Bilal El

Cécile Mignon-Ravix

Pierre Cacciagli

Emmanuelle Buhler

Bruria Ben Zeev

Laurent Villard
email: laurent.villard@univ-amu.fr

Molecular characterization of a 1p36 chromosomal duplication and in utero interference define ENO1 as a candidate gene for polymicrogyria

Keywords: chromosomal rearrangement, 1p36 monosomy, polymicrogyria, in utero electroporation

While chromosome 1p36 deletion syndrome is one of the most common terminal subtelomeric microdeletion syndrome, 1p36 microduplications are rare events. Polymicrogyria (PMG) is a brain malformation phenotype frequently present in patients with 1p36 monosomy. The gene whose haploinsufficiency could cause this phenotype remains to be identified. We used high-resolution arrayCGH in patients with various forms of PMG in order to identify chromosomal variants associated to the malformation and characterized the genes included in these regions in vitro and in vivo. We identified the smallest case of 1p36 duplication reported to date in a patient presenting intellectual disability, microcephaly, epilepsy and perisylvian polymicrogyria (PMG). The duplicated segment is intrachromosomal, duplicated in mirror and contains two genes: ENO1 and RERE, both disrupted by the rearrangement. Gene expression analysis performed using the patient cells revealed a reduced expression, mimicking haploinsufficiency. We performed in situ hybridization to describe the developmental expression profile of the two genes in mouse development. In addition, we used in utero electroporation of shRNAs to show that Eno1 inactivation in the rat causes a brain development defect. These experiments allowed us to define the enolase 1 (ENO1) gene as the most likely candidate to contribute to the brain malformation phenotype of the studied patient and consequently a candidate to contribute to the malformations of the cerebral cortex observed in patients with 1p36 monosomy.

Introduction

Rearrangements of the terminal region of chromosome 1 are particularly frequent and represent up to 20% of all unbalanced rearrangements reported to cause intellectual deficiency and birth defects in the human genome [START_REF] Angelo | Further delineation of nonhomologous-based recombination and evidence for subtelomeric segmental duplications in 1p36 rearrangements[END_REF]. Terminal deletions of chromosome 1 account for up to 67% of all rearrangements on this chromosome [START_REF] Saito | Bilateral perisylvian polymicrogyria, periventricular nodular heterotopia, and left ventricular noncompaction in a girl with 10[END_REF] and are causing one of the most frequent subtelomeric microdeletion syndrome: 1p36 monosomy (MIM 607872) with a frequency of 1 in 5,000 [START_REF] Gajecka | Recurrent interstitial 1p36 deletions: Evidence for germline mosaicism and complex rearrangement breakpoints[END_REF]. No common breakpoints have been identified and these terminal deletions have a highly variable size, ranging from 1 to 12,9Mb [START_REF] Heilstedt | Physical map of 1p36, placement of breakpoints in monosomy 1p36, and clinical characterization of the syndrome[END_REF][START_REF] Greco | Electroclinical features of epilepsy associated with 1p36 deletion syndrome: A review[END_REF]. The severity of the phenotype is generally correlated to the size of the deletion and most patients with 1p36 monosomy suffer from intellectual disability (ID), growth, cardiac and hearing defects, facial dysmorphism, hypotonia, epilepsy and microcephaly [START_REF] Kang | Identification of proximal 1p36 deletions using array-CGH: a possible new syndrome[END_REF][START_REF] Battaglia | Further delineation of deletion 1p36 syndrome in 60 patients: a recognizable phenotype and common cause of developmental delay and mental retardation[END_REF].

The widespread usage of high-resolution array-based comparative genomic hybridization (array CGH) in clinical settings has revealed smaller interstitial deletions in 1p36 [START_REF] Redon | Tiling path resolution mapping of constitutional 1p36 deletions by array-CGH: contiguous gene deletion or "deletion with positional effect" syndrome?[END_REF][START_REF] Rosenfeld | Refinement of causative genes in monosomy 1p36 through clinical and molecular cytogenetic characterization of small interstitial deletions[END_REF][START_REF] Buck | Minimal genotype--phenotype correlation for small deletions within distal 1p36[END_REF]. The molecular characterization of these small rearrangements has allowed to correlate the clinical signs of the patients with the gene content of the deleted segments. Using this strategy, several candidate genes were proposed such as KCNAB2 for epilepsy [START_REF] Heilstedt | Loss of the potassium channel beta-subunit gene, KCNAB2, is associated with epilepsy in patients with 1p36 deletion syndrome[END_REF], SKI for cleft palate [START_REF] Colmenares | Loss of the SKI proto-oncogene in individuals affected with 1p36 deletion syndrome is predicted by strain-dependent defects in Ski-/-mice[END_REF], MMP23 for abnormal cranial suture closure [START_REF] Gajecka | Delineation of mechanisms and regions of dosage imbalance in complex rearrangements of 1p36 leads to a putative gene for regulation of cranial suture closure[END_REF] or GABRD for the neuropsychiatric and neurodevelopmental phenotypes [START_REF] Windpassinger | The human gamma-aminobutyric acid A receptor delta (GABRD) gene: molecular characterisation and tissue-specific expression[END_REF]. However, there are discrepancies on some phenotype genotype correlations [START_REF] Rosenfeld | Refinement of causative genes in monosomy 1p36 through clinical and molecular cytogenetic characterization of small interstitial deletions[END_REF] and a possibility exists that position effects could also cause part of the 1p36 phenotype whereby genes located outside of the deleted segments will cause some of the observed phenotypic characteristics [START_REF] Redon | Tiling path resolution mapping of constitutional 1p36 deletions by array-CGH: contiguous gene deletion or "deletion with positional effect" syndrome?[END_REF].

Although non-allelic homologous recombination was proposed as a mechanism for the 1p36 deletions [START_REF] Saito | Bilateral perisylvian polymicrogyria, periventricular nodular heterotopia, and left ventricular noncompaction in a girl with 10[END_REF][START_REF] Shaw | Implications of human genome architecture for rearrangementbased disorders: the genomic basis of disease[END_REF], there are very few reported duplications of this region. Only two cases of isolated duplications have been reported [START_REF] Heilstedt | Molecular and clinical characterization of a patient with duplication of 1p36.3 and metopic synostosis[END_REF][START_REF] Giannikou | Further delineation of novel 1p36 rearrangements by array-CGH analysis: narrowing the breakpoints and clarifying the "extended" phenotype[END_REF] in addition to duplications associated with more complex rearrangements [START_REF] Khan | Acute lymphoblastic leukemia in a patient with constitutional chromosome 1pter-p36.31 duplication and 1q43-qter deletion[END_REF][START_REF] Tonk | Molecular cytogenetic characterization of a familial der(1)del(1)(p36.33)dup(1)(p36.33p36.22) with variable phenotype[END_REF]. Duplications are more difficult to identify using classical cytogenetic techniques and 1p36 duplications might cause a milder phenotype [START_REF] Heilstedt | Molecular and clinical characterization of a patient with duplication of 1p36.3 and metopic synostosis[END_REF].

