PhD Kyle A Metz

PhD Xinchen Teng

PhD Isabelle Coppens

PhD Heather M Lamb

FIBMS, CSci Bart E Wagner

Jill A Rosenfeld

MS Xianghui Chen

MS Yu ; Zhang

BS Hee Jong Kim

BS Michael E Meadow

Jill ; Rosenfeld

Jong

Tim ; Wang

MS Jill A Rosenfeld

BS Tim Sen Wang

MD Edda D Haberlandt

Glenn W Anderson

MD Esther Leshinsky-Silver

PhD Weimin Bi

MD, PhD Thomas C Markello

MD Marsha Pratt

MBBS Nawal Makhseed

MD Adolfo Garnica

MS,LCGC Noelle R Danylchuk

MD Thomas A Burrow

MD Parul Jayakar

PhD Dianalee Mcknight

MD Satish Agadi

ND Hatha Gbedawo

PhD Christine Stanley

MD Michael Alber

MS Isabelle Prehl

MD,PhD Katrina Peariso

MB, ChB Min Tsui Ong

MD Santosh R Mordekar

MD Michael J Parker

MD Daniel Crooks

MD Pankaj B Agrawal

MD Gerard T Berry

MD Tobias Loddenkemper

PhD Yaping Yang

MD, PhD Gustavo H B Maegawa

PhD Abdel Aouacheria

PhD Janet G Markle

PhD James A Wohlschlegel

MD Adam L Hartman

PhD J Marie Hardwick
email: hardwick@jhu.edu

KCTD7 deficiency defines a distinct neurodegenerative disorder with a conserved autophagy-lysosome defect

Keywords: KCTD7, progressive myoclonic epilepsy 3 (EPM3), neuronal ceroid lipofuscinosis type

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

Introduction

Autism, schizophrenia, dystonia, epilepsy and other disorders have been linked to several members of the gene family KCTD (potassium channel tetramerization domain). [START_REF] Escamilla | Kctd13 deletion reduces synaptic transmission via increased RhoA[END_REF][START_REF]of the Psychiatric Genomics C. Biological insights from 108 schizophrenia-associated genetic loci[END_REF][START_REF] Mencacci | A missense mutation in KCTD17 causes autosomal dominant myoclonus-dystonia[END_REF][START_REF] Van Bogaert | Mutation of a potassium channel-related gene in progressive myoclonic epilepsy[END_REF] All KCTD family proteins have an N-terminal BTB domain that is most similar in sequence to voltage-gated potassium channel tetramerization (T1/BTB) domains, hence the name KCTD. [START_REF] Teng | Genome-wide consequences of deleting any single gene[END_REF] However, KCTD proteins appear unlikely to form channels as they lack predicted transmembrane domains and any direct interaction with potassium channels remains uncertain. Although KCTDs could indirectly alter channel properties, the gene name is potentially misleading and has caused some diagnostic challenges with implied treatments for channelopathies. [START_REF] Oyrer | Ion Channels in Genetic Epilepsy: From Genes and Mechanisms to Disease-Targeted Therapies[END_REF] Patients with KCTD7 mutations have been diagnosed with progressive myoclonic epilepsy (EPM3), [START_REF] Van Bogaert | Mutation of a potassium channel-related gene in progressive myoclonic epilepsy[END_REF][START_REF] Kousi | Novel mutations consolidate KCTD7 as a progressive myoclonus epilepsy gene[END_REF][START_REF] Krabichler | Novel Mutation in Potassium Channel related Gene KCTD7 and Progressive Myoclonic Epilepsy[END_REF][START_REF] Lemke | Targeted next generation sequencing as a diagnostic tool in epileptic disorders[END_REF][START_REF] Farhan | Linkage analysis and exome sequencing identify a novel mutation in KCTD7 in patients with progressive myoclonus epilepsy with ataxia[END_REF][START_REF] Moen | Pathogenic variants in KCTD7 perturb neuronal K+ fluxes and glutamine transport[END_REF][START_REF] Seaby | Progressive myoclonic epilepsy with Fanconi syndrome[END_REF] neuronal ceroid lipofuscinosis 14 (CLN14), [START_REF] Staropoli | A Homozygous Mutation in KCTD7 Links Neuronal Ceroid Lipofuscinosis to the Ubiquitin-Proteasome System[END_REF] or opsoclonus-myoclonus syndrome (OMS). [START_REF] Blumkin | A compound heterozygous missense mutation and a large deletion in the KCTD7 gene presenting as an opsoclonus-myoclonus ataxia-like syndrome[END_REF] We sought to further define the clinical syndrome resulting from KCTD7 deficiency, to distinguish deleterious from normal variants in the general population, and to assign a function to the uncharacterized KCTD7 protein based on insights gained from our studies of the related protein in yeast, Whi2. KCTD family proteins are relatively uncharacterized, but one common theme has begun to emerge. A subset of KCTD family proteins may be components of cullin 3 (CUL3) ubiquitin ligase complexes. [START_REF] Pinkas | Structural complexity in the KCTD family of Cullin3-dependent E3 ubiquitin ligases[END_REF] CUL3 uses BTB-containing adaptor proteins from other protein families to recruit cargo proteins for ubiquitination. Several KCTD family proteins including KCTD7 have been reported to bind CUL3, [START_REF] Staropoli | A Homozygous Mutation in KCTD7 Links Neuronal Ceroid Lipofuscinosis to the Ubiquitin-Proteasome System[END_REF][START_REF] Azizieh | Progressive myoclonic epilepsy-associated gene KCTD7 is a regulator of potassium conductance in neurons[END_REF] consistent with structure modeling for other KCTDs. [START_REF] Pinkas | Structural complexity in the KCTD family of Cullin3-dependent E3 ubiquitin ligases[END_REF] In this capacity as potential cargo adaptors, several other KCTD family proteins have been suggested to target specific cargo proteins for degradation, [START_REF] Escamilla | Kctd13 deletion reduces synaptic transmission via increased RhoA[END_REF] although most await confirmation. A similar role for KCTD7 is consistent with lysosome pathway defects in several other EPM and CLN disorders, [START_REF] Mole | Genetics of the neuronal ceroid lipofuscinoses (Batten disease)[END_REF][START_REF] Carcel-Trullols | Cell biology of the NCL proteins: What they do and don't do[END_REF] and other neurodegenerative processes. [START_REF] Nixon | The role of autophagy in neurodegenerative disease[END_REF] However, the molecular and cellular consequences of KCTD7 deficiency are not known. This project was prompted by our genome-wide yeast genetic screen that uncovered the KCTD-like BTB-containing protein Whi2 of Saccharomyces cerevisiae. [START_REF] Teng | Genome-wide consequences of deleting any single gene[END_REF] Yeast Whi2 is reportedly a general stress response factor, [START_REF] Radcliffe | Deregulation of CLN1 and CLN2 in the Saccharomyces cerevisiae whi2 mutant[END_REF] and has a contested role in mitophagy. [START_REF] Mendl | The scaffold protein Atg11 recruits fission machinery to drive selective mitochondria degradation by autophagy[END_REF]22 Yeast Whi2 and autophagy regulator Atg6 (human Beclin 1) were identified in our screen for factors required to respond to low amino acid availability, [START_REF] Teng | Genome-wide consequences of deleting any single gene[END_REF]23 a condition known to induce catabolic processes such as autophagy in yeast and mammals. [START_REF] Gonzalez | Nutrient sensing and TOR signaling in yeast and mammals[END_REF] Because yeast Whi2 shares sequence similarity and a common domain architecture with human KCTD proteins, [START_REF] Teng | Genome-wide consequences of deleting any single gene[END_REF] we sought to gain new insight into the disease mechanisms due to KCTD7 mutations. We found that both yeast Whi2 and human KCTD7 are

Methods

Patient data collection

Clinicians were provided a form for deidentified data (Table S1, IRB exempt status).

