. Crosstalk [START_REF] Meesters | A survey of perceptual evaluations and requirements of threedimensional TV[END_REF], as one of the main distortions in 3D display may induce unnatural blur in stereoscopic images, of which the perceptual impact is directly related to the amount of screen disparity and may accelerate the difficulty to accommodation, which then leads to visual discomfort [START_REF] Lambooij | Visual Discomfort Associated with 3D Displays[END_REF]. However, other studies show that ratings of visual strain remain constant with increasing crosstalk [START_REF] Seuntiёns | Perceptual attributes of crosstalk in 3D images[END_REF].

Most of the causes described above have been well documented for the case of still stereoscopic images. However, the number of studies of visual discomfort on moving stereoscopic images is comparably small. The object's motion in stereoscopic videos can be categorized in in-depth and planar motion. Some studies showed that motion in depth, i.e., the magnitude of binocular disparity varying over time, could play an important role in visual discomfort, and it might be more important in determining visual discomfort than the absolute magnitude of the binocular disparity [START_REF] Speranza | Effect of disparity and motion on visual comfort of stereoscopic images[END_REF] [START_REF] Yano | A study of visual fatigue and visual comfort for 3D HDTV/HDTV images[END_REF]. In [START_REF] Yano | Two factors in visual fatigue caused by stereoscopic HDTV images[END_REF], the authors concluded that the visual fatigue occurred when the stereoscopic images involved a motion component in depth even if they were displayed within the range of depth of field. Meanwhile, no visual fatigue was found in lateral motion images. However, this conclusion was given under the condition that the velocity of lateral motion was varied over time. It could not give a quantitative analysis of the relationship of the lateral motion velocity and visual fatigue.

In this study, the relationship between visual discomfort and the planar motion at different depth levels was investigated. In the subjective experiment, the stereoscopic stimuli contained a foreground and a background. The foreground object moved in a certain depth plane, with 5 different disparity levels. The velocity of the object stayed the same in one sequence, and there were a total number of 3 different levels (Slow, Medium, Fast) for all stimuli. The background was positioned behind the screen of a fixed distance. The Paired Comparison method was used in this task. The observers were asked to select the stimulus which made them feel more uncomfortable. After analyzing the subjective results, two models for visual discomfort concerning velocity and disparity were constructed. These quantitative models were compared with the subjective results, and showed high correlation with viewers' perception. The preferred model was chosen based on performance as well as algorithmic complexity. It can be used as an index for other related researches, e.g., stereoscopic image quality assessment, stereoscopic image coding.

This paper is organized as follows. In section 2, the experimental setup is presented. In section 3, the results are validated and in Section 4, a deep analysis of the results is done which also includes a detailed discussion. In Section 5, a model is proposed which was fitted to the data. Section 6 concludes the paper.

EXPERIMENTAL SETUP

Experimental design

It is often accepted that 60-70 minutes of arc is the comfort threshold for static disparities [START_REF] Lambooij | Visual Discomfort and Visual Fatigue of Stereoscopic Displays: A Review[END_REF]. Meanwhile, some researchers also use depth of focus (DOF) to calculate the comfortable viewing zone [START_REF] Chen | New Requirements of Subjective Video Quality Assessment Methodologies for 3DTV[END_REF], which refers to the range of distances in image space within which an image appears in sharp focus and is given in terms of diopters (D) (a value of ±0.2D is suggested). To investigate how planar motion affects the visual discomfort at different disparity levels, five binocular disparity levels for the foreground were chosen in this experiment. Three of the angular disparity levels were within the comfortable viewing zone [START_REF] Chen | New Requirements of Subjective Video Quality Assessment Methodologies for 3DTV[END_REF], two were outside it. These can be expressed in degrees of visual angle [START_REF] Holliman | 3D Display Systems[END_REF], as shown in Fig. 1. The binocular angular disparity can be calculated by the following equations ( 1) and ( 2 A In this study, the five angular disparity levels were 0, ±0.65, and ±1.3 degree (+ means crossed, -means uncrossed),assuming that the interpupillary distance was 65mm and the viewing distance was about 90 cm. A background was placed at a fixed position which was behind the screen at a distance of about 46.28cm, with the angular disparity of -1.4 degree. Fig. 2 shows the disparities used in the stimuli and their relationship with comfortable viewing zone. Three velocity levels which represented slow, medium and fast were used in the experiment.

φ β α = - (1)
B φ γ α = - (2)

Stimuli

To avoid the influence of other factors on visual discomfort, we used computer-generated stereoscopic sequences for precise control. The stereoscopic sequences consisted of a left-view and a right-view image which were generated by the MATLAB psychtoolbox [START_REF] Brainard | The Psychophysics Toolbox[END_REF] [START_REF] Pelli | The videotoolbox software for visual psychophysics: Transforming numbers into movies[END_REF]. Each image contained a foreground and a background. A black Maltese cross with 480×480 pixels was used as the foreground object as it contained both high and low spatial frequency components. This was supposed to limit the influence of one particular spatial frequency in the experiment. The Maltese cross moved along a trajectory which was a circle with center point at the center of the screen, and a radius of 300 pixels. The motion direction was anti-clockwise. The reason to choose a circle as the trajectory was that it could avoid the step impulse that came from a sudden change of the motion direction, which may cause unexplained effects of visual discomfort. As the trajectory was a circle, the velocity was expressed in degree/s. The three velocity levels were 71.8, 179.5 and 287.2 degree/s, which represented slow, medium and fast, respectively. The background was generated by adding salt & pepper noise on a black image, and then filtered by a circular averaging filter. The reason that we used this kind of image as the common background of all stimuli was that it could preclude all of the monocular cues on stereopsis. Additionally, to give the viewers a reference of the trajectory a black circle which was the same as the moving track of the object was placed on the background. So, for viewers, the stimuli appeared to be composed of two parts: the salt&pepper-like background with a black circle on it, and a moving Maltese cross on a depth plane with a certain velocity.