Given the small number of duplications identified in 1p36 and their heterogeneity, it is currently not possible to define a syndrome and the only constant phenotypic traits in carriers are non-specific features such as microcephaly, developmental delay and growth retardation [START_REF] Tonk | Molecular cytogenetic characterization of a familial der(1)del(1)(p36.33)dup(1)(p36.33p36.22) with variable phenotype[END_REF].

Here, we report one of the smallest 1p36 duplication identified to date. This rearrangement occurred in a patient presenting with intellectual deficiency, microcephaly, epilepsy and bilateral perisylvian polymicrogyria (PMG). The same patient also presents a small 12p13.1 duplication. PMG has frequently been described in patients with the 1p36 deletion syndrome [START_REF] Dobyns | Consistent chromosome abnormalities identify novel polymicrogyria loci in 1p36.3, 2p16.1-p23.1, 4q21.21-q22.1, 6q26-q27, and 21q2[END_REF][START_REF] Saito | Polymicrogyria and infantile spasms in a patient with 1p36 deletion syndrome[END_REF] but the gene responsible for this phenotype has not been identified. Conversely, no PMG has ever been described in patients carrying a rearrangement of the 12p13.1 region [START_REF] Dobyns | Consistent chromosome abnormalities identify novel polymicrogyria loci in 1p36.3, 2p16.1-p23.1, 4q21.21-q22.1, 6q26-q27, and 21q2[END_REF]. We thus focused our analysis on the 1p36 region. We characterized the rearrangement in detail, performed in situ hybridization to describe the developmental profile of expression of the two genes contained in the duplicated region in 1p36 and we studied the consequences of the inactivation of these two genes in vivo using in utero electroporation.

These experiments allowed us to define the enolase 1 (ENO1) gene as the most likely contributor to the brain malformation phenotype of the studied patient and a candidate contributor to the malformations of the cerebral cortex observed in patients with 1p36 monosomy.

Materials and Methods

Our project obtained ethical approval from Comité de Protection des Personnes Sud-Méditerranée and signed informed consent was obtained from study participants.

Array CGH

Array CGH was performed using NimbleGen 3x720K Whole Genome arrays (Roche Nimblegen, Madison, WI), providing an average resolution of 2.5 kilobases. The microarrays were hybridized and scanned on an Agilent scanner. Data were then extracted using NimbleScan software v2.5 and analysed with SignalMap v1.9 software. Regions showing an abnormal dosage were compared with regions already known to represent non-pathogenic copy number variations (CNV) listed in the database of genomic variants (http://dgv.tcag.ca/gb2/gbrowse/dgv2_hg38). CNVs that were totally included in already reported regions in at least two unrelated healthy individuals, using non-BAC arrays, were considered probably benign and were not studied further. The genomic variant was submitted to the Global Variome shared LOVD and is accessible at the following URL: https://databases.lovd.nl/shared/individuals/00285820

Classical and quantitative PCR

Classical PCR was used to determine the orientation of the duplicated segment using primers designed at each extremity of the duplicated segments, in both orientations.

Expression analysis was performed using cDNAs prepared with RNAs obtained from different human tissues (BD Biosciences, Palo Alto, California, USA) or total RNA of patient and healthy control individuals extracted from lymphoblasts using TRIzol reagent (Invitrogen Corp., Carlsbad, CA, USA). Reverse transcription (RT) reactions were performed using 5μg total RNA. PCR reactions were performed in 20μl of Superscript II reaction buffer (Invitrogen, Carlsbad, California, USA) containing 0.01 M dithiothreitol, 7.5 ng/μl of dN6, 20U of RNase inhibitor (Invitrogen), 10mM dNTP and 200 U of Superscript II reverse transcriptase (Invitrogen) for 1h at 42°C. All classical PCR reactions were performed using the T1 thermocycler (Whatman-Biometra, Goettingen, Germany) in a total volume of 50μl, containing 1X PCR buffer, 0.2mM dNTPs, 1.5mM MgCl2, and 1U Taq polymerase. Real-time PCR reactions on genomic DNA or cDNA were performed using the LightCycler 480 system (Roche, Indianapolis, IN, USA) using the SYBR Green I Master Kit (Eurogentec, Seraing, Belgium) with 2μl of cDNA and 200nM of each PCR primer. Each reaction was performed in triplicate. All primer sequences are detailed on Supplementary Table 1.

Fluorescent in situ hybridization

For FISH analysis, BAC (RP11-651L10 on 1p36.23) and fosmid (G248P81726 on 12p13.1).

DNAs were labelled with Spectrum Orange d-UTP using a nick translation kit (Abbott Diagnostic, Rungis, France) according to the instructions of the manufacturers. Hybridization was performed using the standard procedures [START_REF] Baart | Screening for aneuploidies of ten different chromosomes in two rounds of FISH : a short and reliable protocol[END_REF]. Chromosomes were counterstained with 4,6-diamino-2-phenylindole (DAPI) and observed using an Axioplan-2 Zeiss fluorescent microscope (Zeiss) and the images captured with a CCD camera (Photometrics « SenSys »).

Information was collected and merged using IPLab Spectrum software (Vysis).

In situ hybridization

In situ hybridization was performed on embryonic and adult mouse tissues using specific antisense riboprobes. Embryos were fixed by immersion in 0.1 M phosphate-buffered saline (PBS), pH7.4, containing 4% paraformaldehyde (2-8 h at 4 °C). Adult mice used for in situ hybridization were transcardially perfused (chilled saline for 1 min followed by 0.1M PBS containing 4% paraformaldehyde for 10 min), then embryos or brains were collected and fixed by immersion in 0.1M PBS, pH7.4, containing 4% paraformaldehyde (24h at 4°C).

Tissues were cryoprotected overnight in PBS containing 20% sucrose and embedded in Optimal Cutting Temperature (OCT) compound (VWR, Fontenay-sous-Bois, France) before freezing. The blocks were then stored at -80 °C until use. Cryostat sections (12 mm) were collected on Superfrost+ slides and stored at -80 °C until used. The antisense and sense riboprobes used in this study correspond to nucleotides 149,622,695-149,622,919 for Eno1 and 149,874,114-149,944,365 for Rere on mouse chromosome 4, and to nucleotides 135,023,416-135,023,736 for Ddx47 on mouse chromosome 6 (NCBI m37). In situ hybridization was performed as previously described [START_REF] Andrieu | Expression of the Prader-Willi gene Necdin during mouse nervous system development correlates with neuronal diffenreciation and P75NTR expression[END_REF] with antisense probes transcribed with T7 or SP6 polymerases, and the digoxigenin-labeled probe was detected using an antidigoxigenin antibody coupled to alkaline phosphatase in the presence of NBT/BCIP (Roche, Basel, Switzerland). Control experiments were performed using corresponding sense riboprobes on adjacent sections, giving either no signal or a uniformly low background as expected.