Electron microscopy

Patient fibroblasts were fixed in 2.5% glutaraldehyde (EM grade; Electron Microscopy Sciences, Hatfield, PA) in 0.1 M sodium cacodylate buffer (pH 7.4) for 1 h at room temperature, and processed by the Yale University Electron Microscopy Core as described. [START_REF] Coppens | Host but not parasite cholesterol controls Toxoplasma cell entry by modulating organelle discharge[END_REF]

PCR verification of KCTD7 mutations in patient fibroblasts

Genomic DNA for KCTD7 exon-2 of primary human fibroblasts was amplified from extracted DNA using 5' Primer (TGGCACCAATCAGACCCCAGGGATTGAAGATGGAGCAGCCC) and 3' Primer (CCCATTTATTAAATTTCATCAATATGCTATCTCCTCTTCTAGG), separated on 1% agarose gel, extracted using Qiagen Gel Extraction Kit, and sequenced at MacrogenUSA using the 5' primer to verify patient mutations in these cell lines.

Plasmids

Wild type KCTD7 ORF was PCR amplified (Invitrogen AccuPrime Pfx) from HEK293 cDNA (Qiagen RT Kit) and cloned into a pSG5-derived vector to create N-terminal tagged proteins. Coding changes were subsequently engineered into KCTD7 and CUL3 expression vectors and verified by Sanger sequencing. pr PGK -GFP-ATG8 was engineered by replacing the ATG8 5' regulatory region with the 5' regulatory region of PGK.

Mammalian autophagy assays

Primary patient fibroblasts (derived from 3 mm axilla skin punch) were shipped/received live and maintained in DMEM, 10 Yeast strains (BY4741) transformed with autophagy reporter plasmids were grown overnight in synthetic SC CSH medium, refed for 1 h in fresh SC CSH medium at 1 OD 600 /ml, washed once and switched to low amino-acid medium SC ME as described. [START_REF] Teng | Genome-wide consequences of deleting any single gene[END_REF] Cells corresponding to 2 OD 600 units were collected, lysed for immunoblot analysis. 5

Results

To better define the disorder caused by KCTD7 mutations, we identified 18

Prevalence of patient KCTD7 mutations in the general population

To address disease causality of KCTD7 variants found in new patients, family pedigrees were constructed for the 15 new patients, revealing all variants were inherited (Fig 3A). For all patients (except published patient-15), 57 unaffected family members with available data were either wild type (2 of 10 sequenced siblings) or heterozygous for patient mutations (41 parents, 8 siblings, and 1 grandparent) based on sequence data, and 5 parents based on kinship records of consanguinity (Table S2). The gene damage index GDI-Phred value for KCTD7 (1.235, medium damage prediction) and the selective pressure assessed by the McDonald-Kreitman neutrality index (0.004, moderate purifying) [START_REF] Itan | The human gene damage index as a gene-level approach to prioritizing exome variants[END_REF] are also consistent with a monogenic autosomal recessive disease with complete penetrance. However, cautious causality assignments may still be warranted for specific KCTD7

variants given ~500-20,000 protein-altering variants per individual. [START_REF] Lek | Analysis of protein-coding genetic variation in 60,706 humans[END_REF] If all 30 KCTD7 variants are disease-causing, each is expected to be rare in the general population. In an effort to catalog normal genetic variation in healthy individuals, the exome aggregation consortium ExAC database of ~60,000 unrelated healthy individuals excluded cancer genomes and cohorts with severe pediatric diseases. [START_REF] Lek | Analysis of protein-coding genetic variation in 60,706 humans[END_REF] Thus, most of the KCTD7 patient variants S2). Among all KCTD7 variant alleles identified, T64A (patient-1, T64A/R211X) is the most frequent in ExAC, where it is reported in 9 individuals (0.010% to 0.019% allele frequency in European and African populations, respectively), and in 16 heterozygous individuals in the aggregate gnomAD database of ~130,000 individuals (Fig 3B). However, this frequency is still rare and to date no homozygotes for any amino acid change in KCTD7 has been reported in healthy individuals. [START_REF] Lek | Analysis of protein-coding genetic variation in 60,706 humans[END_REF] Some disease variants reported to cause other CLN disorders (e.g. autosomal recessive CLN1 and CLN6) were subsequently challenged because of their prevalence in the ExAC database; for example CLN6 variant R252H. [START_REF] Sleat | Analysis of large-scale whole exome sequencing data to determine the prevalence of genetically-distinct forms of neuronal ceroid lipofuscinosis[END_REF] However, this CLN6 variant remains a disease candidate as none of the 21 normal individuals with this variant is homozygous. 33

KCTD7 patient mutations cause altered protein behavior

To acquire further evidence that the most prevalent KCTD7 variant T64A (patient-1) is pathogenic, we sought a functional assay. As KCTD7 has no established biochemical activities, we tested for altered subcellular localization as an alternative strategy. S2).

Providing further evidence that patient mutations can alter interactions with CUL3, coimmunoprecipitation assays revealed that the N-terminal BTB-containing region but not the C- The only patients predicted to have functionally benign variants by PolyPhen2, SIFT and PROVEAN also have less debilitating disease (sibling patients-9/-10, Table S1). However, their nucleotide change corresponding to G105E (c.314G>A) is located at the exon2-intron junction and is predicted to affect normal splicing (SpliceSiteFinder-like, MaxEntScan, NNSPLICE, GeneSplicer and Human Splicing Finder). Other KCTD7 patient variants have discordant functional predictions between the different algorithms (Table S2), reinforcing that pathogenicity predictions are inherently limited without 3D structures and functional biochemical assays.

KCTD7 heterozygosity in other neurological disorders

We also identified 16 novel heterozygous KCTD7 variants in 18 additional unrelated individuals with phenotypes related to bi-allelic KCTD7EPM3 patients. These heterozygous patients with predominantly unsolved disease etiologies (mean onset age 8.6 years) have neurological phenotypes including developmental delays, seizures, disease progression, movement disorders and/or intellectual disabilities (Table S3). Only one of these 18 variants occurs more frequently than T64A in the population (Y86H occurs in 53 normal heterozygotes). [START_REF] Lek | Analysis of protein-coding genetic variation in 60,706 humans[END_REF] Two of the 18 are also found in EPM3 patients with bi-allelic KCTD7 mutations, R121L and R153H. However, any role for heterozygous KCTD7 variants as genetic modifiers is not known.