As there were 3 levels of velocity and 5 levels of angular disparity, there would be totally 15 stimuli for the experiment. An example of the stimuli is shown in Fig. 3, in which the foreground object is placed in front of the screen with an angular disparity of 1.3 degree. Fig. 3. An example of a stereoscopic image in the experiment. The foreground object is moving at the depth plane with a disparity of 1.3 degree. The background is placed at the depth plane with a disparity of -1.4 degree. The motion direction of the Maltese cross is anti-clockwise.

Apparatus

The stereoscopic sequences were displayed on a Dell Alienware AW2310 23-inch 3-D LCD screen (1920×1080 full HD resolution, 120Hz), which featured 0.265-mm dot pitch. The display was adjusted for a peak luminance of 50 cd/m 2 when viewed with the active shutter glasses. The graphics card of the PC was an NVIDIA Quadro FX 3800. Stimuli were viewed binocularly through the NVIDIA active shutter glasses (NVIDIA 3D vision kit) at a distance of about 90 cm, which was approximately three times of the picture height. The peripheral environment luminance was adjusted to about 44 cd/m 2 . When seen through the eyeglasses, this value corresponded to about 7.5 cm/ m 2 and thus to 15% of the screen's peak brightness as specified by ITU-R BT.500 [START_REF]Methodology for the subjective assessment of the quality of television pictures[END_REF].

Viewers

Because the task of this experiment needs a high level of concentration throughout the whole process, we decided to conduct an experts-only subjective experiment. We confirmed that the participants understood the necessities to pay close attention to the task they were asked. Ten experts in 3-D perception, coding, quality assessment and subjective experiments participated in the experiment. Eight experts are male, two are female. Their ages ranged from 24 to 43 years old. All have either normal or corrected-to-normal visual acuity. The visual acuity test was conducted with a Snellen Chart for both far and near vision. The Randot Stereo Test was applied for stereo vision acuity check, and Ishihara plates were used for color vision test. All of the viewers passed the pre-experiment vision check.

Assessment Method

In our study, the paired-comparison method was chosen as it is a well-known method in the field of psychophysics [START_REF] Gescheider | Psychophysics: The Fundamentals, Third Edition[END_REF]. In the experiment, the viewers watched a pair of stimuli at one trial, and then they were asked to select the one which made them feel more uncomfortable. A total of 210 pairs were presented in each individual subjective experiment. This number corresponds to the presentation of all combinations of 15 stimuli except for equal presentation on the left and right side. In particular, it contains the same condition with the stimulus order inversed as the first onset of a stimulus might have caused a bias on the feeling of visual discomfort. The presentation order of the whole 210 pairs was randomly permuted for each viewer.

Procedure

The subjective experiment contained a training session and a test session. In the training session, there were five pairs of stimuli. At the beginning, the viewers were told that they will watch a series of stereoscopic motion images. They were asked not to stare at the moving object all the time, but watch the whole screen of the stereoscopic sequence under test. Then, they should select the one which made them feel more uncomfortable, concerning e.g., eye strain, headache, etc. As it was not technically possible to display a pair of stimuli on two screens, the viewers had to use two keys to switch between the pair of stimuli on one screen. There was no time limit for the display of stimuli, so the trial won't continue until the viewers made their decision by pressing a specified button. After the explanation of the experiment, the viewers were asked to do the test by themselves. During the training session, all questions of the viewers were answered. We made sure that after the training session, all of the viewers knew about the process and task of this experiment clearly.

In the test session, the task and procedure were the same as the training session except 210 pairs of stimuli were compared. As the duration of the whole test was different due to individual difference of each viewer, and to avoid visual fatigue caused by long time watching affecting the experimental results, the viewers were asked to have a 15 minutes break after 30 minutes of the test.

In total 15 data sets were acquired as 5 experts participated twice in the experiment but on a different day.

