JP6443938B2 - Coffin-Siris syndrome detection method - Google Patents

Description

  The present invention relates to a method for detecting Coffin-Siris syndrome.

  Coffin-Siris syndrome (MIM% 135900, sometimes abbreviated as “CSS”) is stunted growth, intellectual disability, small head, peculiar facial features, and the absence of nail on the fifth finger of the limb. It is a rare congenital anomaly syndrome characterized by formation (Non-patent Document 1). The majority of cases are sporadic and the inheritance is autosomal dominant inheritance. There are reports of about 100 cases in Japan.

  Although the diagnosis of CSS is diagnosed by a combination of multiple symptoms, the manifestation of symptoms varies from patient to patient and is not easy to diagnose. Even if a genetic abnormality is suspected after birth, it may take several years or more to diagnose with CSS.

  The inventors of the present application have previously clarified that a gene encoding a constituent subunit of a SWI / SNF complex is a responsible gene of CSS (Non-patent Document 2, Patent Document 1). However, the number of CSS cases that can be explained by the responsible gene is about 50% of the total, and there are still many cases in which the genetic cause has not yet been elucidated.

International Publication No. 2013/103094

G. S. Coffin, E. Siris, Am. J. Dis. Child. 119, 433-439 (1970). Nat Genet .; 44 (4): 376-8, 2012

  It is an object of the present invention to provide a novel means that enables genetic diagnosis of CSS cases that cannot be explained by the reported responsible gene.

  The inventors of the present invention conducted complete exome sequencing of 92 patients with CSS, and as a result, discovered two types of SOX11 gene de novo mutations from two unrelated patients. Identified the SOX11 mutation as a pathogenic mutation in CSS and completed the present invention.

That is, the present invention is a method for detecting Coffin-Siris syndrome, which comprises examining whether a target organism has a mutation in the SOX11 gene using a sample separated from the organism, wherein at least one of the SOX11 genes A method of detecting Coffin-Silis syndrome when a mutation in the HMG domain of SOX11 is detected in alleles of the present invention.

  The present invention provides a new index for CSS gene diagnosis. As the responsible gene of CSS, the subunit genes SMARCB1, SMARCA4, SMARCA2, SMARCE1, ARID1A and ARID1B of the SWI / SNF complex have been reported by our group, but SOX11 was newly identified as the responsible gene. As a result, the breadth of CSS genetic diagnosis has expanded. The present invention greatly assists a definitive diagnosis of CSS in a medical institution.

(a) SOX11 mutation in CSS patients. Two missense mutations within the HMG domain (black box) occurred at evolutionarily conserved amino acids. (b) Crystal structure of mouse SOX4 HMG domain bound to DNA. The helix is indicated by a ribbon and the loop is indicated by a thread. DNA is shown as a bar. The amino acid residues at the mutation sites are shown in a space-filling model. In the middle and right figures, some of the amino acid residues involved in the hydrophobic core around the mutation site are further shown in a space-filling model. The amino acid numbers in the figure are those for mouse SOX4, and the amino acid numbers for human SOX11 are shown in parentheses. Hydrogen bonds are indicated by black dotted lines. The molecular structure was drawn using PyMOL. (c) Free energy change due to the mutation shown in the figure was calculated using FoldX software. (d) The result of measuring the transcriptional activity of the GDF5 promoter (−448 / + 319) in HeLa cells by luciferase reporter assay. The lower panel shows the result of immunoblot analysis of the extract from the transfected HeLa cells. NC is negative control, WT is wild-type SOX11, S60P is p.Ser60Pro mutant SOX11, Y116C is p.Tyr116Cys mutant SOX11. It is the result of investigating the expression of SOX11 gene in various human tissues. SOX11 gene expression was measured by TaqMan quantitative real-time PCR assay using cDNA from tissues derived from human adult (right) and fetus (left). Error bars indicate the standard deviation of 3 independent experiments. Relative evaluation was performed using the expression level in the kidney as a reference (1x). “Muscle” is skeletal muscle. Specific expression in the brain was observed in both fetuses and adults, and high expression in the heart was confirmed in adults. (a) Sox11-MO alone or in vitro transcribed human SOX11 (hSOX11) mRNA (WT is wild type; S60P is p.Ser60Pro, Y116C is p.Tyr116Cys) was injected into embryos. Rescue of mortality at 24 hpf was confirmed only in the WT hSOX11 mRNA injection test group. Data are mean ± SD. * P <0.05 by Student's t test. ns is not significantly different. (b) Embryos injected with MO and construct were classified as normal, mild and severe at 24 hpf. The phenotype of embryos injected with sox11a / b MO was partially rescued by WT hSOX11 mRNA. (c) Head size ratio at 48 hpf of embryos injected with control-, sox11a-, sox11b-, and sox11a / b-MO (n = 10) (the average value of control-MO is 1). The midbrain width was measured. Data are mean ± SD. * P <0.01 for Student's t test. (d) Brain cell death in 30 hpf MO-injected embryos observed with acridine orange staining (side view). Sox11 morphant had increased cell death in the CNS. Scale bar is 100 μm. The graph on the right is a graph showing the results of quantifying the acridine orange intensity in morphants (n = 8-10). Data are mean ± SD. * P <0.001 for Student's t test. (e) Rear view of a maximum projection image immunolabeled with anti-HuC / D antibody (upper) and anti-acetylated tubulin antibody (lower) at 48 hpf. In the embryos injected with sox11a- and sox11a / b-MO, the lateral association of the hindbrain was disrupted (asterisk in the figure). Scale bar is 100 μm. It is the result of having confirmed the exogenous human SOX11 transcript in the rescue experiment. Positive control (PC) (plasmid DNA derived from WT human SOX11 in pEF6 / V5-His B construct), control MO, sox11 MO and hSOX11 mRNA were injected into 24 hpf embryos, and RNA extracted from each embryo was sampled RT-PCR was performed using human SOX11 (hSOX11) primer and zebrafish EF1-α primer. In each rescue experiment, an exogenous hSOX11 transcript was identified. It is the result of having detected the apoptosis of the brain cell in the zebrafish which suppressed sox11. Apoptotic cells in 30 hpf zebrafish brain were detected by TUNEL staining. The photographs shown in the figure are representative examples of embryo images injected with control-MO, sox11a-MO, sox11b-MO, and sox11a / b-MO. Scale bar is 100 μm. Clear brain cell apoptosis was observed with each morphant.