Conversely, we cannot rule out the possibility that non-KCTD7 variants act as genetic modifiers of more complex traits affecting onset age or other variations between bi-allelic KCTD7 patients. Patient-9 (G105E/G114E) also has a heterozygous pathogenic variant in GALC that is reported in patients with autosomal recessive, late onset neurodegenerative Krabbe disease, and a heterozygous variant of unknown significance (VUS) in ARID1A, a conserved gene responsible for autosomal dominant intellectual disability (Coffin-Siris syndrome). Patient-14 (R112C/R112C) has mutations in three other genes linked to epilepsy, including a homozygous predicted damaging VUS in the glutamate receptor GRIN2A. These and other noted variants in bi-allelic KCTD7 deficient patients (Table S1) are currently not predicted to be disease-related, but only on the basis that all were inherited from one heterozygous parent and therefore insufficient to cause disease. overfed with oleic acid. [START_REF] Lee | Nutrient-sensing nuclear receptors coordinate autophagy[END_REF] The persistence of lipid droplets in brain tissue could potentially reflect an underlying defect related to lipophagy, a form of autophagy that is required for utilization of lipid stores as an energy source and that requires components of the autophagy pathway. [START_REF] Seo | AMPK and vacuole-associated Atg14p orchestrate mulipophagy for energy production and long-term survival under glucose starvation[END_REF] Bi-allelic KCTD7 mutations that define a diagnosis of EPM3, also define the diagnosis of neuronal ceroid lipofuscinosis 14 (CLN14) based on a study of patient-24 (R184C/R184C). [START_REF] Staropoli | A Homozygous Mutation in KCTD7 Links Neuronal Ceroid Lipofuscinosis to the Ubiquitin-Proteasome System[END_REF] That study reported characteristic ultrastructural features of fingerprint-like profiles and granular osmiophilic deposits (GROD) in fibroblasts and neurons from a skin biopsy and in lymphocytes. [START_REF] Staropoli | A Homozygous Mutation in KCTD7 Links Neuronal Ceroid Lipofuscinosis to the Ubiquitin-Proteasome System[END_REF][START_REF] Anderson | Human pathology in NCL[END_REF] However, we did not observe these or other features considered to be characteristic of neuronal ceroid lipofuscinosis, such as cytosomes, rectilinear profiles (RLP) or curvilinear morphologies described for other CLN subtypes. [START_REF] Anderson | Human pathology in NCL[END_REF] Clinical records indicate that new patient-1 also lacked such ultrastructural features in lymphocytes, skin and rectal biopsy histology, recommended sites for detection of neuronal ceroid lipofuscinosis. [START_REF] Anderson | Human pathology in NCL[END_REF] Although detection can be challenging, [START_REF] Anderson | Human pathology in NCL[END_REF] electron microscopy and/or light microscopy of skin and muscle biopsies from published patients-4, 5, 11, 15, 26, and 30/31, [START_REF] Kousi | Novel mutations consolidate KCTD7 as a progressive myoclonus epilepsy gene[END_REF][START_REF] Krabichler | Novel Mutation in Potassium Channel related Gene KCTD7 and Progressive Myoclonic Epilepsy[END_REF][START_REF] Moen | Pathogenic variants in KCTD7 perturb neuronal K+ fluxes and glutamine transport[END_REF] as well as new patients-1, 20, 22 and 28 also lack these specific features (Table S1). In addition, no storage material resembling other lysosomal storage disorders was detected in neurons, vascular endothelial or smooth muscle cells of patient-1. Broader diagnostic criteria may be needed to include KCTD7 as a CLN disorder.

Evidence for lysosome-pathway defects without characteristic neuronal ceroid lipofuscinosis

Cultured skin fibroblasts naturally lack some of the lysosomal substrates enriched in the brain such as specific glycosphingolipids, and are therefore not expected to reveal some brain pathologies.

However, fibroblast cultures are useful for evaluating other autophagosome-lysosome functions and Other prominent features that distinguish both patient fibroblasts from age-and passagematched control fibroblasts include hybrid structures resembling late single-membrane or partially double-membrane autolysosomes. [START_REF] Nixon | Extensive involvement of autophagy in Alzheimer disease: an immuno-electron microscopy study[END_REF] cell lines. [START_REF] Nixon | Extensive involvement of autophagy in Alzheimer disease: an immuno-electron microscopy study[END_REF][START_REF] Vergarajauregui | Autophagic dysfunction in mucolipidosis type IV patients[END_REF][START_REF] Velikkakath | Mammalian Atg2 proteins are essential for autophagosome formation and important for regulation of size and distribution of lipid droplets[END_REF] Although mitochondrial organelles were not sufficiently preserved in patient biopsy preparations, most cultured fibroblasts from both patients contained a fraction of mitochondria with internal, closed double membrane structures presumed to reflect cristae malformations (Fig 6C).

These malformations are somewhat reminiscent of mitochondria in mitofilin-deficient cells with concentric cristae. [START_REF] John | The mitochondrial inner membrane protein mitofilin controls cristae morphology[END_REF] Ultrastructural abnormalities were prevalent in patient cells but absent in control cells (Fig 6D). The apparent accumulation of several abnormal organelles in patient-derived fibroblasts and in a brain biopsy is consistent with a defect in the phagolysosome pathway.

Conserved autophagy defects as a potential mechanism of disease pathogenesis

We initiated this KCTD7 project based on insights gained from studying the poorly characterized yeast protein Whi2, which shares sequence similarity with KCTD7 and harbors a homologous BTB structural domain. [START_REF] Teng | Genome-wide consequences of deleting any single gene[END_REF] Yeast lacking WHI2 are sensitive to multiple cell stresses and fail to halt the cell cycle in response to low amino acid levels in the media, conditions known to induce autophagy. [START_REF] Teng | Genome-wide consequences of deleting any single gene[END_REF][START_REF] Radcliffe | Deregulation of CLN1 and CLN2 in the Saccharomyces cerevisiae whi2 mutant[END_REF]23 Taken together with our ultrastructural findings in patient samples, we asked if whi2-deficient yeast have a general autophagy defect using an established reporter for yeast Atg8, [START_REF] Graef | Mitochondria regulate autophagy by conserved signalling pathways[END_REF] homolog of mammalian autophagy marker LC3. In low amino acid medium, the autophagyresponsive ATG8 promoter is induced to express the reporter, and during autophagy flux undergoes lysosomal/vacuolar processing that cleaves the protease-sensitive Atg8 moiety from the more stable GFP protein in wild type cells. [START_REF] Graef | Mitochondria regulate autophagy by conserved signalling pathways[END_REF] In striking contrast to wild type, whi2-deletion strains are profoundly To extend these findings from yeast, the skin-derived fibroblast lines from patients-3 and -4

with BTB domain mutations were analyzed for autophagy defects. The accumulation of endogenous lipidated/mature LC3-II following treatment with chloroquine to inhibit lysosome function that otherwise degrades LC3-II. [START_REF] Mizushima | How to interpret LC3 immunoblotting[END_REF] The functional consequences of autophagy deficiency were assessed by determining the effects of KCTD7 on neurite outgrowth triggered by serum withdrawal in mouse neuroblastoma N2a cells. The maturation of N2a cells to produce extensive neuron-like processes requires autophagy, as knockdown of conserved Beclin 1/Atg6 blocks neurite extension. [START_REF] Zeng | Roles of autophagy and mTOR signaling in neuronal differentiation of mouse neuroblastoma cells[END_REF] We found that partial knockdown of endogenous mouse Kctd7 severely reduces neurite outgrowth triggered by serum withdrawal, indicating a critical role for KCTD7 in neurite maturation (Fig 8F andG).