In utero electroporation of small hairpin RNAs (shRNAs)

shRNA sequences were designed using sequence of the common regions of the 3'UTR and 5'UTR of rat and mouse transcripts. They correspond to positions 1659_1680 and 1668_1688 of transcript ENSRNOT00000024106 for shEno1-1 and shEno1-2, respectively and positions 852_874 and 6227_6249 of transcript ENSRNOT00000024443 for shRere-1

(5'UTR) and shRere-2 (3'UTR) respectively. The fragments were amplified using PCR and cloned into the mU6pro vector [START_REF] El Waly | Nhej1 deficiency causes abnormal developement of the cerebral cortex[END_REF]. Inserts were sequenced to confirm the absence of the variant. In order to test the efficacy of the shRNAs, rat 3T3 cells were transfected with each shRNA and the levels of Eno1 or Rere expression were measured using quantitative PCR, taking into consideration the percentage of transfected cells.

Electroporations were performed at E15.5 in WISTAR rats (Janvier, Le Genet Saint Isle, France). Pregnant females were anesthetized with a mixture of ketamine (100 mg/kg) and Xylazine (10 mg/kg) and the uterine horns were exposed. 0.5 to 1μl of DNA solution was injected into one lateral ventricle of each embryo using pulled glass capillaries and a microinjecter (Picospritzer ΙΙ). The DNA solution contained 0.5mg/ml of a pCAG-RFP plasmid either alone or with 1.5mg/ml of shRNA, with fast green 2mg/ml (Sigma). Embryo heads across the uterine wall were placed between tweezer-type electrodes (NEPA Gene). Five electric pulses (50 V; 50 ms) were delivered at 950 ms intervals, using an electroporator BTX ECM 830 (BTX Harvard Apparatus). Uterine horns were placed back and the abdomen sutured. Rat embryos were deeply anaesthetized with pentobarbital (100 mg/kg) and transcardially perfused (with 4 % paraformaldehyde for 10 min). The brains were removed, post-fixed for 5 h, and cut into coronal sections using a vibratome (Leica). Sections were permeabilized (0.1 % Triton X-100 PBS), and labelled with 4,6-Diamino-2-phenolindole dihydrochloride (DAPI), in order to visualize the nuclei.

Cells dissociation and sorting

Brains were extracted 3 days after co-electroporation with either shEno1, shRere or shScramble and pCAG-GFP plasmids. Subventricular zones and cortical plates were dissected manually. Cells were dissociated using the MACs « Adult Brain Dissociation Kit, mouse and rat » (Miltenyi Biotec, Paris, France). Cells were gated based on forward scatter, side scatter and trigger pulse width. Autofluorescent cells were excluded based on their nonspecific signal in the Amcyan channel. Cells were further gated as GFP+ (Green Fluorescent Protein). Cells were sorted using FACS (Fluorescence-Activated Cell Sorting) into lysis buffer (10:1 mix of Resuspension Buffer and Lysis Enhancer from Cells Direct one-step qRT-PCR kit, Thermofisher, Massachusetts, USA). After sorting, tubes were immediately frozen on dry ice, then stored at -80°C until processing for RT-qPCR. RT-qPCR results were the means of 3 wells per condition per gene.

Results

Case report

The patient, a 35 years old woman, was born at 32 weeks after uneventful delivery. From 8 months of age developmental delay was noticed gradually becoming consistent with bilateral opercular syndrome including severe motor dysphasia, difficulties in chewing and swallowing, continuous drooling, accompanied by hemiparesis, microcephaly and progressive kyphoscoliosis. Although cognition was not formally assessed, the patient showed severe expressive speech dyspraxia and was studying in a special school system. She was never independant in her daily skills and had poor communication skills. She could write simple words, could communicate with communication board using more than 100 signs and was able to do calculation up to 10. Seizures started at 3 years of age and included GTCs, absences and drop attacks. Repeated EEG's showed bilateral synchronous and independent slow spike wave discharges leading to the diagnosis of symptomatic Lenox-Gastaut syndrome. Seizures were and are resistant to all available anti-epileptic drugs, ketogenic diet but show partial response to vagal nerve stimulation. Currently the woman is wheelchair bound because of severe kyphoscoliosis, recurrent drop attacks and progressive spasticity. Recurrent aspirations related to drooling and abnormal chewing and swallowing led to G-tube insertion. No cognitive deterioration is noted. MRI showed abnormal temporoparietal gyration which is consistent with bilateral polymicrogyria (Figure 1).

Identification and characterization of the 1p36.23 and 12p13.1 duplications

We have studied the patients' DNA using high resolution comparative genomic hybridization (CGH) on arrays providing a mean resolution of 2,5 kilobases. This analysis detected 33 common copy number variants (CNV) and 5 CNVs that had not previously been reported (Supplementary Table 2). One CNV (loss 1q21.2) was located in an intergenic regions. We focused on candidate protein-coding genes directly impacted by the rearrangements.

The four remaining CNV (Supplementary Table 2) contain transcription units. Using quantitative PCR on genomic DNA of the patient and her mother (the paternal DNA is not available), we showed maternal inheritance for two of them (Supplementary Table 2) and they were not considered further. The last two correspond to duplications that were not inherited from the mother : a 200 kilobases duplication in 1p36. [START_REF] Andrieu | Expression of the Prader-Willi gene Necdin during mouse nervous system development correlates with neuronal diffenreciation and P75NTR expression[END_REF] In order to identify the chromosomal location of the duplicated fragments in the patient cells, we performed fluorescent in situ hybridization (FISH) using bacterial artificial chromosomes (BAC) and fosmid probes. This analysis revealed that the two duplication events were intrachromosomal and were located at the original locus (Figure 2B). Next, we designed a series of PCR primers in order to amplify the junction fragments of the two duplication events and to determine the orientation of the duplicated segments (Supplementary Table 1). We amplified also a part of the TBC1D24 gene like a positive control for the quality of the DNA. This PCRs gave us a band around 700pb for the 1p36 duplication and a band around 1200 pb for the 12p13.1 duplication. These bands were sequenced and this strategy allowed us to precisely map the duplications breakpoints (Figure 3A) and to show that the 1p36.23 region was duplicated in mirror while the 12p13.1 region was duplicated in tandem. The 1p36.23 duplication starts at nucleotide 8,663,118 and ends at nucleotide 8,863,991 of chromosome 1 (GRCh38 Hg38). A comparison with previously published cases reveals that this region is the smallest 1p36 rearrangement reported to date (Figure 3B). The 12p13.1 duplication starts at nucleotide 12,797,698 and ends at nucleotide 12,843,060 on chromosome 12 (GRCh38 Hg38).