  SOX11 (SRY (sex determining region Y) -box 11, NCBI Gene ID: 6664) is a transcription factor involved in early embryogenesis. GDF5, which undergoes transcriptional regulation by SOX11, is a cell growth factor, and is known to play an important role in articular cartilage differentiation and proliferation and joint formation. The SOX11 protein is a 441 residue protein, and the gene is composed of one exon. The region from amino acids 48 to 118 is an HMG domain involved in sequence-specific DNA binding, and is evolutionarily highly conserved (Jauch, R., Ng, CK, Narasimhan, K. & Kolatkar , PR Biochem J 443, 39-47 (2012)). The nucleotide sequence of the SOX11 gene and the amino acid sequence of the encoded SOX11 protein are known, and are registered in GenBank, for example, with an accession number of NM_003108. SEQ ID NOs: 1 and 2 in the sequence listing of the present application show the base sequence of the coding region of SOX11 and the encoded amino acid sequence. SEQ ID NO: 3 shows the SOX11 genomic sequence including a part of 5′-UTR and 3′-UTR. Positions 56 to 1381 of SEQ ID NO: 3 are coding regions in exon 1.

  In the present invention, whether or not the target living body has a mutation in the SOX11 gene is examined using a sample separated from the living body. SOX11 gene mutations include mutations that delete at least part of the SOX11 protein in addition to changes (substitutions, deletions, insertions) in a few amino acid residues of the encoded SOX11 protein. Sequence changes are encompassed, including mutations that delete all or part of the SOX11 gene region. For example, missense mutation, nonsense mutation, frameshift mutation, inframe deletion or insertion mutation (deletion or insertion of one or more amino acids) due to base substitution, deletion, insertion, duplication, etc. in the exon or intron region , Mutations that cause splicing abnormalities, or microdeletions of chromosomal regions containing the SOX11 gene, but are not limited thereto. The genomic sequence of the SOX11 gene shown in the sequence listing and the amino acid sequence of the encoded SOX11 protein are typical examples of normal SOX11 sequences. The presence or absence of mutations in the SOX11 gene is determined based on the SOX11 gene shown in the sequence listing. The sequence can be used as a reference, and can be judged by comparison with the reference sequence.

  When at least one harmful mutation is present in at least one allele of the SOX11 gene, the CSS of the target organism is detected. The detection of harmful mutations in the SOX11 gene is a great aid for physicians in the definitive diagnosis of CSS. “Detrimental mutation” or “pathogenic mutation” is a mutation that impairs the transcriptional activity of SOX11, and includes a mutation that inhibits binding to a target DNA. Whether a certain SOX11 gene mutation impairs the transcriptional activity of SOX11 can be easily examined by a well-known reporter assay. An example of a specific method is as described in the examples below, but a brief description is as follows. That is, for example, the promoter region of the GDF5 gene that undergoes transcriptional regulation by SOX11 is linked upstream of a reporter gene such as a luciferase gene, and this construct is introduced into a suitable cultured cell. A construct that expresses a mutant SOX11 protein having an arbitrary mutation is separately prepared, and this construct is introduced into the aforementioned cultured cells, and the mutant SOX11 protein is expressed in the cultured cells. If the mutant SOX11 protein retains transcriptional activity, the expression of the reporter gene is confirmed.If the transcriptional activity is impaired, the expression of the reporter gene is not reduced or confirmed. It is possible to examine whether the mutation is a mutation that impairs the transcriptional activity of SOX11.