Discussion

Manifestations of KCTD7 mutations

Guided by our findings in yeast, we investigated the clinical and cellular consequences of KCTD7 mutations. Bi-allelic KCTD7 mutations cause an early onset (16.8±6 months) progressive myoclonic epilepsy previously ascribed only to older children. [START_REF] Mole | Genetics of the neuronal ceroid lipofuscinoses (Batten disease)[END_REF][START_REF] Carcel-Trullols | Cell biology of the NCL proteins: What they do and don't do[END_REF] Two reported phenotypes for KCTD7/EPM3 patients were not found in the new patient cohort reported here. Clonic eye movements reported for patient-3 diagnosed with opsoclonus-myoclonus syndrome (OMS) [START_REF] Blumkin | A compound heterozygous missense mutation and a large deletion in the KCTD7 gene presenting as an opsoclonus-myoclonus ataxia-like syndrome[END_REF] were not detected though not formally tested. We also did not detect specific neuronal ceroid lipofuscinosis pathology reported for patient-24, the founder case for CLN14 designation, although this patient potentially had more severe disease (onset 8 mo, died 17 yr). 13

Causality of KCTD7 variants

The low frequency in the general population of each of the 30 unique KCTD7 patient variants, including T64A, and the lack of homozygous patient variants in healthy individuals, [START_REF] Lek | Analysis of protein-coding genetic variation in 60,706 humans[END_REF] indicate that these 30 variants are causal for EPM3/CLN14. This disorder occurs worldwide and the patients in this study have diverse ancestry (e.g. Moroccan, Syrian Sephardi, European, Native American, French Canadian). Genome sequence data from the general population suggest at least 0.05% of healthy unrelated individuals may carry a heterozygous pathogenic KCTD7 variant. [START_REF] Lek | Analysis of protein-coding genetic variation in 60,706 humans[END_REF] The relatively higher than average arginine content of the KCTD7 protein (7.3% vs. 4-6%) may contribute to the mutation frequency (12 of 30 variants change an Arg, Table S1) given that arginine codons have the highest proportion of CpG sequences and CpG mutations (affecting DNA methylation) are by far the most prevalent in the population. [START_REF] Lek | Analysis of protein-coding genetic variation in 60,706 humans[END_REF] Additional rare heterozygous KCTD7 variants were identified in 17 additional patients with related yet clinically distinct disorders (Table S3), although any contribution to disease is unknown.

However, when considering the genetic complexities of neurobehavioral disorders such as autism, other genetic modifiers with discernable clinical phenotypes may exist. KCTD7 sibling patients-9/10

were diagnosed with autism. The KCTD7-related KCTD13 gene at 16p11.2 is thought to have a role in a small subset of autism cases. [START_REF] Escamilla | Kctd13 deletion reduces synaptic transmission via increased RhoA[END_REF] Any manifestations of heterozygous KCTD7 mutations in late onset disorders analogous to heterozygous loss of progranulin in older adults with frontotemporal dementia/FTD versus bi-allelic mutations that cause lipofuscinosis CLN11 [START_REF] Ward | Individuals with progranulin haploinsufficiency exhibit features of neuronal ceroid lipofuscinosis[END_REF] are unexplored. Based on yeast studies that first identified the KCTD-like yeast Whi2, 5 KCTD7 variants could potentially compensate for more deleterious mutations. Spontaneous mutations in the analogous gene WHI2 can compensate in part for the more detrimental lack of mitochondrial fission factor Fis1 or several other genes. [START_REF] Teng | Genome-wide consequences of deleting any single gene[END_REF]23 Biological and biochemical roles for KCTD7

The biochemical function of KCTD7 is not known and little is understood about the other 24 human family members (KCTD1-21, TNFAIP1, KCNRG, SHKBP1 and BTBD10). Inspired by our finding that yeast Whi2 is required to suppress cell growth in low amino acid conditions, [START_REF] Teng | Genome-wide consequences of deleting any single gene[END_REF]23 it was reasonable to consider that whi2-deficient yeast were also defective for autophagy induction.

Therefore, we tested for an evolutionarily conserved function between Whi2 and KCTD7 in autophagy. We found that both yeast Whi2 and KCTD7 are required for normal basal autophagy and low nutrient-induced autophagy (Figs 7 and8). Both yeast Whi2 and the KCTD7 homolog, KCTD11, were recently shown to suppress TORC1/mTORC1, a known inhibitor of autophagy. [START_REF] Chen | Whi2 is a conserved negative regulator of TORC1 in response to low amino acids[END_REF] The same study failed to detect an effect of KCTD7 on TORC1/mTORC1 activity in yeast and primate COS7 cells. However, a role for KCTD7 in autophagy-lysosome function is consistent with our ultrastructural studies revealing abnormal autophagosome-lysosome structures, mitochondrial cristae and supernumerary lipid droplets potentially reflecting impaired lipophagy (Fig 5 and6). Related pathologies are observed with aging-related lysosomal dysfunction and progressive decline in chaperone-mediated autophagy rates in late-onset Alzheimer disease, 37 perpetuated by oxidation of partially degraded macromolecules derived from mitochondria, glycosphingolipids and other components in autolysosomes resulting in reactive oxygen species that interact with lysosomal iron. [START_REF] Kurz | Redox activity within the lysosomal compartment: implications for aging and apoptosis[END_REF] Consistent with our finding of abnormal mitochondrial cristae in patient-derived cells, one study investigated the role of yeast Whi2 in mitophagy (a subtype of autophagy). They reported that the spontaneous WHI2 mutation in FIS1 knockout strains, rather than loss of the FIS1 mitochondrial fission gene, causes a defect in the degradation of yeast mitochondrial organelles. [START_REF] Mendl | The scaffold protein Atg11 recruits fission machinery to drive selective mitochondria degradation by autophagy[END_REF] However, a subsequent study challenged this conclusion, reporting that mitochondrial fission mediated by FIS1 rather than WHI2 is required for normal mitophagy. 22 Thus, the question remains open.

A possible role for KCTD7 in protein turnover is consistent with having an N-terminal BTB proteins can serve as adaptor proteins that retrieve substrates for the CUL3 ubiquitin ligase complex. [START_REF] Genschik | The emerging family of CULLIN3-RING ubiquitin ligases (CRL3s): cellular functions and disease implications[END_REF] Interesting, several other KCTD family members were identified in screens for cullin-ARIH1 complex components, [START_REF] Scott | Two Distinct Types of E3 Ligases Work in Unison to Regulate Substrate Ubiquitylation[END_REF] consistent with serving as an E3 ubiquitin ligase adaptor. CUL3 has many critical roles in cells, and a role for CUL3 in autophagy has gained recent attention. CUL3 and its BTB-Kelch adaptor protein KLHL20 were found to prevent overzealous autophagy by direct ubiquitination and degradation of ULK1, a key upstream positive regulator of autophagy induction. [START_REF] Liu | Cul3-KLHL20 Ubiquitin Ligase Governs the Turnover of ULK1 and VPS34 Complexes to Control Autophagy Termination[END_REF] Although KCTD family proteins have not been demonstrated to have a molecular role in autophagy, defective autophagy-lysosome pathways are consistent with their causal roles in neurodegenerative disorders. S1) and from published cases as cited by reference number. 4, 7-11, 13, 14 S2).