VERIFICATION OF TEST RESULTS

Validation analysis on raw data

According to the experimental design of 3 velocity levels and 5 disparity levels, there were totally 15 stimuli. Each stimulus was compared with every other stimulus, and for each pair two trials were carried out because the sequence of the occurrence also matters. Therefore, there were 15×14=210 trials for the whole experiment. 210 comparison results and 15 stimuli which were not compared to themselves could be arranged in a 15×15 matrix with the diagonal value being 0.5. A three-dimensional matrix M(i,m,n) is used to express each individual subjective experimental results, as shown in Fig. 4. The row represents the stimulus of the first presentation, and the column represents the stimulus of the second presentation. For the ith individual subjective experiment, M(i,m,n) represents the number of times stimulus m is selected over stimulus n. For example, in the ith individual subjective experiment, the pair of stimuli was stimuli m and n, and stimulus m is presented first. If the observer choose stimulus m as more uncomfortable, then,

M(i,m,n)=1, otherwise M(i,m,n)=0.
For each individual subjective experiment, the matrix M is binary without considering the diagonal elements. Before any further data analysis about the relationship of visual discomfort to binocular angular disparity and velocity, we checked the validity of the raw data regarding the following aspects: 1) Screening of the observers. To remove the data that stemmed from the observers who were inclined to give random answers in the experiment. 2) Verification of each pair condition. There might be a pair of stimuli in which most of the viewers always chose the one presented first. The influence of the presentation orders on visual discomfort, and the interaction effects of the pair of stimuli on visual discomfort can be verified in this process. Ideally, for all pairs of stimuli, the observer's answers should not depend on the presentation orders. Thus, it can be expressed by the equation M(i,m,n)+M(i,n,m)=1. A statistical method was used to verify it. The Student's-t-Test was performed on each individual subjective experimental results, which correspond to the elements of the upper and lower triangular matrices of M for each subjective experiment, and expressed as M iu and M il respectively, as Fig. 5 (1) shown. The question thus changed to if M iu and 1-M il were obtained from a Gaussian process with a common mean value. When the hypothesis was verified by the Student's-t-Test, it can be concluded that the observer gave consistent answers in this individual subjective experiment. Otherwise, the data from this individual experiment might not be valid and should be rejected. The whole process that utilizes Student's-t-Test will be referred to as "Consistency test" in this paper.

Similarly, the correctness of each pair condition would be checked by the "Consistency test". It was performed on S mn and 1-S nm , where S mn was a binary vector and expressed as [M (1,m,n), M(2,m,n),…, M(t,m,n)] assuming there were totally t individual subjective experiments, as shown in Fig. 5(2). It represented all of the observers' selections one by one for the trial that stimulus m and n were compared, and stimulus m was presented first.

In our experiment, the Student's-t-Test was performed at 5% significance level. Both, the screening of observers and the verification of each pair condition passed the "Consistency test".

Evaluation of the Thurstone model scores

As the raw data had passed the "Consistency test", it indicated that the presentation order didn't influence the experimental results. We reconstructed the matrix for individual subjective experimental results as follows: the value in the row m and column n counted the number of times that stimulus m was selected over stimulus n. The presentation order was not considered any more. E is used to express this new matrix. It should be noted that E(i,m,n)+E(i,n,m)=2 (Each pair was compared twice). G is used to express the new matrix for the global subjective experimental results, in which ( , ) ( , , )

i G m n E i m n = ∑
. F is used to express the matrix in which the row m and column n represent the proportion that stimulus m is selected over stimulus n in all subjective experiments, and ( , ) ( , , ) 2

G m n F i m n t = × .
The Thurstone model [START_REF] Thurstone | A law of comparative judgment[END_REF][17] is frequently used in fitting paired comparison data and ranking data (after rankings are transformed into paired comparisons). Thus, the Thurstone model was fitted to the matrix G to get the Thurstone scores. Then the data was linearly rescaled in the interval of [0, 1]. Score 0 corresponded to the condition which was closest to the 2-D conditions in this study and the viewers experienced the least visual discomfort. Score 1 corresponded to the condition in which viewers experienced the most visual discomfort in the experiment.

The Thurstone scores of all stimuli for visual discomfort are shown in Fig. 7. Firstly, we checked how well the Thurstone scores of all stimuli fitted to the global subjective experimental results. M Thurstone (m,n) is used to represent if the Thurstone score of stimulus m is higher than that of stimulus n. Thus, M Thurstone is a binary matrix without considering the diagonal elements. To compare the matrix M Thurstone with the global subjective experimental results, the matrix F is changed to a binary matrix by thresholding the value which is below the threshold 0.5 to 0 and above the threshold to 1. This binary matrix is expressed by F C . Two evaluation methods were used to check the agreement of the Thurstone scores with the global subjective experimental results. The first one was the "Consistency test". It was performed on the M Thurstone and F C . And they passed the "Consistency test". Secondly, an "Agreement test" was conducted on the Thurstone scores and the global subjective experimental results, which means calculating the proportion that the value in each position of M Thurstone was the same with the corresponding value in F C . The "Agreement test" result was 0.96, which indicated that the Thurstone scores were highly consistent with the observers' subjective perception.

ANALYSIS OF RESULTS ON DISPARITY AND VELOCITY

The effects of disparity and velocity on visual discomfort

As there were a foreground object and a background in the stimulus, the relative disparity between the foreground object and the background was used to analyze their effects on visual discomfort. The binocular angular disparity of the background was -1.4 degree, thus the 5 relative angular disparity levels of the foreground object were 0.1, 0.75, 1.4, 2.05, 2.7 degree. Fig. 7 and Fig. 8 show the effects of relative disparity and velocity on visual discomfort. The vergence-accommodation conflict is one of the well accepted reasons that may result in visual discomfort. Thus, in our experiment, it was expected that the stimuli with large magnitude of angular disparity should induce more visual discomfort since the vergence-accommodation conflict was large in this situation. However, as shown in Fig. 7,it's not the case, especially for the stimuli whose relative disparity was 0.1 degree but the binocular disparity were far beyond the comfortable viewing zone, their ratings of visual discomfort were much lower than the stimuli with 0 degree of binocular angular disparity. The vergenceaccommodation conflict might have a smaller effect on visual discomfort in this situation. On the opposite, it could be clearly found that the ratings of visual discomfort increased with the relative angular disparity. Thus in our study, the relative angular disparity might have a predominant effect on visual discomfort and it might outweigh the vergence-accommodation conflict.