  Within amino acid residues that are evolutionarily highly conserved in the SOX11 protein, especially within the HMG domain (amino acids 48 to 118, positions 142 to 354 in the base sequence shown in SEQ ID NO: 1) These mutations are likely to be harmful mutations in SOX11, which is an indicator of CSS. Mutations that delete one or more amino acid residues with high evolutionary conservation in the HMG domain, or mutations in which such amino acid residues are substituted with amino acids having different properties are usually harmful mutations. Rare base mutations that are not registered in well-known databases regarding the diversity of base sequences such as NCBI dbSNP and 1000 Genomes Project. Can be considered a harmful mutation that is an indicator of CSS.

  Various prediction tools that can examine whether or not an amino acid substitution mutation in a gene is a pathogenic mutation are known. For example, SIFT (http://sift.jcvi.org/), PolyPhen (http://genetics.bwh.harvard.edu/pph/), PolyPhen-2 (http://genetics.bwh.harvard.edu/ pph2 /), Mutation Taster (http://www.mutationtaster.org/), Align GVGD (http://agvgd.iarc.fr/agvgd_input.php), etc. are known. It is also possible to determine whether the actually detected SOX11 mutation is a harmful mutation using such a known prediction tool. In SIFT, if the score is less than 0.05, the substitution is predicted to be intolerant (affects changes in protein function). PolyPhen is predicted to be pathogenic if the score exceeds 2.0. PolyPhen-2 scores between 0.000 (most likely benign) to 0.999 (most likely harmful), and pathogenic when the decision based on the score is possibly damaging Sex mutations are strongly suggested. Mutation Taster classifies as pathogenic or polymorphic. In Align GVGD, a class score is evaluated in the range of Class C0 (low possibility) to Class C65 (high possibility), and a mutation of class score C55 or higher indicates a pathogenic mutation.

  The mutation shown in the following Table 1 is a pathogenic mutation of CSS newly identified by WES analysis for CSS patients in the Examples. All of these occur at amino acid residues with high evolutionary conservation in the HMG domain of SOX11 (Fig. 1a), which are not found in the healthy population and are pathogenic mutations as assessed by the prediction tool described above. This mutation has been strongly suggested. However, these two types of mutations are examples of SOX11 gene mutations that serve as CSS indicators in the present invention, and the scope of the present invention is not limited to these specific examples.

  A mutation in the SOX11 gene can be detected by analyzing the nucleotide sequence using a nucleic acid sample such as genomic DNA or RNA. In particular, it is most reliable and desirable to analyze a genomic sequence using a genomic DNA sample. Nucleic acid samples such as genomic DNA can be easily prepared from peripheral blood, oral mucosa swabs, and the like by conventional methods. In addition, various prenatal genetic testing methods are known, and it is possible to examine whether a fetal SOX11 gene mutation exists. For example, methods of collecting and examining cells from the fetus (using amniotic fluid, villi, umbilical cord blood), non-invasive testing methods of examining fetal genetic mutations using fetal cells mixed in maternal blood, in vitro Various methods such as a method using one cell of a fertilized fertilized egg (pre-implantation diagnosis) are known. In the non-invasive testing method, a maternal blood sample containing fetal cells corresponds to “a sample separated from a living body”, and a fetus corresponds to a “target living body”.

  The amino acid sequence of a protein can be affected not only by the region coding for the protein but also by mutations in the intron and UTR region, but in genetic testing, usually the coding region and its vicinity several tens to several hundred bases, for example, 30 to 50 bases In general, inspection is performed including a certain degree of adjacent non-coding regions. In the present invention, the sequence analysis may be performed for the coding region in the exon of the SOX11 gene and the non-coding region in the vicinity thereof. When detecting mutations by analysis of genomic sequence, refer to SEQ ID Nos. 1 and 3 in the Sequence Listing of the present application and the genomic sequence of SOX11 gene available from publicly known databases, design primers appropriately, and use genomic DNA samples Therefore, sequencing may be performed by a conventional method. Mutations can be identified in detail by determining the base sequence of the SOX11 gene on the target biological genomic DNA and comparing it with the wild-type reference sequence. By analyzing the determined base sequence using known software such as SeqScape (registered trademark), mutation detection and profiling can be easily performed.