F

Supporting Materials

Table S1. Supporting clinical data Table S2. KCTD7/EPM3 genetic data Table S3. KCTD7 heterozygous variant data

  Conserved roles for human KCTD7 and yeast Whi2 Word count: Title [104 char], Running title [46 char], Abstract (248 words), Introduction [424 words], Discussion [1065 words], Body (Introduction through Discussion) [4,780], 8 Figures [2 color

figures, 4

 4 figures, 4 figures provided in both color and B&W, no tables], 50 References

  autophagy in low nutrients, further supported by the accumulation of lipid bodies, defective mitochondria and abnormal autolysosomes.

  (10 of 14) are from previously unpublished patients (Table

  The N-terminal amino acids 1-149 of wild type KCTD7 containing the BTB domain, when expressed in HeLa (or other) cells, forms unusual flowing filament-like structures of unknown relevance in the cytoplasm and forms similar smaller structures in the nucleus (Fig 4A). Taking advantage of these elaborate structures to distinguish the effects of patient mutations, we found that expression of T64A(1-149) abolishes these cytoplasmic structures and instead localizes predominantly in a nuclear ball-and-stick pattern (Fig 4A). Two other BTB domain mutations from published patients cause other distinguishable morphologies. L108M (patients 11-13) increases the occurrence of mini-circles at filament termini (Fig 4A, arrows), and D115Y (patient-15, lacking family genetics) causes massive filament-like structures (Fig 4A), which is likely a cause or consequence of protein stabilization (Fig 4B).When co-expressed with CUL3, an E3 ubiquitin ligase component and reported binding partner of KCTD7,[START_REF] Staropoli | A Homozygous Mutation in KCTD7 Links Neuronal Ceroid Lipofuscinosis to the Ubiquitin-Proteasome System[END_REF][START_REF] Azizieh | Progressive myoclonic epilepsy-associated gene KCTD7 is a regulator of potassium conductance in neurons[END_REF] both wild type and D115Y, and to a lesser extent L108M, were capable of recruiting CUL3 from its more diffuse localization to KCTD7 structures, except the T64A mutant that only rarely co-localizes with CUL3 in fuzzy nuclear spots (Fig4C and D). Thus T64A, as well as L108M and D115Y are likely to alter KCTD7 function, consistent with functional-effect prediction algorithms PolyPhen2, SIFT and PROVEAN, and L108M and D115Y are predicted damaging by two of these algorithms (Table

  is required for CUL3 interaction, consistent with a role for KCTD7 as a CUL3 adaptor. Furthermore, the patient BTB domain mutations tested in full-length KCTD7 (R70W, L108M and likely R84Q) impair binding to CUL3 (Fig4E).

12 Electron 1 (

 121 microscopy analysis of a frontal lobe brain biopsy from new patient-T64A/R211X) at age 8 years revealed neuronal pathology (Fig 5A) that was absent in the brain of a neurologically normal 6.7 yr child (not shown). The prominent brain pathology in patient-1 was the electron-dense lipofuscin (lysosomes) (Fig 5A). Similar structures are commonly observed in normal aging brain but not in children. A potential feature of KCTD7 brain lipofuscin is the late autophagosome-like structures (Fig 5B-D, arrows) engorged with both electron-dense lysosomes and electron-lucent structures presumed to be lipid droplets (Fig 5A-D, arrowheads). Soft lipid droplets are compressed between lysosomes or bulging against a delimiting membrane (Fig 5C and D, arrowheads). Others have observed similar structures in autophagy-deficient cells, and in cells

  ultrastructure. In contrast to an age-and passage-matched control, prominent features of earlypassage skin-derived fibroblast cultures from patient-3 and patient-4 with BTB domain mutations include the supernumerary lipid droplets often in close proximity to mitochondria and ER (Fig 6A).

13 Some

 13 lipid droplets appeared to be engulfed in membrane-bound structures (Fig 6A, arrowheads), reminiscent of the lipid droplets in patient brain, possibly suggesting a stalled catabolic process.

  These hybrid structures contain local electron-densities typical of lysosomes (Fig 6B, black arrows) that are associated with larger autophagosome-like structures containing residual undegraded material (Fig 6B, white arrows) and occasional electron dense lipid whirls (Fig 6B). Similar hybrid structures are also characteristics of diverse neurodegenerative disorders including mucolipidosis type IV, Alzheimer's disease, CLN types and autophagy-deficient

  defective for autophagy induction and flux (Fig 7A). Similar but less dramatic results were obtained with an autophagy flux-specific reporter expressed by the constitutive PGK promoter, indicating that Whi2 is required for normal autophagy induction and flux after switching to low amino acids (Fig 7 B). This autophagy defect was not due to a general defect in reporter expression or global protein free GFP expressed via an autophagy-independent promoter (ADH1) was expressed indistinguishably in wild type and whi2-deficient yeast (Fig 7C).

  Consistently, LC3-II accumulated less efficiently in low-passage patient fibroblasts versus age-and passage-matched controls following chloroquine treatment to assess basal autophagy flux (Fig 8A and B), and when autophagy was induced by withdrawing amino acids and glucose (Fig 8B and C). Autophagy induced by more severe starvation was also significantly lower in patient fibroblasts assessed by conversion of LC3I to LC3II (Fig 8E). Thus, both WHI2-deficient yeast and patient cells have a defect in autophagy based on Atg8/LC3 assays following nutrient depletion. To confirm the origins of our fibroblast cell lines, genomic DNA isolated from patient and control cells used in these studies was sequenced, revealing the expected variants for patients-3 and -4.

Fig 1 .

 1 Fig 1. Genetics and clinical features for all KCTD7 patients. (A) KCTD7 protein changes and clinical features for 37 new and published patients numbered by KCTD7 variant position and color-coded by protein region as in panel B. Initial DNA sequencing strategies: whole genome (WGS), whole exome (WES), clinical diagnostic sequencing panel (DSP), genome-wide linkage/autozygosity mapping (GWL), Sanger sequencing (Sngr), and the heat map of clinical features for all new patients are derived from deidentified clinical data (TableS1) and from

21 Fig 3 .

 213 Fig 3. KCTD7 family pedigrees and mutation frequency. (A) Pedigrees of 11 new families from Fig 1A with 18 novel KCTD7 variants in 15 bi-allelic patients; birth order unknown for family 12. (B) All patient variants listed in the ExAC 32 and gnomAD sequence databases http://gnomad.broadinstitute.org/ for the correct KCTD7 transcript ENST00000275532 (TableS2).

Fig 4 .Fig 5 .Fig 6 .Fig 7 .