From Fig. 7 it can be seen that viewers were more likely to experience visual discomfort when the velocity was high. Thus it is suggested that for real-world applications, it might be necessary to reduce the depth budget for fast motion sequences while the depth budget for slow motion sequences may be increased. Fig. 7 also shows that some stimuli may have a similar effect on visual discomfort. For example, there were four different conditions that their Thurstone scores were close to 0.7. In the following, the notation '(relative disparity, velocity)' is used to express the stimulus. They were (0.1, 287.2), (0.75, 287.2), (2.05, 179.5) and (2.05, 71.8). It seems that the stimulus which had a large relative disparity and a slow velocity had a similar effect on visual discomfort when compared to the stimulus which had small relative disparity but fast velocity. For a more precise analysis of what kind of stimuli may have similar effects on visual discomfort, we checked the viewers' experimental results, i.e. the matrix F, to find in which cases their values were near 0.5, we defined this range as [0.45 0.55]. The results are shown in Table I, where the stimuli were expressed by '(relative disparity, velocity)'. It seems important to note that besides the stimuli pairs which had similar velocities and relative angular disparities, the table also confirms the hypothesis above: A stimulus which has large relative disparity and slow velocity might have a similar effect on visual discomfort compared to a stimulus which has small relative disparity but fast velocity. Thus, for viewers, slow moving stereoscopic sequences with large depth budget and fast moving stereoscopic sequences with small depth budget would result in a similar degree of visual discomfort. Fig. 8 depicts that the ratings of visual discomfort increase with the velocity for each relative disparity level. It can be found that besides the line with the relative disparity of 0.1 degree, the other 4 lines are nearly parallel. This indicates that the relative disparity might have a small influence on the slope of the visual discomfort scores for different velocities, except very small relative disparities.

Thus, the offset of the scores of two different relative disparity levels was nearly constant. For example, for the relative disparities of 0.75 and 1.4 degree, the distances of the two lines (as shown in Fig. 8) at different velocities were almost equal. Thus, the two stimuli which had two different relative disparities but the same velocities might give the viewers a similar degree of the perceived differences in visual discomfort, no matter how velocity changed. It should be noted that the conclusion above didn't fit to the case of very small relative disparity, e.g., 0.10 degree; the differences of the scores between 0.1 degree and other relative disparities were significantly different at different velocities.

Discussion

In this study, the experimental results showed that viewers might experience more visual discomfort when the foreground object was moving in a depth plane with larger relative angular disparity. The vergence-accommodation conflict might not be a predominant factor in this study. It could be explained that due to the existence of a background, there would be two vergence points for the viewers in the stimuli, one was the background, and the other was the foreground object. When watching the stimulus, the viewers' attention may switch between the background and the object. The larger of the relative distances between the foreground and background, the larger of the abrupt change of the amount of vergence-accommodation mismatch when switch from one object to another, which might be a cause of visual discomfort.

The experimental results also showed that for a fixed relative disparity level, viewers feel more visual discomfort when the object moves faster. Furthermore, if there were two stimuli, the viewers might experience a similar degree of visual discomfort when one was with large relative disparity and slow velocity and the other was with the small relative disparity but fast velocity. And, the two stimuli which had two different relative disparities but the same velocity might give the viewers a similar degree of the perceived differences in visual discomfort when velocity changed.

OBJECTIVE VISUAL DISCOMFORT MODEL

Construction of visual discomfort models

From the normalized Thurstone score it could be found that the visual discomfort was affected by the relative disparity and velocity of the object in the stimuli. To better identify their relationship, a visual discomfort model should be constructed. As the relationship of visual discomfort to relative disparity and velocity was nearly linear, the simplest model would be the one in Equation ( 3), which will be noted as Model 1:

1 2 3 Q a v a d a = ⋅ + ⋅ + (3)
where Q represents visual discomfort, 1 a , 2 a and 3 a are coefficients, v is the velocity and d is the relative angular disparity.

However, it might be possible that the relative disparity and velocity have an interaction effect on visual discomfort. For each velocity level, the relationship between visual discomfort and relative disparity was nearly linear. Thus, the visual discomfort can be expressed as:

1 2 ( ) ( ) Q f v d f v = ⋅ + (4
) In our experiment, a linear function seems to be the most appropriate fitting method. So, f 1 and f 2 are modeled as linear functions of velocity. Finally, the visual discomfort can be described by Equation [START_REF] Meesters | A survey of perceptual evaluations and requirements of threedimensional TV[END_REF], where b 1 , …, b 4 are all constant and we define this as Model 2.