  The SOX11 gene mutation of interest in the present invention is mainly a hetero mutation. Methods for detecting heteroduplexes using denaturing high performance liquid chromatography (dHPLC) and High Resolution Melt methods are known as methods for detecting heteroduplexes (Bde Juan I et al. (2009)). Breast Cancer Res Treat 115: 405-414, Takano et al. (2008) BMC Cancer 8:59, etc.). However, the SOX11 gene is a gene consisting of a single exon, the size of the coding region on the genome is as small as about 1300 bp, and it is easy to determine the base sequence of the entire coding region on the genome. Therefore, in the present invention, it is convenient and preferable to determine the nucleotide sequence of the coding region of the SOX11 gene from a genomic DNA sample and examine the presence or absence of mutation.

  The deletion of the gene region can be confirmed by an analysis using a DNA microarray, or a conventional method such as an in situ hybridization method or a Southern hybridization method. Examination of the deletion of the gene region by these methods may be performed before or after sequencing, and may be performed as necessary.

  A method for examining the copy number of a gene in a genome using a DNA microarray is known, and can be carried out using a commercially available array, an array prepared by a conventional method, and known software. For example, WO2009 / 084472 describes a method for identifying microscopic deletions of chromosomes by CGH (comparative genomic hybridization) using an array loaded with thousands of BAC clones. Genomic DNA is collected from a patient and a healthy person, and the patient DNA is Cy5-labeled and the healthy person's DNA is Cy3-labeled, and a probe prepared by replacing the labeled dye is hybridized with the array, and a commercially available scanner and The presence or absence of fine deletion and its position can be identified by digitizing the signal from the label using software (for example, GenePix4000B and GenePixPro6.0 of AXON) and performing analysis by a conventional method. In addition, methods using Affymetrix's GeneChip Human Mapping 250K Nsp Array and CytoScan HD array are also known.

  In the in situ hybridization method, cells of a target organism are collected and a chromosome sample is prepared. A probe is prepared by labeling a DNA that specifically hybridizes with the target SOX11 gene region, and the probe is hybridized with the chromosome sample. Deletion of the target gene can be detected by examining the presence or absence of a signal from the probe. The labeling of DNA is not particularly limited, but is usually performed using a radioisotope or a fluorescent dye (Cy5, Cy3, FITC, etc.), and a fluorescent dye is more commonly used. When a fluorescently labeled probe is used, this method is called FISH method. For those skilled in the art, DNA probes that specifically hybridize with the target SOX11 gene region can be designed with appropriate primers by referring to the SOX11 gene genomic sequence shown in the sequence listing of this application and the genomic sequences registered in known databases. However, it can be prepared by performing PCR using normal genomic DNA as a template. In addition, a clone such as a BAC clone containing the target SOX11 gene region can be labeled and used as a probe. Clones such as BAC clones containing human genomic DNA are also commercially available and are readily available. The DNA used for the probe may cover the entire coding region of the target gene or may cover only a part of the coding region.

  In the Southern hybridization method, a genomic DNA sample of a target organism is cleaved with an arbitrary restriction enzyme, electrophoresed on an agarose gel or the like, and the DNA is transferred to a membrane. On this membrane, a DNA probe prepared by labeling a DNA that specifically hybridizes with the target SOX11 gene region is hybridized. By examining the presence or absence of the detected band, the deletion of the target gene can be detected. Even a mutation such as a point mutation can be detected by this method because the size of the detected band changes when the mutation causes a change in the restriction enzyme site. DNA used as a probe can be prepared in the same manner as described above, and the labeling of the probe is not particularly limited, but is usually performed using a hapten such as a radioisotope or digoxigenin.

  Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited to the following examples.

[Materials and methods]
Patients and clinical data All patients were diagnosed with CSS by the attending physician's clinical geneticist. DNA samples were isolated from peripheral blood leukocytes by conventional methods. Informed consent was obtained from all participants regarding the publication of the experimental protocol and patient face in the literature. This study was approved by the Ethics Committee of Yokohama City University School of Medicine.