 4567 Fig 4. Patient mutations in the BTB domain affect cullin 3 (CUL3) interactions. (A) Immunofluorescence microscopy of N-terminal HA-tagged KCTD7-BTB (amino acids 1-149) in Kyoto HeLa cells transfected 18 h and stained with 1:1000 anti-HA (Santa Cruz HA Y-11) and 1:2000 anti-Myc (Calbiochem Ab-1 OP10L) (similar results without tag and in other cell types tested). Representative of >3 independent experiments. (B) Immunoblots (12% SDS-PAGE, PVDF) of samples described for panels A, C and D probed for anti-HA (1:1000; Santa Cruz Y11), anti-Myc (1:2000; Calbiochem), and anti-actin as loading control (1:10,000; MP Biomedicals 691001), representative of two independent experiments each with both WT and inactive K712R mutant CUL3 yielding similar results. (C) Immunofluorescence microscopy of Kyoto HeLa cells transfected with N-terminal 6Myc-tagged CUL3 (K712R) alone and detected with anti-Myc (similar results for wild type 6Myc-CUL3). (D) Parallel samples to panels B and C co-transfected with HA-KCTD7(1-149) and N-terminal 6Myc-cullin3(K712R) and dual-stained with anti-Myc and anti-HA. Individual gray scale and color merged immunofluorescence microscopy images shown. Representative of >3 independent experiments per condition. Scale bar = 10 µm in all panels. (E) Co-immunoprecipitation of WT 6Myc-Cul3 from HEK293 whole cell lysates (WCL) after transient co-transfection with 3His-3Flag-KCTD7 using anti-Flag M2 affinity matrix for immunoprecipitation (IP). The strength of the interaction between CUL3 and KCTD7 variants was quantified as a ratio of the IP cMyc signal to the IP Flag signal and normalized to the WT KCTD7/CUL3 ratio in the total IP. Representative of 3 independent experiments is shown. **P<0.000001, *P<0.03

Fig 8 .

 8 Fig 8. Conservation of autophagy defect in KCTD7 patient fibroblasts. (A) Immunoblot of control age-/passage-matched human fibroblasts and fibroblasts derived from patient-3 and -4 (passage 6-8) for endogenous autophagy protein LC3 (1:1000 anti-LC3, Cell Signaling 2775) and HSP90 loading control (BD Biosciences 610419) detected with HRP-conjugated secondary antibodies (GE Healthcare, 1:20,000), developed using ECL-Prime (GE Healthcare). Compare LC3-II levels before versus after 1 h with 15 µM chloroquine (Sigma C6628) or carrier control to assess basal autophagy (arrows). (B) Quantified data for panel A, pooling 5757 and 498 control cells versus patient cells from 3 independent experiments, each performed in duplicate or triplicate for each of the 4 cell lines (n=11 per condition) and presented as the ratio of LC3-II levels +/-15 µM chloroquine after correcting for loading; mean+/-SE, 2-tailed t-test, p = 0.00065. (C) Autophagy assay as in panel A except cells were treated 3 h +/-glucose, +/-amino acids, +/-100 nM Bafilomycin A1 (Enzo). (D) Quantification for panel C presented as mean+/-SE of LC3-II levels adjusted to relative loading controls and calculated as the fold change over control 5757 in full medium for 6-8 replicates in 4 independent experiments. Two-tailed t-test, *p<0.05. (E) Autophagy assay as in panel A except cells were incubated in amino acid-free/serum-free Earle's balanced salt solution (Thermo Fisher). The change in LC3-II/LC3-I ratios relative to time 0 is presented as mean+/-SE for 3 independent experiments; 2-tailed t-test for control-5757 vs. patient-3 at 2 h and 3 h (p=0.002 and p=0.018), and patient-4 (p=0.023 and p=0.020, respectively). (F) Light microscopy (Nikon TE200) of control and mouse Kctd7-specific shK7.1 shRNA knockdown (TRCN0000069304 transfected with FuGENE or Lipofectamine2000) in N2a mouse neuroblastoma cells refed +/-serum 48 h to monitor neurite outgrowths (arrows). (G) Immunoblot confirmation of endogenous Kctd7 knockdown by shK7.1 with 1:1000 anti-KCTD7 (Abcam ab83237) and anti-HSP90 (BD Biosciences 610419) loading control. Lysates and immunoblots were prepared as described for panel A, except visualized using Amersham Hyperfilm ECL (GE Healthcare).

  Fig1 Patients 101x159mm (300 x 300 DPI)

Fig 8

 8 Fig 3 Genetics 107x168mm (300 x 300 DPI)

KCTD7 mutations are associated with a progressive neurodegenerative disorder

	novel mutations
	in 15 patients (11 families) from several sequencing centers and institutions (Fig 1A). A total of 30
	novel KCTD7 variants from 37 new and published patients can be grouped into three protein regions,
	the N-terminal BTB domain, a C-terminal cluster and a less defined middle region (Fig 1B). All 37
	patients have homozygous or compound heterozygous variants in KCTD7 (23 missense, 3 stopgain F and 4 frameshifts). However, their association with disease does not constitute proof of pathogenicity for each patient variant. To address this point, we first characterized the clinical syndrome. o r
	P
	Overt seizures mark the recorded age of onset for 76% of KCTD7 patients, many with e e
	accompanying developmental delays and movement disorders, predominantly ataxia, tremors and r
	dyskinesia (Fig 1A). The remaining 24% first develop movement disorders or developmental delays prior to seizure onset. Genetic testing for KCTD7 variants at earlier ages could potentially identify R e
	more patients with movement disorders before seizure onset. Other prominent clinical features v
	include the loss of normal developmental milestones achieved in early childhood, difficulty walking, loss of speech and fine motor skills, and severe cognitive decline. While EEG findings are often positive, brain MRI is typically normal at onset but may detect diffuse or focal brain atrophy after i e w
	disease progression, for example as observed for new patient-1 (T64A/R211X) but not for new
	patient-2 (Table S1). Patient-2 underwent a complete corpus callosotomy at age 6.9 years, and was

seizure-free for at least 18 months, with some improvement in motor control. All patients progressed to develop myoclonic epilepsy, and all with available data developed movement disorders. Most become wheelchair-bound and non-verbal, and 6 of 37 died at ages 3-18 years (Fig 1A

, asterisks

).

The few ambulatory patients now in their 20's (patients-9, -10 and -19) have significant motor and cognitive deficits and exhibit autism, obsessive compulsive disorder or schizophrenia, in addition to epilepsy (Table

S1

).

Early onset without retinal degeneration distinguishes KCTD7 patients 9

Distinguishing KCTD7/EPM3 patients from related disorders is the early age of onset, consistent with published case reports (Fig

2

). Average onset age for all 37 patients is ~17 months, range 5-24 months except one of a sibling pair lacking C-terminal residue W289 with disease onset reported at 36 months (patient-37) (Fig

1A

). No gender bias is present for incidence (49% males) although males tended to be diagnosed at a younger age (mean onset 15.1 mo males, 19.1 mo females). This early onset age for KCTD7/EPM3 patients does not overlap with other early onset myoclonic epilepsies (EPM1A, EPM2, EPM4), with the exception of infantile CLN1 (onset 6-24 months) caused by mutations in lysosomal enzyme PPT1 (Fig

2

).