We next considered the gene content of the two duplicated regions. The duplicated segments contain the RERE and ENO1 genes on chromosome 1 and the DDX47 gene on chromosome 12. Based on the characterization of the duplication breakpoints performed above, we observed that the RERE and ENO1 genes had been interrupted by the chromosome 1 duplication while the DDX47 gene was entirely contained within the duplication on chromosome 12. We used a panel of mRNAs prepared from various human tissues to detect ENO1, RERE or DDX47 transcripts. Because several alternative transcripts exist for the three genes, we designed PCR primers allowing the amplification of all isoforms (Supplementary Table 1). We found that the three genes have a ubiquitous expression (Supplementary Figure S1A). Given that the three transcripts were detected in lymphocytes, we used quantitative PCR to quantify the expression levels of the three transcripts using mRNA prepared from lymphoblasts of the patient (Supplementary Table 1). This analysis revealed that the ENO1 gene was under-expressed (54% of wild type level) in the cells of the patient while DDX47 was overexpressed (216% of wild type level) (Figure S1B). We were not able to quantify the expression level of RERE because we observed that the expression of this gene is highly variable in control cell lines (data not shown).

Expression of ENO1 and RERE in mouse development

In order to document the spatio-temporal expression of the three genes, we used in situ hybridization in mouse embryos and in the adult mouse brain (Figure 4I The Ddx47 transcripts were difficult to detect in the different development stages. We observed a faint mRNA expression in the urogenital region at E12.5 (Figure 4I.A). No signal was observed in the central nervous system, neither at any embryonic stages nor in the adult CNS.

Neuronal migration evaluation

In order to observe the consequences for brain development of a downregulation the two genes located in 1p36, we used in utero electroporations in rat embryos. We first designed several small hairpin (sh) RNAs targeting the two genes (shEno1-1 and shEno1-2, shRere-1 and shRere-2). These shRNAs were designed to target the 3' and/or 5' untranslated region (UTR) of the rat Eno1 and Rere transcripts because subsequent experiments were performed in that species. In order to test the efficacy of the shRNAs, rat 3T3 cells were transfected with each shRNA and the levels of Eno1 or Rere expression downregulation were measured using quantitative PCR, taking into consideration the percentage of transfected cells. We selected the most efficient shRNAs for each gene: 69,2% and 45% reduction of Eno1 expression compared to wild type levels for shEno1-1 and shEno1-2 respectively, 4,8% and 49,7% reduction of Rere expression compared to wild type levels for shRere-1 and shRere-2 respectively.

Next, we performed in utero electroporations with either the shEno1-1 shRNA or shRere-2 and a red fluorescent protein (RFP) plasmid or the RFP plasmid alone. Embryos were electroporated at E15.5. Embryos were analysed at E20, 5 days post-electroporation. In embryos electroporated with the RFP plasmid alone, with the scramble shRNA or with shRere, 98.3% (SEM=0.24% ; n=3 brains), 97.9% (SEM=0.1% ; n=7 brains) and 97.7% In order to test the efficacy of the shEno1-1 and the shRere-2 in vivo, we performed in utero electroporation in Wistar rat embryos using either the shEno1-1 or shRere-2 and a Green Fluorescent Protein (GFP) plasmid (pCAG-GFP) or the GFP plasmid alone. Embryos were electroporated at E15.5. In order to see the effect of each shRNA on gene expression during the cell migration, brains were extract at E18, 3 days post-electroporation. Cells were dissociated from 2 shScramble (control), 10 shEno1 and 11 ShRere dissected brains. After dissociation, we FACS sorted cells based on the GFP+ fluorescence compared with GFP negative control cells. After the FACs sorting, we tested the expression of Eno1 and Rere by RT-qPCR (Supplementary Table 1). The expression of the Eno1 gene was reduced by 41% after the electroporation of the shEno1 however the expression of the Rere gene was reduced by 56% after the electroporation of the shRere (Supplementary Figure S2). In conclusion, the downregulation of the Rere gene expression by 56% in the electroporated neuroblasts does not perturb their migration to the cortical plate, however, the downegulation of the Eno1 gene expression by 41% in the electroprated neuroblasts disturbs their migration to the cortical plate and blocks 92% of them in the intermediate zone.

Discussion

The search for chromosomal rearrangements in patients presenting a malformation of the cerebral cortex is a powerful approach to identify new candidate genes. We used this strategy to study a patient presenting bilateral polymicrogyria. Polymicrogyria (PMG) is a malformation of the cerebral cortex characterized by a loss of the normal microscopic and macroscopic architecture with the appearance of numerous small and unfolded gyri and disorganization of cortical layering [START_REF] Dobyns | Consistent chromosome abnormalities identify novel polymicrogyria loci in 1p36.3, 2p16.1-p23.1, 4q21.21-q22.1, 6q26-q27, and 21q2[END_REF]. PMG can be caused by genetic variants. It is genetically heterogeneous and variants in several genes were reported to be the cause of the malformation [START_REF] Stutterd | Polymicrogyria : A common and heterogeneous malformation of cortical development[END_REF].

We then identified two duplications in our patient. Despite that duplication could change the structure of the genome, the topologically associated domains (TADs) organisation and therefore the gene expression [START_REF] Kyukwang | Characterization of Structural Variations in the Context of 3D Chromatin Structure[END_REF], we focused on protein-coding genes directly impacted by the rearrangements. The first duplication concerns the 12p13.1 chromosomal region and contains a single gene called DDX47, entirely duplicated. This gene encodes a poorly described DEAD box protein member having a putative role as an RNA helicase [START_REF] Strausberg | Mammalian Gene Collection Program Team. Generation and initial analysis of more than 15,000 full-length human and mouse cDNA sequences[END_REF]. We found that DDX47 is overexpressed in the lymphoblasts of the patient, as expected following the duplication of the entire transcription unit. However, expression analysis in mouse embryonic development shows that the DDX47 transcript is mainly detected in the urogenital system. The absence of similar cases in genomic variant databases, the absence of linkage to 12p13 of a disease associated with a malformation of the cerebral cortex, combined to the expression pattern of DDX47, did not supported an obvious role for this transcript in cortical development and we did not studied this gene further. The second duplication concerns the 1p36.23. This small 200 kilobases duplication contained two interesting genes, ENO1 and RERE. Sequencing revealed the presence of two similar regions surrounding the duplication which could be the cause of duplication following homologous recombination. The first region is located between the base pairs 8663091 and 8663103 (CTATTTGGGCGGG) of the RERE gene and the second region is located between the base pairs 8863979 and 8863991 (CTCTTTGGGTTGG) of the ENO1 gene.