Evaluation of the fitted models

To evaluate the performance of these models on predicting the scores of visual discomfort, two methods were employed. Firstly, the RMSE of the fitted scores and the Thurstone scores were calculated. Then, the "Agreement test" was conducted on the individual subjective experimental results and the global subjective experimental results with the fitted two models. The "Agreement test" results for individual subjective experiment are shown in Fig. 11 and Fig. 12, for Model 1 and Model 2 respectively. The comparison results of the two models are shown in Table II. It could be found that there was no significant difference between the two models. When considering the complexity, Model 1 is slightly preferable for use as an index for the stereoscopic image related researches, e.g., stereoscopic image quality assessment, visual discomfort, or 3-D video coding.

SUMMARY AND CONCLUSIONS

In this study, the experimental results provided some new findings. Firstly, the relative angular disparity between the foreground and the background might be more significant in determining the visual discomfort than the binocular angular disparity of the foreground. The vergence-accommodation conflict might not significantly affect the visual discomfort in this study. Secondly, the planar motion with faster velocity results in more visual discomfort. Thirdly, a stimulus which has large relative disparity and slow velocity might have a similar effect on visual discomfort compared to a stimulus which has small relative disparity but fast velocity. Finally, the differences of the visual discomfort scores between two stimuli with different disparities were nearly constant, on which the velocity might have a quite small effect.

To quantify the effects of relative angular disparity and velocity on visual discomfort, two visual discomfort models were constructed. Both of them showed high correlation with subjective perception on visual discomfort. The preferred one with less algorithmic complexity can be used as an index for some related researches, e.g., stereoscopic image quality assessment, stereoscopic image coding.

It should be noted that there was a limitation in our experiment. The velocity and disparity level were comparably coarse which may not give a precise measurement about their effects on visual discomfort. Furthermore, in this study, we only investigated the effects of velocity and disparity on visual discomfort. Some other factors that may result in visual discomfort as well, e.g., the size of the object, the background type, the motion trajectory will also be investigated in the future.

  binocular angular disparities for A and B. Note that the positive value represents the crossed disparity, such as the point A; the negative value represents the uncrossed disparity, such as the point B.

Fig. 1 .

 1 Fig. 1. The definition of the binocular angular disparity, where F is the fixation point.

Fig. 2 .

 2 Fig. 2. The relationship of the foreground and the background position and the comfortable viewing zone

  ,m,1) M(i,m,2) M(i,n,1) M(i,n,2)

Fig. 4 .

 4 Fig. 4. An example of the matrix M for the ith individual subjective experimental results. The row represents the stimulus of the first presentation, and the column represents the stimulus of the second presentation.M(i,m,n) represents the number of times stimulus m is selected over stimulus n in the ith individual subjective experiment and can thus be either 0 or 1.

Fig. 5 .

 5 Fig. 5. The matrices used in the raw data validation test. (1) The diagram of the upper and lower triangular matrix used for screening of the observers. (2) The diagram of the vector used for the verification of each pair condition.

Fig. 6 .

 6 Fig. 6. The "Agreement test" results for each individual subjective experiment results and the Thurstone scores. The dashed line represents the mean of all results. After verifying the Thurstone scores based on the global experimental results, we checked the agreement of the Thurstone score with the individual subjective experimental results. The "Agreement test" was performed on E for each individual subjective experiment and M Thurstone . The results are shown in Fig. 6. Their mean value was 0.7914 and the standard deviation was 0.0833, which indicated that most of observers agree with the Thurstone score.

Fig. 7 .

 7 Fig. 7. Normalized Thurstone scores for visual discomfort. The different lines represent the different velocity levels, where slow, medium and fast represent 71.8, 179.5 and 287.2 degree/s. The outer two dashed lines represent the upper and lower limits of the comfortable viewing zone. The dashed line in the middle represents the position of screen plane.

Fig. 8 .

 8 Fig. 8. Normalized Thurstone scores for visual discomfort. The x-axis represents the velocity of the object, the y-axis represents the normalized Thurstone scores and the different lines represent the different relative disparity levels.TABLE I THE STIMULI PAIRS WHICH HAVE SIMILAR RATINGS ON VISUAL DISCOMFORT Number Stimuli Pairs (relative disparity, velocity) 1 (0.75, 287.2) & (2.05, 179.5) 2 (0.1, 287.2) & (2.05, 179.5) 3 (0.1, 179.5) & (0.75, 71.8)

  A curve fitting was performed on the normalized Thurstone scores in our experiment, the predicted coefficients for the two models were 0.0018,0.2102, -0.0477 for 1 a , 2 a , 3 a and 0.3110, 0.0026, -0.0006, -0.1888 for b 1 , b 2 , b 3 , b 4 , respectively. Fig.9and Fig.10show the regression results of the two models for visual discomfort. The solid lines show the predicted visual discomfort scores for different relative disparity and velocity, the dashed lines denote the normalized Thurstone scores in this experiment.

Fig. 9 .

 9 Fig. 9. The predicted visual discomfort of Model 1 in function of disparity and velocity. Solid lines represent fitted scores. Dashed lines represent original normalized Thurstone scores.

Fig. 10 .

 10 Fig. 10. The predicted visual discomfort of Model 2 in function of disparity and velocity. Solid lines represent fitted scores. Dashed lines represent original normalized Thurstone scores.

Fig. 11 .

 11 Fig. 11. The "Agreement test" results of each individual subjective experimental result on the fitted Model 1. The dashed line represents the mean of all results, which is 0.7822.