[START_REF] Williams | Diagnosis of the neuronal ceroid lipofuscinoses: an update[END_REF][START_REF] Franceschetti | Progressive myoclonic epilepsies: definitive and still undetermined causes[END_REF]

However, KCTD7 patients uniformly lacked the characteristic CLN1-associated retinal abnormalities at onset and associated blindness (Fig

1A

).

[START_REF] Carcel-Trullols | Cell biology of the NCL proteins: What they do and don't do[END_REF][START_REF] Williams | Diagnosis of the neuronal ceroid lipofuscinoses: an update[END_REF]

These findings shift the age downward for considering the diagnosis of EPM3, which is typically diagnosed in childhood or adolescence. KCTD7 patients are also distinguished from other related disorders. They have more severe cognitive decline and earlier onset than patients with a BTB domain variant in Kv3.1/ KCNC1 (onset age 3-15yr),

[START_REF] Oliver | Myoclonus epilepsy and ataxia due to KCNC1 mutation: Analysis of 20 cases and K+ channel properties[END_REF]

but a later average onset than infantile spasms due to autosomal dominant mutations in DNM1 (onset typically 4-7 months) or autosomal recessive mutations in TBC1D24 (GTPaseactivating ARF6-binding protein) that cause pleiotropic neurologic disorders with myoclonic seizures (median onset 2-3 months, often at birth) (Fig

2

).

[START_REF] Balestrini | TBC1D24 genotype-phenotype correlation: Epilepsies and other neurologic features[END_REF][START_REF] Von Spiczak | DNM1 encephalopathy: A new disease of vesicle fission[END_REF]

  The constituents of lipofuscin granules in patient brain appear to be residual bodies derived from lysosomes and could potentially be produced by a partial degradation of unsaturated lipids. Brain biopsies for future patients may provide more valuable information. The ultrastructural features in patient samples together with defective autophagy responses in patient fibroblasts and the corresponding yeast deletion strain are consistent with the possibility that an autophagy-lysosome pathway defect underlies the disease caused by biallelic KCTD7 mutations. Although lysosome pathway defects are implicated in a growing number of neurological disorders, each can manifest differently, presumably owing to the molecular details not yet known. New therapies will be needed that enhance autophagosome-lysosome function without worsening defective bottlenecks downstream in the pathway.

	F
	o r
	P
	e e
	r
	R e
	v
	i e w
	These patients are characterized
	by movement disorders and developmental delays that may precede onset of intractable myoclonic
	seizures more often than appreciated based on recorded parental comments. The few ambulatory
	patients exhibit severe cognitive and psychosocial impairments. All patients exhibit disease
	progression indicative of an underlying degenerative process despite apparently normal initial

developmental milestones. Ultrastructural analysis of a brain biopsy or skin fibroblasts from a total of three different patients revealed shared features, most notably the accumulation of lipid droplets and abnormal phagolysosomes containing undegraded material. Misshapen mitochondrial cristae 15 membranes were also prominent features of skin fibroblasts where sample preparation is more amenable to organelle preservation (Figs

5 and 6

).

  Note, L108M patient 13 was confirmed by the authors to be distinct from patients-11 and -12. Estimated age of onset (months) for males vs.

	females is not significant, p=0.055 (two-tailed t-test). *Deceased. (B) Map of human KCTD7
	isoform-1 (289 amino acids) encoded on 4 color-shaded exons (scale units = 10 residues); 30 patient
	variants are grouped in color-coded clusters as in panel A. BTB domain (transparent box); missense
	mutations (solid line), nonsense mutations (dashed), frameshift (double line), mutations occurring in
	>1 family (diamond), different amino acid mutation at the same position in >1 family (square).
	Fig 2. Earlier disease onset distinguishes KCTD7/EPM3 from other myoclonic epilepsies.
	Range (bars) and median (line) age of disease onset for early-onset disorders with myoclonic seizures.
	TBC1D24 calculated from Supplementary data in Balestrini et al., 29 DNM1, 30 CLN1/CLN3, 17
	KCNC1, 28 MERRF, 50 EPM1A/EPM2/EPM4, 27 sialidosis/mucolipidosis-I
	https://emedicine.medscape.com/article/948704-overview, and for KCTD7/EPM3 from Fig 1A.

Acknowledgements

We thank Drs. Rachel Kneen and Lorraine Potocki for clinical information, Drs. Constantin D'Ydewalle and Charlotte Sumner for assistance with qRT-PCR on patient fibroblasts, medical illustrator Heidi Hildebrandt for the mutation map (Fig 1B), and Drs. Jodi Nunnari and Joseph Heitman for kindly providing the autophagy plasmids pr ATG8 -GFP-ATG8 and pr ADH1 -GFP, respectively. This work was supported by NIH R01 NS083373 (JMH), NIH R01 NS037402 (JMH), CURE Epilepsy Foundation (JMH), NIH K08 NS070931 (ALH), NIH R01 GM089778 (JAW), National Natural Science Foundation of China 31401197 (XT), Natural Science Foundation of Jiangsu Province BK20140318 (XT), and Jiangsu Key Laboratory of Neuropsychiatric Diseases BM2013003 (XT). This work was performed while ALH was a full-time employee of Johns Hopkins University.

Author Contributions

KAM, XT, ALH and JMH contributed to the conception and design of the study; KAM, XT, IC, HML, BW, JAR, XC, YZ, HJK, MEM, TSW, EDH, GWA, ELS, WB, TCM, MP, NM, AG, NRD, PJ, DM, SA, HG, CS, MA, IP, KP, TAB, MTO, SRM, MJP, DC, PBA, GTB, TL, YY, AA, JAW, ALH and JMH contributed to the acquisition and analysis of data; KAM, XT, IC, HML, BW, GHBM, JGM, ALH and JMH contributed to drafting the text and preparing the figures.

Potential Conflicts of Interest:

The authors have declared that no conflict of interest exists.


Patrick M Dansette

Assia Hessani

Catherine Boisson-Vidal

phenyl Cytochrome P450 dependent metabolism of fluindione in vitro in rat and human microsomes and in vivo in rat

INTRODUCTION : Fluindione (2-(4'fluoro-phenyl)-phenylindan1,3-dione) is an anti-vitamin K (AVK) marketed in 1967 in France and in Luxembourg (1-3). It is still the leading AVK in France in 2016 (around 800.000 patients, 82% of the market). However very little is known on the metabolism of this drug. In order to better understand the pharmacokinetics of this drug (4,5), we started to study the in vitro and in vivo metabolism of fluindione. The results below show a series of new metabolites both in vitro and in vivo.

METHODS:

In vivo : Sprague Dawley rats weighting 200-400 g were injected IP with fluindione (5 mg/mL solution in sodium bicarbonate) at 5 or 20 mg/kg. Blood was obtained at the tail at selected time and urine was collected.