We showed that ENO1 expression was reduced by 50% in the patient cells mimicking the haploinsufficiency caused by a monosomy. Expression analysis in mouse development revealed a strong expression of both transcripts in the central nervous system. Interestingly, 1p36 monosomy has been associated with bilateral polymicrogyria [START_REF] Dobyns | Consistent chromosome abnormalities identify novel polymicrogyria loci in 1p36.3, 2p16.1-p23.1, 4q21.21-q22.1, 6q26-q27, and 21q2[END_REF][START_REF] Saito | Polymicrogyria and infantile spasms in a patient with 1p36 deletion syndrome[END_REF]. Although 1p36 deletions are common, early descriptions did not provide cerebral imaging results [START_REF] Greco | Electroclinical features of epilepsy associated with 1p36 deletion syndrome: A review[END_REF][START_REF] Shapira | Chromosome 1p36 deletions: the clinical phenotype and molecular characterization of a common newly delineated syndrome[END_REF][START_REF] Faivre | Prenatal detection of a 1p36 deletion in a fetus with multiple malformations and a review of the literature[END_REF][START_REF] Slavotinek | Monosomy 1p36[END_REF].

More recently however, correlations between 1p36 rearrangements and polymicrogyria were reported. In a large study of 64 patients presenting a deletion of 1p36 [START_REF] Dobyns | Consistent chromosome abnormalities identify novel polymicrogyria loci in 1p36.3, 2p16.1-p23.1, 4q21.21-q22.1, 6q26-q27, and 21q2[END_REF], 20% of the cases had polymicrogyria. Combined with three additional reports [START_REF] Gajecka | Recurrent interstitial 1p36 deletions: Evidence for germline mosaicism and complex rearrangement breakpoints[END_REF][START_REF] Saito | Polymicrogyria and infantile spasms in a patient with 1p36 deletion syndrome[END_REF][START_REF] Shiba | Neuropathology of brain and spinal malformations in a case of monosomy 1p36[END_REF], these studies allowed to define a putative minimal critical region of 3.8 Mb in 1p36 for polymicrogyria localized between 1Mb and 4.8Mb from the 1p telomere (see Figure 3B). The 1p36 rearrangement described here is the smallest identified to date in association with polymicrogyria. Although it lies outside of the minimal critical region defined previously [START_REF] Dobyns | Consistent chromosome abnormalities identify novel polymicrogyria loci in 1p36.3, 2p16.1-p23.1, 4q21.21-q22.1, 6q26-q27, and 21q2[END_REF], it contains the RERE gene, proposed to be a candidate gene for polymicrogyria [START_REF] Saito | Polymicrogyria and infantile spasms in a patient with 1p36 deletion syndrome[END_REF]. RERE encodes a member of the arginine/glutamic acid repeat-containing protein family. This protein was shown to be a nuclear receptor co-regulator interacting with NR2F2 and NR2E1 [START_REF] Vilhais-Neto | Rere controls retinoic acid signalling and somite bilateral symmetry[END_REF][START_REF] Wang | Histone deacetylase-associating Atrophin proteins are nuclear receptor corepressors[END_REF] and to interact with histone deacetylases in the mouse embryo [START_REF] Shiba | Neuropathology of brain and spinal malformations in a case of monosomy 1p36[END_REF][START_REF] Yanagisawa | Protein binding of a DRPLA family through arginine-glutamic acid dipeptide repeats is enhanced by extended polyglutamine[END_REF], but its function is not completely elucidated [START_REF] Yanagisawa | Protein binding of a DRPLA family through arginine-glutamic acid dipeptide repeats is enhanced by extended polyglutamine[END_REF]. Different mouse models with null or hypomorphic RERE alleles have been produced [START_REF] Zoltewicz | Atrophin 2 recruits histone deacetylase and is required for the function of multiple signaling centers during mouse embryogenesis[END_REF][START_REF] Kim | An allelic series of mice reveals a role for RERE in the development of multiple organs affected in chromosome 1p36 deletions[END_REF][START_REF] Kim | Mouse model reveals the role of RERE in cerebellar foliation and the migration and maturation of Purkinje cells[END_REF]. These models revealed that a deficiency of RERE leads to a wide range of developmental defects including the brain, heart and kidney. One of these was described with abnormal cerebellum development [START_REF] Kim | Mouse model reveals the role of RERE in cerebellar foliation and the migration and maturation of Purkinje cells[END_REF] but without neuronal migration defects in the cerebral cortex. Moreover, in human, a new genetic syndrome was recently described in RERE variant cases [START_REF] Fregeau | De novo mutations of RERE cause a genetic syndrome with features that overlap those associated with proximal 1p36 deletions[END_REF][START_REF] Jordan | Genotype-phenotype correlations in individuals with pathogenic RERE variants[END_REF]. It is characterized by neurodevelopmental disorder that may be accompanied by brain, eye and/or heart anomalies, but none of the 19 individuals reported in the Jordan et al and Fregeau et al. studies had any kind of polymicrogyria.

We were not able to assess the expression level of RERE in the cells of our patient because of the large variability of RERE expression in normal human lymphoblasts. However, taking into account the absence of neuronal migration defect in mouse models or in human RERE syndrome, as well as in our own in utero electroporation experiments, we do not favour a role of RERE variants in the polymicrogyria phenotype observed in the duplicated patient. The absence of a neuroblasts migration defect in heterozygous Rere +/-mice is comparable to our results obtained with the shRere decreasing Rere expression by 50.3%. Nonetheless, RERE deficient embryos that carry both a null or a hypomorphic allele of Rere have been described to have Purkinje cell migration defects [START_REF] Zoltewicz | Atrophin 2 recruits histone deacetylase and is required for the function of multiple signaling centers during mouse embryogenesis[END_REF][START_REF] Kim | Mouse model reveals the role of RERE in cerebellar foliation and the migration and maturation of Purkinje cells[END_REF].

Rather, our results indicate that ENO1 encoding α-enolase, is a good candidate to contribute to that phenotype. Enolase is a glycolytic enzyme involved in production of ATP. The functional enzymes are dimers composed of subunits encoded by separate genes designated ENO1, ENO2 and ENO3 for α, γ and β subunits, respectively. Type αα-enolase is the predominant form and is found in wide range of tissues. In the developing brain, α dimers switch towards γ-enolase subunits expression while neurons undergo terminal differentiation [START_REF] Joseph | Enolase activity and isoenzyme distribution in human brain regions and tumors[END_REF].