  Patient 1 (female) had a normal pregnancy and was born at 38 weeks of gestation. Weight at birth is 2,340 g (-1.9 SD), height is 45 cm (-2.2 SD), and front and rear diameter circumference (OFC) is 30.5 cm (-1.8 SD). Low muscle tone, difficulty in feeding (especially in the neonatal period), delayed growth. Standing 5 months, sitting 11 months, walking 1 year 11 months. At 1 year and 7 months, a meaningful word was given. The developmental index at age 3 is 57. Abdominal ultrasonography confirmed that the left kidney was rather small. The patient has hypomorphism in the center of the face, narrowing of the eyelids, binocular sequestration, upward cleft eyebrows, long eyelashes, low nose root, short nose with nostrils up, short person, open mouth, thick lips and low position It had a peculiar facial feature characterized by the auricle, and dysplasia of the distal phalanx with low nail formation (particularly the fifth finger) was also observed. Other features were hairy, long eyelashes and rich hair. At the age of 4 years and 8 months, the height was as low as 92.1 cm (-2.9 SD), and growth hormone deficiency was suspected, but the results evaluated in the stimulation test were normal. At 10 years old, the height is 119 cm (-2.8 SD), the weight is 20.1 kg (-1.8 SD), and the OFC is 47.3 cm (-3.3 SD). Although I am in a special support class at school, I can go to school on my own for about an hour and a half and communicate with my classmates in some language.

  Patient 2 (female) was referred to a genetic clinic for evaluation of short stature at the age of 16. She was born in normal pregnancy but was born at low weight (1.75 kg, -4SD). Although the developmental milestones were normal, parents always felt that my child was behind other children. Academic performance is low, IQ is 70-80. I was in regular class, but I had a hard time passing the class exam every year. Although she was short, she did not have a rough face. The eyelids are small, the orbital bumps are hypoplastic, and no drooping eyelids are observed. The nose was long, the nose was hypoplastic and the nose column was overhanging. The hair was dark, coarse and sparse and grew to the back. The fourth and fifth folds were short, all fingernails were low-formed, and the fingers were tapered. The 4th and 5th heels of both feet and the 3rd heel of the right foot were significantly less formed. There is tortosis on the third, fourth and fifth heels of the right foot and the third and fourth heels of the left foot. A skeletal examination was performed, but no abnormalities were found on the radiograph. Bone age is 13-14 years and follicle stimulating hormone is 1.57 IU / L (normal value: <5 IU / L). Ultrasonography at age 16 before menarche showed uterine dysplasia and abnormal rotation of both kidneys. No secondary sexual characteristics were recognized until menarche at age 17. Patient 2 is now 17 years old, still short, 141 cm (-5 SD) tall, 31.3 kg (-3 SD) and OFC 50.5 cm (-4.5 SD).

Whole Exome Sequence Analysis Whole Exome Sequence Analysis (WES) has been reported (Saitsu, H. et al. Nat Genet 45, 445-9, 449e1 (2013); Tsurusaki, Y. et al. Clin Genet (2013). DOI : 10.1111 / cge.12225). Trio-based WES was conducted for two families. That is, 3 μg of genomic DNA was sheared using Covaris 2STM system (Covaris, Woburn, Mass., USA), and SureSelect XT Human All Exon V4 or V4 + UTRs (Agilent Technology, Santa Clara, Calif., USA) Used and partitioned according to instructions. The exon-enriched DNA library was sequenced with HiSeq2000 (Illumina, San Diego, Calif., USA) with a 101-bp paired-end read and a 7-bp index read. Runs were performed with 4 samples per lane (2.5 pM each, including different indices). Image analysis and base calling were performed using HiSeq Control Software / Real Time Analysis and CASAVA 1.8.2 (Illumina). Mapping to the human genome hg19 was performed using Novoalign (http://www.novocraft.com/main/page.php?s=novoalign). The lead after alignment was treated with Picard (http://picard.sourceforge.net) to remove PCR duplicates. Mutation calls were made using Genome Analysis Toolkit 1.5-21 (GATK) with Best Practice Variant Detection with the GATK v3 and annotated with ANNOVAR (2012Feb23). Common mutations (MAF ≧ 0.01) registered in dbSNP135 were excluded.

Mutation Filtering All mutations within the exon and splice site ± 2 bp range from dbSNP137, 1000 Genomes Project, The National Heart Lung and Blood Institute Exome Sequencing Project Exome Variant Server (NHLBI-ESP 6500), and laboratory Mutations registered in any of the mutation databases (control exome data derived from 408 persons) and mutations located within the segment duplication were excluded, and heterozygous non-synonymous mutations were extracted. Mutations were verified by Sanger sequencing.

Structural modeling and free energy calculation
SWISS-MODEL server 5 (Kiefer, F., Arnold, K., Kunzli, M., Bordoli, L. & Schwede, T. Nucleic Acids Res 37, D387-92 (2009)) is the closest structure to human SOX11 The crystal structure of the HMG domain of mouse Sox4 that binds to DNA (Protein Data Bank code 3U2B) was selected. To investigate the identified missense mutations, free energy changes due to mutations were calculated using FoldX software (version 3.0) (Guerois, R., Nielsen, JE & Serrano, L. J Mol Biol 320, 369-87 (2002). Schymkowitz, J. et al. Nucleic Acids Res 33, W382-8 (2005)). The calculation was repeated three times, and the obtained data was expressed as an average value and a standard deviation.