In Vitro : Rat or human liver microsomes or recombinant Cytochrome P450 (CYP) (Supersomes Gentest) were incubated with fluindione (100 and 200 µM) for 10 to 60 min in presence of NADPH generating system. Reactions were stopped with one volume of CH 3 CN/AcOH 9:1, centrifuged at 13000 g and supernatants were analyzed by HPLC/MS on a LCQ Advantage system or an Exactive Orbitrap (Thermo) using negative ESI. The metabolite M2 was obtained from semipreparative incubations, purified by HPLC and analyzed by NMR on a Bruker 500 MHz. Recombinant human P450: The formation of M2 was studied using insect cell microsomes expressing recombinant CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4 and 3A5. CYP1A2, 2B6, 2C9, 2D6, 3A4 and 3A5 could make this metabolite. CYP2C9 and CYP1A2 had best affinities (about 30 µM) as shown in Table1. Bruker 500 MHz. Synthesis: 13 C 6 labeled fluindione was synthetized from 13 C 6 labeled phthalic anhydride (6). 3-(4-Fluoro-phenyl)-4-hydroxy-coumarin (7), 3-(4-Fluorophenyl)-4-hydroxy-isocoumarin (8), 2-(4-hydroxyphenyl)-indane-1,3-dione ( 9) and 2hydroxy-fluindione (10) were synthetised by modification of known methods.

RESULTS :

In vivo : Rats were treated by 5 and 20 mg/kg fluindione 13 Plasma and Human Urine: A poly-medicated volunteer took orally 10 mg of fluindione. Fluindione could be detected in plasma at 1, 2 and 6 hours. However the metabolites could only be found in very low amount (limit of detection).

Synthesis of some potential metabolites and 13 C 6 labeled fluindione:

In order to characterize M2, the chemical synthesis of several candidate molecules was performed: 2-hydroxy-fluindione, 2-(4'-fluorophenyl)-4-hydroxy-coumarin, 2-(4'-fluorophen yl)-4-hydroxy-isocoumarin, 4'-hydroxy-phenindione . Metabolite M2 did not correspond to any of the synthetized products. However M1 was identified as 4'-hydroxy-phenindione . -These oxidations are P450 dependent and human recombinant CYP2C9 and CYP1A2 have good affinities for formation of M2 also formed by CYP3A4. In vivo in rat, the primary metabolites are found in plasma, but in urine one find the sulfate esters of M2 and M3.

-A tentative experiment in a human volunteer showed only fluindione and M2 in plasma and only traces of fluindione in urine.

-The metabolism of fluindione is mediated by several cytochrome P450 which explain why CYP2C9 is not a genetic determinant for its posology.

-Finally the structure of M2 and M3 remain to be characterized.

Fluindione Metabolite M2

Fluindione

  metabolism of fluindione in vitro in rat and human microsomes and in vivo in rat. Patrick M. Dansette 1 , Assia Hessani 1 , Virginie Siguret 2,3 , Catherine Boisson-Vidal 2 , Jean-Luc Bouche r 1 and Blandine Dizier 2 with the participation of master students : Carole Berndt, Caroline Moreau, Idir Hamdidouche, Slimane Moktari, Antoine Bienne, Shannon Pecnard and Victoria Rutman. 1 LCBPT, CNRS UMR 8601, Université Paris Descartes, Sorbonne Paris-Cité, PARIS, France 2 INSERM UMR-S-1140 "Innovations thérapeutiques en hémostase" Faculté de Pharmacie, Université Paris Descartes, Sorbonne Paris-Cité, PARIS, France 3 Service d'hématologie biologique (Pr A. Veyradier) -Hôpital Lariboisière, PARIS, France Human liver microsomes : Incubation with human liver microsomes showed that metabolite M2 was formed in presence of NADPH. Control without NADPH were blank. Its formation was inhibited by benzyl imidazole (50 µM) implicating cytochrome P450 catalysis. An impurity, 2-hydroxy-fluindione slowly increased on storage of incubations. It's a known oxidation product of fluindione by air.

  C 6 / 12 C 1/1 mixture Plasma : Analysis of plasma showed doubly labeled compounds with a doublet in the mass spectrum : fluindione F (m/z = 239/245, C 15 H 9 O 2 -) and metabolites: M1 (m/z = 237.0558/243.0760) corresponding to defluorination and hydroxylation, and identified by comparison with an authentic to 2-(4hydroxyphenyl)-indane-1,3-dione and M2 (m/z = 255.0467/ 261.0668) corresponding to C15H8O3F thus addition of an oxygen to fluindione. M2 was less polar than fluindione and had a UV spectrum nearly superimposable with that of fluindione (see below). A minor metabolite M3 also less (m/z = 253.0499/259.0700) corresponding to C15H9O4 thus fluindione defluorinated and dihydroxylated . Urine : Analysis of urine showed the same metabolites in low amount plus series of more polar metabolites. They corresponded to sulfate esters: S2 was relatively abondant ( m/z = 335.0006, C15H8O6FS ) its MS and MS2 showed fragmentation at m/z = 255.0446 with loss of SO 3 . S3 had a m/z = 333.0046 in MS and its MS2 spectrum showed fragmentation at m/z = 253.0499 with loss of SO 3 . Thus we believe that S2 and S3 are the sufate esters of M2 and M3.

Figure 2 :

 2 Figure 2 : Selected mass and UVspectra of fluindione and its metabolites

  Incubation of rat liver microsomes with 100 µM fluindione showed formation of a major metabolite identical to M2 with a m/z = 255.0446 and a UV spectrum identical to that of fluindione. A semi-preparative incubation using 60 mL incubation medium allowed to isolate by HPLC about 0.3 mg of metabolite. Its proton NMR spectrum showed a disymetrical molecule with all the ring A proton different. The fluorophenyl ring was not modified. We could not abtain a 13C spectrum for further identification. M2 was compared to synthetic 3-(4-fluoro-phenyl)-4-hydroxy-coumarin, 3-(4-fluoro-phenyl)-4hydroxy-isocoumarin, 2-(4-hydroxy-phenyl)-indane-1,3-dione and 2-hydroxyfluindione which were all different. Thus until now we don't have the structure of M2.

Figure 1 :

 1 Figure 1 : Proton NMR spectra of fluindione and metabolite M2 References: 1) Molho D. and Boschetti E. (1963), French Patent 1.369.396 2) Geiger K et al.US patent (1963), US patent 3,090,813. 3) Shapiro L. et al. (1961) J Org Chem 26, 3580-81. 4) Comets E, et al. (2012) Clin Pharmacol Ther. 91(5):777-86 and references therein. 5) Moreau C, et al. (2014) Thromb Haemost. 111(4):705-12. 6) Oskaja, V.and Vanags, G. (1961) Lat PSR Zin Akad Vestis3, 67-76. 7) Zhou ZZ et al. (2013) Chem Pharm Bull (Tokyo) 61(11), pp 1166-1172. 8) Faizi DJ, et al. (2016) J Am Chem Soc. 138(7):2126-9. 9) Marinov M. et al. (2014) J. Structural Chem. 55, 3, 446-455.

  .

  metabolized in rat and human microsome to oxidized metabolites corresponding to either an oxidation of the B ring of the indanedione (M2, m/z=255), or oxidation of the fluorophenyl with defluorination (M1, m/z =237) and both reactions (M3, m/z = 253).

Table 1 : Kinetic parameters for M2 formation by human P450 and liver microsomes

 1

	CYP or microsomes	1A2	2B6	2C9	2D6	3A4	Pool HLM
	Km (µM)	40	489	33	656	311	143
	Vm (nmol/min/nmol P450)	2.2	3.7	3.2	5.9	8.9	0.3
	Vm/Km (L /min/nmol)	55	7.6	100	9	29	2