The ENO1 transcript levels were reduced by 50% in the cells of the patient with the 1p36 duplication, mimicking monosomy. It is worth noting that 8 heterozygous loss of function variants (excluding the splice variants that can sometimes preserve a reading frame) are found in the 120292 alleles of the control population in the Genome Agreggation Database (gnomAD). This may suggest that ENO1 dysfunction could contribute to a neuronal migration defect in combination with other factors, rather that acting as a classical monogenic disease causing gene. Should this be true, ENO1 haploinsufficiency would contribute to an oligogenic phenotype. Also, it is possible that the minimal critical region defined in 2008 by Dobyns and colleagues [START_REF] Dobyns | Consistent chromosome abnormalities identify novel polymicrogyria loci in 1p36.3, 2p16.1-p23.1, 4q21.21-q22.1, 6q26-q27, and 21q2[END_REF] contains regulatory elements acting on ENO1 and/or other distant transcripts.

The analysis of its expression in mouse development revealed a strong expression in the cerebral cortex during embryogenesis compatible with a role during brain development. Most interestingly, in utero electroporation of interfering Eno1 shRNAs revealed that the migration of electroporated neurons is severely impaired during the early phases of cerebral cortex development. According to the previously mentioned oligogenic hypothesis, an interaction between Eno1 and Rere could participate in polymicrogyria, even though the downregulation of Eno1 alone prevents 92% of neuroblasts from reaching the cortical plate and causes a severe abnormal migration phenotype. Taken together, these elements strongly suggest that ENO1 must be considered as a contributor gene to the phenotype of malformation of the cerebral cortex in the 1p36 region and that it could be screened for variants in patients with polymicrogyria of unknown origin.

  and a 44 kilobases duplication in 12p13.1, arr[GRCh38]1p36.23(8654490_8663239)_(8864067_8868341)x3, 12p13.1(12789530_12797985)_(12842155_12855828)x3 (Figure 2A).

  ). During the development, Eno1 is expressed at embryonic stage E14.5 in the inner (ventricular) side of the neopallial cortex (Figure 4I.B). The expression of this gene subsequently decreases after E14.5. In the adult brain, we detected a strong expression of Eno1 in the cornus ammoni (CA) regions CA2 and CA3 of the hippocampus and a moderate expression in the cerebral cortex (Figure 4I.D). The Rere mRNA was detected at E14.5 in the central nervous system, with a strong expression in the neopallial cortex and the midbrain (Figure 4I.B). The expression in the neopallial cortex appears to be higher in the outer (pial) side than the inner (ventricular) side. The transcripts of Rere are detected to a lesser extent at later embryonic stages (E17.5) (Figure 4I.C).

(

  SEM=0.31% ; n=5 brains) of electroporated neurons reached the cortical plate, respectively (Figure 4II.A, 4II.B and 4II.C). On the contrary, in embryos electroporated simultaneously with shEno1-1 and the RFP plasmid, only 8% of electroporated neurons (SEM=1.18% ; n=3 brains) reached the cortical plate and a large number of RFP positive neurons were still visible in the intermediate zone (IZ) at E20 (Figure 4II.D). A summary of the results of these experiments is presented in Figure 4II.E

Figure 1 .

 1 Figure 1. Magnetic resonance imaging of the patient brain showing bilateral perisylvian polymicrogyria (arrows). Representative sagittal fast spoiled gradient echo (FSPGR) image (A), axial FSPGR image (B) and coronal fast spin echo (FSE) T2 image (C) sections are shown. Notice asymmetry of location and extent of abnormal cortex on both hemispheres and asymmetric ventricles possibly explaining the patients' hemiparesis in addition to her opercular symptoms and signs.

Figure 2 .Figure 3 .

 23 Figure 2. A. Schematic representation of the genomic duplications identified in the patient on chromosomes 1 and 12, following human genome build 38 (GRCh38). Grey bars show the duplicated regions, based on the first and the last duplicated oligonucleotides on the CGH array. Exons are represented as black or grey boxes and an arrow indicates the position of the translation initiation codon and the orientation of transcription. B. Fluorescent in situ hybridization performed on metaphases of the patient with probes RP11-651L10 (BAC clone on chromosome 1) and G248P81726 (fosmid clone on chromosome 12), both spanning the duplicated regions. These probes appear as red signals and white arrows show the duplicated chromosomes

Acknowledgements

The authors thank the Centre de Ressources Biologiques of La Timone Children's Hospital (Karine Bertaux, Cécile Mouradian and Annachiara Desandre Giovannoli) for assistance with the samples used in this study and Virginie Bascunana and Dr. Françoise Watrin for technical assistance.

identify uncertainly delimited deletions. The grey box denotes the previously published minimal critical regions for PGM, located between 1 and 4.8 Mb on chromosome 1.


Bogdan Iorga

Virginie Mouriès

Philippe Savignac

Carbanionic displacement reactions at phosphorus : synthesis and reactivity of 5,5-dimethyl-2-oxo-2-(1,3-dithiane-2-yl)-1,3,2dioxaphosphorinane

We describe the formation and reactivity of the lithiated carbanion of 5,5-dimethyl-2oxo-2-(1,3-dithian-2-yl)-1,3,2-dioxaphosphorinane 3b. The derived carbanion 2b is prepared in quantitative yield by internal quench condensation of 1,3-dithiane and 2-chloro-5,5-dimethyl-2oxo-1,3,2-dioxaphosphorinane 1b with two equivalents of LDA at low temperature. The carbanion 2b is reacted with a variety of ketones in one-carbon homologation reaction and a comparison with other reagents is done. chlorophosphates / dithiane / LDA / lithiated phosphonate esters / Horner-Wadsworth-Emmons reaction Réactions de substitution nucléophile sur le phosphore

Introduction

There is a considerable interest in the synthesis of functional phosphonates, since they have been demonstrated as important precursors in organic synthesis and material science. Several convenient and practical routes to these reagents have been summarized recently 1 . They can be classified into four main categories of reactions : the Michaelis-Arbuzov 2 , Michaelis-Becker 3 and Kinnear-Perren 4 reactions and the addition of trivalent phosphorus reagents at unsaturated carbon 5 . By contrast, generation of a C-P bond by carbanionic displacement of a good leaving group attached at an electropositive quinquevalent phosphorus center has not been yet widely exploited for the preparation of functionalized phosphonates. Our early efforts in this area involved the nucleophilic attack of nitrile-stabilized anions on bis(dimethylamino)chlorophosphate 6 and the reaction of alkyllithiums on trialkylphosphates 7 . More recently, we reported the synthesis and some reactions of diethyl (2-pyridyl)-methylphosphonate [START_REF] Carran | Phosphorus Sulfur Silicon Relat Elem, in press (9) a) Carey FA, Court AS[END_REF] which was efficiently accomplished by condensation of the lithiated carbanion of 2-methylpyridine with diethyl chlorophosphate. The carbanionic displacement of chlorine from phosphorus occurs readily at low temperature to generate the carbon-phosphorus bond. An attractive feature in this reaction is the transient formation of an α-phosphorylated carbanion which is of significant synthetic utility and can be exploited for further reactions and especially for the one-carbon homologation of ketones under the Horner-Wadsworth-Emmons conditions. We now wish to present in details the synthesis and reactivity of 5,5-dimethyl-2-oxo-2-(1,3-dithiane-2-yl)-1,3,2-dioxaphosphorinane 3b prepared according to the same tactic. To the best of our knowledge, there has been no report concerning formation and reactivity of 3b. Several thioacetals of formylphosphonates are known, 3a, c, d (Scheme 1) and we have undertaken to compare the new 3b with diethyl 1,3-dithian-2-yl phosphonate 3a.