Analysis of SOX11 expression in human tissues TaqMan quantitative real-time PCR using cDNA derived from adults (Human MTC Panel I, # 636742, Clontech, Mountain View, CA, USA) and fetuses (Human Fetal MTC Panel, # 636747, Clontech) Was done. Predesigned TaqMan probes for human SOX11 (Hs00167060_m1, Life Technologies, Carlsbad, Calif., USA) and human β-actin (ACTB, 4326315E, Life Technologies) were used. PCR was performed using Rotor-Gene Q (QIAGEN, Valencia, Calif., USA), and the expression level was normalized to the internal standard gene ACTB according to the 2- ^ΔΔCt method. Renal expression was used as a standard (1x).

Expression vector
The SOX11 open reading frame (ORF) clone was purchased from Promega (Tokyo, Japan), and SOX11 mutants (c.178T>C; p.Ser60Pro and c.347A>G; p.Tyr116Cys) were KOD-Plus. -Produced by site-directed mutagenesis using Mutagenesis Kit (TOYOBO, Osaka, Japan). Wild-type (WT) and mutant SOX11 cDNA were amplified by PCR using In-Fusion PCR Cloning Kit (Clontech Laboratories) and cloned into mammalian expression vector pEF6 / V5-His B (Life Technologies). It was also cloned into the animal expression vector p3xFLAG-CMV-14 (Sigma, St Louis, MO, USA). As the GDF5 promoter, the 5′-flanking sequence (−448 / + 319, SEQ ID NO: 7) of the GDF5 gene was PCR amplified and cloned into pGL3-basic vector (Promega). All construct sequences were confirmed by Sanger sequencing.

Immunostaining Mouse neuroblastoma 2A (Neuro-2A) was cultured in Dulbecco's modified Eagle's medium (DMEM) -high glucose GlutaMAX supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin (Life Technologies) . Neuro-2A cells were seeded in 24-well plates 24 hours prior to transfection. Each expression construct (200 ng) was transfected into Neuro-2A cells using X-tremeGENE 9 DNA Transfection Reagent (Roche Diagnostics, Indianapolis, Ind.). Twenty-four hours after transfection, cells were fixed in 4% paraformaldehyde / PBS for 15 minutes at room temperature and permeabilized in 0.1% Triton X-100 / PBS for 5 minutes at room temperature. Detection of SOX11 protein with V5-6xHis tag at the C-terminus uses mouse anti-V5 primary antibody (1: 200; Life Technologies) and Alexa Fluor 546 goat anti-mouse IgG secondary antibody (1: 1,000; Life Technologies) It was done. Smears were encapsulated in VAPItashield mounting medium with DAPI (Vector Lab, Burlingame, Calif., USA). Confocal images were acquired using a FLUOVIEW FV1000-D microscope (Olympus, Tokyo, Japan).

Luciferase assay
HeLa cells were cultured in DMEM-high glucose supplemented with penicillin (50 units / ml), streptomycin (50 μg / ml) and 10% FBS. ATDC5 cells were cultured in DMEM / Ham's F-12 (1: 1) supplemented with the above antibiotics and 5% FBS. Cells were seeded in 24-well plates 24 hours prior to transfection. TransIT-LT1 (Takara, Otsu, Japan), pGL3 reporter (500 ng / well), effector (250 ng / well) and pRL-SV40 Performed with internal standard (6 ng / well) vector. Cells were harvested 24 hours after transfection, and luciferase activity was measured using a Picker Gene Dual Sea Pansy Luminescence Kit (Toyo B-Net, Tokyo, Japan). Production of WT and mutant SOX11 protein was assessed by immunoblot analysis using anti-FLAG M2 HRP monoclonal antibody (1: 3,000; Sigma) according to the manufacturer's instructions.

Morpholino microinjection Antisense translation inhibition Morpholino (MO) sox11a-MO (5'-CGCTGTTGTCCGTTTGCTGCACCAT-3 ', SEQ ID NO: 4), sox11b-MO (5'-CTGTGCTCCGTCTGCTGCACCATGT-3', SEQ ID NO: 5) (Gadi, J. et al. J Biol Chem 288 (35): 25400-13 (2013)) and standard control-MO (5′-CCTCTTACCTCAGTTACAATTTATA-3 ′, SEQ ID NO: 6) were obtained from GeneTools (Philomath, Oregon, USA) and finished. The embryos at the 1-2 cell stage were injected at a concentration of 0.1 mM or 0.2 mM. Rescue assay prepares human SOX11 mRNA with cap structure transcribed in vitro from pEF6 / V5-His B construct using mMessage mMachine RNA Synthesis Kit (Ambion, Carlsbad, CA) according to manufacturer's instructions And injected into 1-cell stage embryos. In each MO knockdown and rescue experiment, embryos derived from the same clutch were used as experimental bodies and controls. About 1 μg of RNA having a cap structure was injected per embryo.