Fig. 12 .

 12 Fig. 12. The "Agreement test" results of each individual subjective experimental result on the fitted Model 2. The dashed line represents the mean of all results, which is 0.7905.

TABLE II .

 II COMPARISON RESULTS OF THE TWO MODELS

	Model	RMSE	Global	Individual Agreement
			Agreement	Mean	Std.
	1	0.05	0.92	0.7822	0.0728
	2	0.03	0.95	0.7905	0.0803

ACKNOWLEDGEMENT

The participation of the observers is gratefully acknowledged. This work has been partly conducted within the scope of the JEDI (Just Explore Dimension) ITEA2 project which is supported by the French industry ministry through DGCIS and the PERSEE project which is financed by ANR (project reference: ANR-09-BLAN-0170).


Marcus Barkowsky
email: marcus.barkowsky@univ-nantes.fr

Patrick Le Callet

ON THE PERCEPTUAL SIMILARITY OF REALISTIC LOOKING TONE MAPPED HIGH DYNAMIC RANGE IMAGES

Keywords: High Dynamic Range, Tone Mapping Operators, Quality of Experience, Objective Image Quality, Subjective Experiment

High Dynamic Range (HDR) images are usually displayed on conventional Low Dynamic Range (LDR) displays because of the limited availability of HDR displays. For the conversion of the large dynamic luminance range into the eight bit quantized values, parameterized Tone Mapping Operators (TMO) are applied. Human observers are able to optimize the parameters in order to get the highest Quality of Experience by judging the displayed LDR images on a realism scale. In the study presented in this paper, two TMOs with three parameters each were evaluated by observers in a subjective experiment. Although the chosen parameter settings vary largely, the chosen images appear to have the same QoE for the observers. In order to assess this similarity objectively, three commonly used image quality measurement algorithms were applied. Their agreement with the preference of the observers was analyzed and it was found that the Visual Difference Predictor (VDP) outperforms the Structural Similarity Index and the Root Mean Square Error. A threshold value for VDP is derived that indicates when two LDR images appear to have the same Quality of Experience.

INTRODUCTION

High Dynamic Range (HDR) images provide a large range of new applications. For example, the higher precision may be used in Wide Color Gamut applications. On the other hand, the higher luminance range can be used for adjusting the contrast locally, similar to the human eye. There are no displays available today which allow displaying HDR images in a native representation. This would require the rendering of the luminance ranging from an object in moonlit night to direct sunlight. However, some displays allow a wider range of luminances and are thus capable of partly rendering HDR images.

In order to display HDR images on the currently available reference displays, a conversion to the frequently used eight bit range by Tone Mapping Operators (TMO) is necessary. A recent overview over the different classes of TMO algorithms and a subjective comparison of eight algorithms can be found in [START_REF] Kuang | Testing HDR Image Rendering Algorithms[END_REF]. A subjective comparison of different TMOs with two real world indoor scenes has been presented in [START_REF] Yoshida | Perceptual evaluation of tone mapping operators with real-world scenes[END_REF]. In [START_REF] Ledda | Evaluation of tone mapping operators using a High Dynamic Range display[END_REF] six TMOs were compared to the appearance of the same scene on an HDR capable monitor in a subjective experiment using an adaptation of the paired comparison method.

While the TMOs already exploit several properties of the Human Visual System (HVS), they still need manual tuning by adjusting the algorithm parameters. In the publications mentioned above, the standard parameters for the TMOs were used. In this study, the participants of a subjective experiment were asked to select the optimal parameter set. A set of images was prerendered that span the useful parameter range. The observers chose those images from the set which provided the most realistic impression. This was considered as a criterion for the best Quality of Experience in this application. No explicit reference by an HDR display or by a real world scene was provided to them.

As conducting a subjective experiment is very time consuming, a prediction of the result by an objective measurement would be advantageous. Towards this goal, a first analysis was performed which might help in reducing the number of observers and understanding the criterions which were used by the observers. The total set of rendered LDR images is split into two classes: Those which were chosen by the observers and those which were not. Taking any of the chosen images as a reference, it can be assumed that the other chosen images are perceptually closer than those which belong to the other class. Three full reference image quality metrics were tested for their ability to discriminate between the two classes.

The paper is organized as follows. In Sec. 2 the preparation of the LDR images and the conditions of the subjective experiment are presented which generated the two subsets. The analysis of the objective image quality algorithms is provided in Sec. 3 before conclusions are drawn in Sec. 4.

SUBJECTIVE EXPERIMENT

In the subjective experiment four synthetic and four natural HDR images were used. The images were scaled to a resolution of 1920 × 1080 pixels. These eight HDR images were processed by two TMOs. For both algorithms the implementation in Qtpfsgui was used [START_REF]Qtpfsgui Project Homepage[END_REF].

The first TMO was published by Mantiuk et al. in [START_REF] Mantiuk | A perceptual framework for contrast processing of high dynamic range images[END_REF]. It uses a contrast representation of the image and exploits the contrast perception of the HVS. The implementation contains three parameters which are termed contrast, saturation and detail. The range of parameters was evaluated and 10 distinct choices for contrast, 8 for saturation and 3 for detail were chosen. Thus, for each of the eight HDR images, a total of 240 LDR images was created.