Scheme 1

Results and discussion

Ketene dithioacetals are versatile synthetic intermediates which may be converted by reductive-hydrolysis to aldehydes 9 or by hydrolysis to carboxylic acids derivatives 10 , and consequently a large number of carbonyl olefination reactions have been designed and developped for their preparation 9a,11,12 . The most widely used methods for the synthesis of ketene dithioacetals involve the Wittig, Horner-Wittig and Horner-Wadsworth-Emmons reactions of α-phosphoryl dithioacetals with carbonyl compounds 12 . The classical synthesis of α-phosphoryl dithioacetals is accomplished in high yields by a Michaelis-Arbuzov process involving 2-chloro-1,3-dithiane and a trialkylphosphite 13 . The preparation of the same compounds via the reaction between 2-lithio-1,3dithiane and chlorophosphines and subsequent oxidation of the resultant phosphine has been reported by Juaristi et al. 14 , then with diethyl chlorophosphate by Comins et al. 15 . The overall yield of this reaction is modest, and frequently a chromatographic purification is necessary.

We anticipated that dithiane phosphonates could be efficiently prepared by condensation of the derived carbanion of 1,3-dithiane with a chlorophosphate under internal quench conditions. Thus, the generated carbanion is immediately trapped by the electrophile before any possible degradation. In addition, this way offers the possibility to choose the ester substituents of the chlorophosphate so as to modify the reactivity of the phosphoryl group. Two chlorophosphates were tested, the 2-chloro-5,5-dimethyl-2-oxo-1,3,2-dioxaphosphorinane 1b, prepared according to the "vacuum process" previously described by us 16 , and the diethyl chlorophosphate 1a which is a commercially available. The cyclic chlorophosphate 1b is obtained on molar scale in nearly quantitative yield and used without any purification (m.p. 106-108°C against 63% yield and m.p. 104.5-106°C after recrystallisation 17 ). The chlorophosphate 1b is soluble in acetone, benzene, THF and dichloromethane but insoluble in water, ether and cyclohexane.

The lithiated phosphonate esters 2a or 2b were directly obtained by simultaneous addition of a mixture of 1,3-dithiane and chlorophosphate 1a or 1b to LDA (2 equiv) in THF at low temperature (internal quench conditions) (Scheme 2). As checked by 31 P-NMR, the result was complete disappearance of the chlorophosphate with clean and complete formation of the stable carbanions 2a (δ 31 P(THF)=45.2) or 2b (δ 31 P(THF)=41.2) which can be kept for a long time at 15°C without decomposition.

Scheme 2

Z 2 P Z 1 O Cl S S Z 2 P Z 1 O Li S S O S S + 2 LDA -78°C,THF 15°C,THF S S Z 2 P Z 1 O H 2 O 1a : 4 a -j 2 a , b 3 a , b O O Z 1 = Z 2 = EtO 1b : Z 1 , Z 2 =

Rdt. (%) 4

Route After some experimentation, we chose to compare the reactivity of 2a (route A, Table 1) and 2b (route B, Table 1) towards cyclic ketones, sterically hindered or not. Early reported preparations of ketene thioacetals 4 using 1a included especially aromatic aldehydes 12d,e , aromatic ketones 12d,e , α,β-unsaturated aldehydes and ketones 12c . Recently, the use of 2-phosphoryl 1,3-dithiane bearing trifluoroethyl group on phosphorus 3c for the direct one-carbon homologation of ketones was described 12a . One of the attractive feature of 3c is due to the strong electron withdrawing character of the trifluoroethyl groups which stabilize the derived carbanion, rendering it less basic, and accelerate the elimination step of the Horner-Wadsworth-Emmons reaction 12a .

We have selected phosphonate 3b because it is known that modification of the structure of the phosphoryl group in the dithioacetal influences the reactivity of the corresponding carbanion towards ketones 12c . Cyclic phosphonates are useful intermediates in organic synthesis owing to the enhanced sensitivity of the phosphorus atom to nucleophilic attack. Effectivelly, on addition of the ketone to 2b at 15°C, an elevation of temperature was observed (10-15°C). By contrast, the same reaction with 2a is not so exothermic. On the other hand, reaction time are shorter with 2b than with 2a. For example, reaction of cyclopentanone with 2b needs about 3h against 12h with 2a 12a . As shown in Table 1, our experimental results demonstrate that the cyclic phosphorylated anion 2b is reactive towards a large variety of cyclic ketones mainly due to its less crowded structure which allow an easier approach of the electrophile. The yields are very high, but decrease significantly when the reaction is accomplished with hindered ketones (4i, j). The evidence is given by the difference of reactivity between norcamphor 4h and camphor 4i derivatives. In addition, our attempts in obtaining the corresponding ketene diacetals from 2,6-dimethyl-cyclohexanone and 1tetralone failed, resulting in complete enolization of the ketone whatever the temperature (-15°C or +15°C).

Conclusion

Consequently, an efficient synthesis of 5,5-dimethyl-2-oxo-2-(1,3-dithian-2-yl)-1,3,2dioxaphosphorinane 3b connected to an easy formation of the corresponding lithiated anion 2b has been realized. On reaction with cyclic ketones, 2b has proved to be a good nucleophile able to afford the corresponding ketene thioacetals 4a-j in high yields. Furthermore, we make a comparison on the opportunity to utilize 5,5-dimethyl-2-oxo-2-chloro-1,3,2-dioxaphosphorinane 1b or diethyl chlorophosphate 1a under Horner-Wadsworth-Emmons conditions. It appears clearly that the title compound is the best suited to the synthesis of ketene thioacetals 4. Moreover the feasibility of the approach encourages the developement of related possibilities for C-P bond formation.