Cell death detection To detect apoptotic cells in live embryos, embryos 30 hours after fertilization (30 hpf) were manually removed from the chorion, and acridine orange (2 μg / ml in egg seawater) at 28 ° C, 1 Incubated for hours. After washing with egg-water for 10 minutes × 6 times, the embryo was anesthetized with tricaine, encapsulated in 2% methylcellulose, and observed with a confocal microscope. Apoptotic cells were examined by TUNEL assay as previously reported (Koshimizu, E. et al. PloS One 6, e17688 (2011)).

Detection and quantification of visible and fluorescent images All animals were photographed under identical conditions using an LSM510 confocal microscope (Carl Zeiss, Jena, Germany). For each animal, acridine orange positive cells were quantified using the selection tool included in Adobe Photoshop. For embryo analysis, the head region defined for each embryo was selected. After pixel selection, the fuzziness setting was set to 0, and the number of selected pixels was calculated by image histogram calculation.

Whole immunohistochemical analysis
For HuC / D staining, 48 hpf embryos were fixed with 4% paraformaldehyde overnight at 4 ° C. and dehydrated with methanol at −20 ° C. For staining with acetylated tubulin, 48 hpf embryos were fixed overnight at 4 ° C. with Dent's fixative (80% methanol, 20% DMSO). These embryos were permeabilized with proteinase K, then post-fixed with 4% paraformaldehyde and washed with PBSTX (PBS containing 0.5% Triton X-100). After treatment with 4% normal goat serum (NGS) in PBSTX for 2 hours at room temperature, mouse anti-HuC / D antibody (1: 500, A21271, Life Technologies Co.) or mouse anti-acetylation in 4% NGS / PBSTX solution Incubated with tubulin antibody (1: 1000, T7451, Sigma) at 4 ° C. overnight. Embryos were washed 5 times with PBSTX for 10 minutes and incubated with goat anti-mouse FITC secondary antibody diluted with 2% NGS / PBSTX for 2 hours at room temperature. After washing for 10 minutes × 5 times, the embryos were encapsulated in 2% methylcellulose and observed using a FLUOVIEW FV1000-D confocal microscope (Olympus).

[result]
Identification of SOX11 mutation by WES
As a result of WES as reported in 92 CSS patients (Tsurusaki, Y. et al. Nat Genet 44, 376-8 (2012); Tsurusaki, Y. et al. Clin Genet (2013)) Two unrelated patients (2.2% of 92) identified two SOX11 gene mutations c.178T> C (p.S60P) and c.347A> G (p.Y116C). 54 patients (58.7%) had mutations in any of the five SWI / SNF subunit genes (data not shown).

  Newly identified amino acid mutations in SOX11 are highly conserved HMG domains involved in sequence-specific DNA binding (Jauch, R., Ng, CK, Narasimhan, K. & Kolatkar, PR Biochem J 443, 39- 47 (2012)), a mutation in an amino acid that was evolutionarily conserved from zebrafish to human (FIG. 1a). Neither of the two mutations found affected the localization of the SOX11 protein to the nucleus (data not shown). No abnormalities in the SWI / SNF subunit genes were found in the two families.

  Two patients had characteristic facial features, small head, stunted growth, hypoxia of the fifth nail and mild intellectual disability (Kosho, T. et al. Am J Med Genet A 161, 1221- 37 (2013)). At school, Patient 1 is in a special support class and Patient 2 is in a regular class, but both have poor academic performance (Table 2, above).

Effects of identified SOX11 mutations on the structure and function of human SOX11
(1) Structural analysis by modeling Mutation site in the crystal structure of mouse Sox4 (Jauch, R., Ng, CK, Narasimhan, K. & Kolatkar, PR Biochem J 443, 39-47 (2012)), an analog of human SOX11 FoldX software (Guerois, R., Nielsen, JE & Serrano, L. J Mol Biol 320, 369-87 (2002); Schymkowitz, J. et al. Nucleic Acids Res 33, W382-8 (2005) ) Was used to calculate the free energy change due to mutation.

  Ser60 is located in the helix structure of the HMG domain (FIG. 1b), and therefore the S60P mutation affects the overall folding of the HMG domain and impairs DNA binding of SOX11. The FoldX results confirm this prediction, and the free energy change due to mutation was high enough to destabilize protein folding (> 10 kcal / mol, Figure 1c) (Khan, S. & Vihinen, M. Hum Mutat 31, 675-84 (2010)).