The second TMO models the characteristics of the cones in the retina and was proposed by Reinhard et al. in [START_REF] Reinhard | Dynamic range reduction inspired by photoreceptor physiology[END_REF]. The parameter space was evaluated with 13 settings for brightness, 10 for chrominance, and 7 for the lightness parameter. This leads to a total of 910 LDR images for each of the eight HDR images.

A subjective experiment was setup that allowed the participants to evaluate the three parameters by using the slider device shown in Fig. 1. Only the first three sliders were used and each corresponded to one parameter. The LDR image that corresponded to the slider position was displayed immediately on a TVLogic LVM-401W reference screen. The room setup and viewing distance corresponded to ITU-R BT.500 and the HDTV testplan of the Video Quality Experts Group (VQEG) [START_REF] Cermak | Test Plan for Evaluation of Video Quality Models for Use with High Definition TV Content[END_REF]. For the two TMO non-overlapping groups of naïve observers were invited, 20 for Mantiuk and 21 for Reinhard. The viewers were screened for acuity and color vision. They were asked to choose the image that provided the most realistic impression as this can be expected to give the highest Quality of Experience for a longer presentation. At least five changes of the sliders and at least one change per slider was required by the assessment program before the observer was allowed to vote. The finally chosen image was seen on the screen while the observer confirmed his choice. A training session preceded the subjective experiment. The chosen image was recorded for each observer and each of the eight HDR images.

PERFORMANCE ANALYSIS FOR IMAGE QUALITY ALGORITHMS

Three image quality algorithms were tested whether they could predict the observers choice. The inputs to all algorithms are two images and the output is a single value which indicates the similarity of the two images. Firstly, the simple Root Mean Square Error (RMSE) measure is used. As The algorithm indicates the probability of change detection for each pixel individually. The freely available implementation also outputs the number of pixels in the image that were alerted to exceed a certain visibility threshold. In this evaluation, the percentage of pixels that exceed the 75% visibility threshold was chosen.

The performance analysis compares the difference measured by the three algorithms to the observers' opinion. The analysis is based on two assumptions. The first assumption is that images chosen by the observers are equal. In our subjective experiment, the term "equal" refers to a notion of "realistic appearance" which is shared by the observers. The second assumption is that images which were not chosen by the observers differ significantly from those which were chosen. This assumption may be weak because only 20 out of 240 for Mantiuk and 21 out of 910 images could be selected. So it has to be considered that a certain number of non-chosen images does not differ significantly. This problem is addressed by the type of analysis performed.

In Table 1 an overview of the number of tested conditions is given. The number of "equal" conditions refers to comparisons in which both images were chosen by observers while the term "dissimilar" refers to comparisons in which only one image was chosen by an observer. The RMSE and SSIM algorithms are commutative, e.g. testing image A versus B reveals the same result as image B compared to image A. The VDP is not commutative so the full matrix of combinations needs to be considered.

For the performance comparison, a Receiver Operating Characteristics (ROC) analysis is performed. The notation follows the one presented in [START_REF] Green | Signal Detection Theory and Psychophysics[END_REF]. In our case, there are two alternatives: Either the images are equal or they are dissimilar.

Mantiuk Reinhard

Fig. 2. ROC analysis displaying the percentage of correct decisions on the y-axis and false alarms on the x-axis.

The subjective experiment is used as ground truth. The two alternatives are denoted as s and n, corresponding to "signal" and "no-signal". In our case, "signal" means that the two images are equal, thus each chosen by an observer. On the contrary, "no-signal" means that only one image was chosen by an observer. Correspondingly, the objective measure should provide the answer whether the images are equal ("S") or dissimilar ("N").

The objective part will be exemplified with the RMSE measure. For any two images given, the RMSE algorithm returns a value that measures the distance between the images. By choosing a threshold which is denoted as t, two classes of images can be generated. If the RMSE value is smaller than the threshold, the images are considered equal, otherwise they are classified as dissimilar. By evaluating the difference between the images chosen by the observers, the probability P (S|s) can be obtained. This probability specifies the number of correct decisions, e.g. when the RMSE is less than the threshold. The same threshold is applied to the RMSE values where the images were found dissimilar in the subjective experiment. The probability P (S|n) is obtained. This is the false alarm ratio: the objective algorithm identifies the images as equal while they were judged to be dissimilar. Please note, that in this particular evaluation, the false alarm ratio also includes the drawback of the second assumption mentioned above. Some conditions which are meant to be equal are classified as dissimilar by the subjective experiment because of the limited number of observers. Thus, all algorithms are affected in the same way by the fact that the measured false detection rate is higher.

In the optimal case, P (S|s) = 1 and P (S|n) = 0. In practice, the values depend on the threshold t. The smaller the value of t, the less images are classified as equal and the lower the two probabilities and vice versa. The relationship can be plotted in a ROC curve.