Experimental section

NMR spectra were recorded on a Bruker AC 200 spectrometer operating at 200 MHz for proton, 50.3 MHz for carbon and 81.01 MHz for phosphorus. 31 P downfield shifts (δ) are expressed with a positive sign, in ppm, relative to external 85% H 3 PO 4 in H 2 O. 1 H and 13 C chemical shifts (δ) are reported in ppm relative to CDCl 3 as internal standard. Coupling constants (J) are given in Hz. The following abbreviations are used: s, d, t, q, p, m for singlet, doublet, triplet, quadruplet, pentuplet and multiplet respectively. Organic solvents were purified by standard procedures. THF was distilled under an inert atmosphere from purple solutions of sodium-benzophenone ketyl. The synthesis of all compounds were carried out under dry nitrogen.

General procedure for the synthesis of 2-phosphoryl-1,3-dithiane (3a) or (3b) An oven-dried, 250 ml, four necked, round-bottomed flask is fitted with an efficient mechanical stirrer, a thermometer, a condenser and a pressure equalizing funnel with a nitrogen inlet. Under a gentle flow of nitrogen the flask is charged with 26.5 ml (42 mmol) of a 1.6M solution of n-BuLi in hexane then 4.45 g (44 mmol) of i-Pr 2 NH in 20 ml THF is gently added at -78°C. At LDA thus formed, a mixture of 1a 3.62 g (21 mmol) and 2.4 g (20 mmol) of 1,3-dithiane or 1b 3.9 g (21 mmol) and 2.4 g (20 mmol) of 1,3-dithiane, dissolved in 50 ml of THF is added at the same temperature. After 30 min. at low temperature, the phosphorylated 1,3-dithiane carbanion 2a or 2b obtained is allowed to warm-up at room temperature and hydrolysed with 50 ml of water. For 3a, the aqueous layer is extracted with Et 2 O (2x20 ml) and the combined organic extracts are dried (MgSO 4 ) and evaporated under reduced pressure to afford the crude product which is purified by a silica gel chromatographic column using hexane as eluent. For 3b the white precipitate is separated from the aqueous layer by filtration and washed with ether. Diethyl 1,3-dithian-2-yl-phosphonate (3a) b.p./torr 129-134°C/0.12 15 31 P NMR (CDCl 3 ): δ +18.7 (s) 1 H NMR (CDCl 3 ): δ 1.38 (t, 3 J HH =7, 6H, CH 3 CH 2 O), 1.88-2.23 (m, 2H, CH 2 of 5-dithianyl), 2.50-2.65 (m, 2H, CH 2(eq) of 4,6-dithianyl), 3.45-3.85 (m, 3H, CH 2(ax) of 4,6-dithianyl and CH of 1dithianyl), 4.28 (p, 3 J HH = 4 J PH =7, 4H, CH 3 CH 2 O) 13 C NMR (CDCl 3 ): δ 16.8 (d, 3 General procedure for the synthesis of ketene thioacetals (4a-j)

The reaction is carried out in the same manner as above. After formation of 2a or 2b at low temperature and warming to 15°C a ketone (19 mmol) dissolved in THF is added. After a few hours (about 2 to 4h) at room temperature, the reaction mixture is hydrolysed with 50 ml of water. The aqueous layer is extracted with Et 2 O (2x20 ml) and the combined organic extracts are dried (MgSO 4 ) and evaporated under reduced pressure to afford the crude product which is purified by a silica gel chromatographic column using hexane as eluent. -1,3-dithiane (4a) Colourless solid, m.p.<45°C

2-Cyclopentylidene

  J PC =5.8, CH 3 CH 2 O), 25.4 (s, CH 2 of 5-dithianyl), 26.5 (s, CH 2 of 4,6-dithianyl), 34.4 (d, 1 J PC =160.0, CH of 1-dithianyl), 64.1 (d, 2 J PC =7.0, CH 3 CH 2 O) MS (CI, NH 3 ), m/z (relative intensity): 257 (M+1, 100), 274 (M+18, 34)5,5-dimethyl-2-oxo-2-(1,3-dithian-2-yl)-1,3,2-dioxaphosphorinane (3b) m.p. 242-244°C 31 P NMR (CDCl 3 ): δ +13.1(s)1 H NMR (CDCl 3 ): δ 1.10 (s, 3H, CH 3 ), 1.15 (s, 3H, CH 3 ), 1.85-2.25 (m, 2H, CH 2 of 5-dithianyl), 2.55-2.70 (m, 2H, CH 2(eq) of 4,6-dithianyl),3.50-3.85 (m, 2H, CH 2(ax) of 4,6-dithianyl), 3.85- 4.05 (m, 2H, CH 2(eq) O), 4.20-4.35 (m, 2H, CH 2(ax) O)13 C NMR (CDCl 3 ): δ 22.0 (s, CH 3 ), 22.2 (s, CH 3 ), 25.5 (s,CH 2 of 5-dithianyl), 26.9 (s, CH 2 of 4,6-dithianyl), 32.7 (d, 1 J PC =154.0, CH of 1-dithianyl), 33.2(d, 3 J PC =6, C(CH 3 ) 2 ), 76.7 (d, 2 J PC =6.5, CH 2 O) MS (CI, NH 3 ), m/z (relative intensity): 269 (M+1, 100), 286 (M+18, 9)

1 H

 1 NMR (CDCl 3 ): δ 1.60-1.85(m, 4H, CH 2 of 3,4-cyclopentyl), 2.15 (m, 2H, CH 2 of 5-dithianyl), 2.35-2.50 (m, 4H, CH 2 of 2,5-cyclopentyl), 2.85-3.00 (m, 4H, CH 2 of 4,6-dithianyl)13 C NMR (CDCl 3 ): δ 25.9 (s, CH 2 of CH 2 of 5-dithianyl), 27.2 (s, CH 2 of 3,4-cyclopentyl), 30.6 (s, CH 2 of 4,6-dithianyl), 33.7 (s, CH 2 of 2,5-cyclopentyl), 113.9 (s, Cq of 1-cyclopentyl), 149.7 (s, Cq of 2-dithianyl) MS (CI, NH 3 ), m/z (relative intensity): 187 (M+1, 100)2-(4-tert-Butyl-cyclohexylidene)-1,3-dithiane (4b)Pale yellow solid, m.p. 66-66.5°C 11d 10-adamantyl),35.6 (s, CH of 1,3-adamantyl), 37.3 (s, CH 2 of 4,6,8,9-adamantyl), 39.2 (s, CH 2 of 4,6-dithianyl), 111.6 (s, Cq of 2-adamantyl), 154.2 (s, Cq of 2-dithianyl) MS (CI, NH 3 ), m/z (relative intensity): 253 (M+1, 100)

Table 1

 1

	A	Route B

Acknowledgments

We would like to thank the CNRS and the Ecole Polytechnique for financial support . B.I. would like to thank the Ecole Polytechnique for financial support.