  Tyr116 forms a hydrophobic core with the side chains of the DNA recognition loop structure (FIG. 1b). The Y116C mutation has a low free energy change (<1 kcal / mol, Fig. 1c) and is unlikely to significantly affect the folding of the HMG domain, but may alter the conformation of the DNA recognition loop structure important for DNA binding There is.

(2) Functional analysis by luciferase assay
Both mutations are located within the HMG domain (Kan, A. et al. BMC Dev Biol 13, 4 (2013)), which is required for SOX11 to regulate GDF5 transcription. According to the luciferase assay in HeLa cells and ATDC5 cells using the GDF5 promoter, the transcriptional activity of each of the two mutant proteins was lower than that of the wild type (WT) (Fig. 1d, ATDC5 cell data (Omitted).

  When the transcription level of SOX11 was examined using a human cDNA panel, it was confirmed that SOX11 was expressed exclusively in brain tissue (fetus and adult) and heart tissue (adult) (FIG. 2). This confirms the role of CSS brain features (Table 2, supra) found in the two patients with SOX11 mutations.

(3) Analysis of the function of sox11 in zebrafish Furthermore, the function of sox11 was examined in zebrafish. The zebrafish genome contains two human SOX11 orthologs, sox11a and sox11b. These orthologs are expressed in all cells until gastrulation and are then restricted to the developing central nervous system (CNS) (Rimini, R. et al. Mech Dev 89, 167-71 (1999) de Martino, S. et al. Dev Dyn 217, 279-92 (2000)). We used morpholino oligonucleotides (MO) (sox11a-MO, sox11b-MO, sox11a / b-MO) as previously reported (Gadi, J. et al. J Biol Chem (2013)). And sox11b was knocked down. Injection of low dose sox11a-MO (1.6 ng) and sox11a / b-MO (1.6 ng) into embryos significantly increased mortality compared to control-MO embryos (˜6.3%) (sox11a-MO ~ 74.2%, sox11a / b-MO; ~ 75.4%) (Figure 3a). When WT human SOX11 mRNA (hSOX11-WT mRNA) was co-injected with sox11a / b-MO, the survival rate of morphants at 24 hpf improved (62% -34%) (FIGS. 3a and 4).

  In order to compare the gross morphology of control-MO-injected embryos and sox11 morphants, the effects of brain cell death were classified into three categories: severe, mild and normal (FIG. 3b). The gross phenotype of sox11a / b double morphant was partially rescued by hSOX11-WT mRNA (the proportion of normal embryos was 5% with sox11a / b-MO alone injected compared with hSOX11-WT mRNA 9% for injection, P <0.05) (Figure 3b).

  The head size at 48 hpf of sox11a, sox11b and sox11a / b morphant was significantly reduced (P <0.00001 for sox11a-MO and sox11a / b-MO, P <0.01 for sox11b-MO) (FIG. 3c). Acridine orange staining and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) confirmed that apoptosis was significantly increased exclusively in microcephaly embryos (FIGS. 3d and 5).

  Immunostaining at 48 hpf was also performed for HuC / D, a marker of early postmitotic and mature neurons, and acetylated tubulin, an axonal marker, for further analysis of neurons. (Figure 3e). In sox11 morphant, HuC / D positive neurons were decreased, especially in the telencephalon and diencephalon (Fig. 3e, upper panel). In anti-acetylated tubulin staining, it was also confirmed that the number of axons was greatly reduced in the forebrain, midbrain and hindbrain of sox11 morphant compared to control-MO-injected embryos (Figure 3e, lower panel). ).

Claims (4)

A method for detecting coffin-Siris syndrome, comprising examining whether a target organism has a mutation in the SOX11 gene using a sample isolated from the organism, wherein at least one allele of the SOX11 gene contains HMG of SOX11 A method wherein Coffin-Siris syndrome is detected when a mutation in the domain is detected.   The method according to claim 1, wherein the method is carried out by examining a genomic sequence using a genomic DNA sample. The method according to claim 1 or 2 , comprising examining whether or not at least one of the following mutations indicative of Coffin-Siris syndrome is present.
(1) Mutation in which the 60th amino acid of SOX11 protein replaces serine with proline
(2) Mutation in which the 116th amino acid of SOX11 protein is substituted from tyrosine to cysteine The method according to claim 3 , wherein the mutations (1) and (2) are the following mutations.
(1) Mutation where T at position 178 (position 233 in SEQ ID NO: 3) of the SOX11 gene coding region is C
(2) Mutation where A at position 347 (position 402 in SEQ ID NO: 3) becomes G in the SOX11 gene coding region