The results for our experiment are shown in Fig. 2. On the left side, the results for Mantiuk are presented. For each percentage of correct decisions chosen on the y-axis, a certain percentage of false alarms has to be accepted. It can be seen that the best performance is provided by the VDP algorithm as it has the lowest false alarm rate in the relevant area which usually ranges from 50% to 90%. The same is true for the Reinhard TMO. The RMSE and SSIM perform slightly better when compared to Mantiuk. Typically, a correct decision percentage of 50%, 75%, or 90% is requested. The resulting false alarm rates and the threshold values that need to be chosen for each algorithm are provided in the first two columns of Table 2. The choice of the threshold depends on the requirements for a particular purpose. For example, when it is preferred to have a low false alarm (FA) ratio, it may be agreed to have a chance of 50% of missing two images that are actually similar. On the opposite, when a manual inspection assures that the falsely alarmed images are sorted out, a large correct detection percentage (CD) might be used, e.g. 90%.

A practical application of these results would require the specification of a single threshold value for the output of the image quality measurement algorithm. This poses a problem which shall be explained with the RMSE algorithm. In Table 2 the threshold value for 90% correct detection and the Mantiuk TMO is given as 0.126. When using the same threshold when assessing images generated with the Reinhard TMO we get only a correct detection of 50%. In order to achieve 90%, a threshold of 0.352 would be necessary. Please note, that this threshold is very high: it indicates that the average difference in pixel values is 35%. Correspondingly, an average difference of 90 for each pixel on an eight bit image is accepted as similar. Exactly the same reasoning is true for the SSIM algorithm. The threshold value of 90% for Mantiuk corresponds to 0.837 which is closest to the 50% threshold for the Reinhard TMO. For obtaining 90% correct decisions, a SSIM threshold of 0.435 would be necessary. This value is usually the result of comparing the reference image to largely degraded images which are voted as "poor" in subjective experiments.

The VDP appears to be more stable as it shows a value of 1.04 for the threshold in Mantiuk and 0.845 in Reinhard. This threshold value is also more comprehensible: About 1% of the pixels in the image exceed a detection threshold of 75%.

In order to further evaluate the robustness of the algorithms and to determine a common threshold, the datasets of chosen and nonchosen data were merged for the two TMOs. The resulting ROC curves are displayed in Fig. 3 and the threshold values are provided in the last column of Table 2. It is apparent, that VDP outperforms RMSE and SSIM. A threshold value of 0.98 for distinguishing between chosen and nonchosen images at 90% correct decision is indicated for the VDP value. The number of false alarms would then be 43%.

CONCLUSIONS

Previously, the development of subjective and objective assessment of image quality was mainly focused on characterizing transmission systems. However, in recent times, there is an increasing demand for judging the visual equivalence of images. One of the main areas is the High Dynmic Range Imagery where the improved accuracy is either used to span a larger range of brightness or to enlarge the color gamut. In this contribution, a subjective assessment methodology was introduced to locate the best parameters for two Tone Mapping Operators. Furthermore, the question was investigated whether the subjective experiment can be predicted by using the current image quality algorithms. The ROC analysis was adapted and applied to the problem. It was shown that the Visual Difference Predictor outperforms RMSE and SSIM, probably due to its modelling of the Human Visual System. The threshold value of 1% for the VDP value is proposed as a criterion when judging whether two image of the same TMO appear similar.

Fig. 1 .

 1 Fig. 1. Slider device used in the subjective experiment

Fig. 3 .

 3 Fig. 3. ROC analysis for the combined datasets of the TMOs Mantiuk and Reinhard.

Table 1 .

 1 Properties of the analysis for each of the eight HDR images Cb, Cr color space was performed[START_REF] Ntia / Its | A3: Objective Video Quality Measurement Using a Peak-Signal-to-Noise-Ratio (PSNR) Full Reference Technique[END_REF]. The second algorithm is the Structural Similarity Index (SSIM)[START_REF] Wang | Image Quality Assessment: From Error Visibility to Structural Similarity[END_REF]. This measurement is based on local statistics. The freely available Matlab implementation was used. As a third algorithm the Visual Difference Predictor (VDP) was chosen in order to include an algorithm which incorporates the modelling of the Human Visual System[START_REF] Mantiuk | Predicting Visible Differences in High Dynamic Range Images -Model and its Calibration[END_REF]. It was specifically written to predict a visible difference and accepts HDR images as well as LDR images. In this evaluation, only the LDR part was used.

	TMO Algorithm	Mantiuk Reinhard
	Number of images presented	240	910
	Number of observers	20	21
	Number of "equal" conditions	380	420
	Number of "dissimilar" conditions	4.400	18.669
	the images are stored in red, green, and blue component, the
	mean squared error of all three planes was calculated. Please
	note, that this differs from the calculations that are usually
	performed for PSNR calculations in that no conversion to the
	Y ,

Table 2 .

 2 Results for the analysis of the image quality algorithms in terms of threshold value (t), correct decision (CD) P (S|s), and false alarm (FA) P (S|n) in percent

	Algorithm	Mantiuk		Reinhard	Combined
		CD FA	t FA	t FA	t
	RMSE	50 75 90	23 0.047 47 0.078 79 0.126	16 0.126 37 0.251 55 0.352	25 0.072 54 0.155 71 0.281
	SSIM	50 75 90	20 0.972 44 0.928 61 0.837	15 0.887 38 0.635 57 0.435	21 0.949 44 0.842 69 0.591
	VDP	50 75 90	14 0.024 28 0.316 41 1.040	15 0.019 30 0.187 44 0.845	15 0.022 29 0.251 43 0.980