Somatic GJA4 gain-of-function mutation in orbital cavernous venous malformations

Sample preparation

For the discovery cohort, frozen OCVM, cerebral cavernous malformation (CCM), and vertebral hemangioma (VH) samples and paired peripheral whole blood samples were obtained at the Department of Neurosurgery, The University of Tokyo (UT). For the validation cohort, OCVM samples, including both frozen and formalin-fixed paraffin-embedded samples from UT, which were independent of the discovery cohort, and the Department of Ophthalmology, Tokyo Medical University (TMU), were obtained. For the prospective study and magnetic-activated cell sorting, freshly resected tissue samples of two OCVM and one conjunctival capillary hemangioma were obtained at TMU. Commercially available DNA extraction kits (QIAamp DNA Micro Kit and QIAamp DNA formalin-fixed paraffin-embedded (FFPE) Tissue Kit, QIAGEN, Venlo, The Netherlands) were used to isolate genomic DNA from the tissues, according to the manufacture’s protocol. Genomic DNA from peripheral blood leukocytes was isolated at SRL Inc. using the DNA Extraction Kit (Talent, Italy). The study protocol was approved by the Human Genome, Gene Analysis Research Ethics Committee at UT (approval number, G10028) and the Ethics Committee at TMU (approval number, T2020-0051). This study was carried out in accordance with the Declaration of Helsinki of the World Medical Association and the principles set out in the Department of Health and Human Service Belmont Report.

Targeted deep sequencing

Targeted deep sequencing was performed using TruSeq V3 kits (Illumina, San Diego, CA). For library preparation, the Agilent SureSelect XT Focused Exome target enrichment kit (Agilent, Santa Clara, CA) was used with DNA from tissue or blood samples. Libraries were sequenced on an Illumina HiSeq2500 instrument with 100 bp paired-end sequencing, with a median coverage of 210 ± 82 × for vascular anomalies and 194 ± 61 × for blood samples. Sequencing reads were mapped to the reference genome (hg19) with the Burrows–Wheeler Aligner and then processed with GATK best practice for Somatic SNVs + Indels (default setting of FilterMutectCalls). The datasets supporting this study were submitted to Japanese Genotype–phenotype Archive (JGA) (accession number JGAS000325).

Sanger sequencing

Bidirectional Sanger DNA sequencing assays were performed to confirm candidate mutations. The sequence of the used primers are as follows: forward primer (CCGTGGTGGGTAAGATCTGG), reverse primer (GCCTGGTCATAGCAGACGTT). PCR was performed using the Veriti 200 thermal cycler (Applied Biosystems, Waltham, MA). KOD DNA Polymerase (TOYOBO Co., Ltd., Osaka, Japan) was used in accordance with the manufacturer’s instructions. Cycle sequencing was performed at FASMAC (Atsugi, Japan) with the ABI Genetic Analyzer 3130XL or ABI DNA Analyzer 3730xL (Applied Biosystems).

ddPCR

Detection of GJA4 c.121G > T (p.Gly41Cys) was performed on the QX200 Droplet Digital PCR system (Bio-Rad Laboratories, Inc., Hercules, CA) at UT. Data was analyzed using QuantaSoft v1.4 (Bio-Rad Laboratories). The sequence of the used primers and probes for ddPCR are as follows: forward primer (TTCCGCATCCTCATC), reverse primer (AGGCCTGGTCATAG), fluorescent wild-type probe (5′-HEX-CCTGGCCTGCGAGTC) and mutation allele probe (5′-FAM-CCTGGCCGGCGAGTC). The 20 μl reaction mix consisted of 10 μl of ddPCR Supermix for Probes (no dUTP) (Bio-Rad Laboratories), 1 μl of Custom ddPCR FAM assay (catalog. no.10031276, Bio-Rad Laboratories), 1 μl of Custom ddPCR HEX Assay (catalog. no.10031279, Bio-Rad Laboratories), genomic DNA (40 ng for magnetic-activated cell sorting [MACS]-sorted cells, frozen tissues, and blood samples, or 40 ng or more for FFPE tissue to obtain sufficient copies of the target from fragmented DNA), and DNase/RNase free water up to a total 20 μl. Cycling conditions for the reaction were 95 °C for 10 min, followed by 40 cycles of 94 °C for 30 s and 56 °C for 1 min, then 98 °C for 10 min, and finally a 4 °C hold on a Bio-Rad S1000 Thermal Cycler. As positive control DNAs for each assay, we used pcDNA6/myc-His A GJA4 wild-type (WT) and—c.121G > T (p.Gly41Cys) plasmids. In addition, distilled water was genotyped with the study samples as non-template controls. ddPCR results of samples that did not have ≥ 500 droplets for GJA4 WT were considered unreliable and excluded. For larger OCVM samples, DNA isolation and ddPCR were performed for two or three replicates. The results of frozen tissue DNA or FFPE tissue DNA with the highest number of GJA4 WT droplets in the absence of frozen tissue were selected as definitive mutation allele frequency (MAF). We set a minimum of 0.5% fractional abundance to call a sample positive (similar to [16]).

Magnetic-activated cell sorting (MACS)

Surgically resected fresh tissues of two OCVMs and one conjunctival capillary hemangioma were cut into pieces and incubated with Collagenase/Hyaluronidase (STEMCELL technologies, Vancouver, Canada) for 30 min at 37 °C with regular agitation; then, vascular endothelial cells were isolated using the CD31 MicroBead Kit, human (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s recommendations. From CD31-positive and CD31-negative cell fractions, we extracted genomic DNA using the QIAamp DNA Micro Kit.

Immunohistochemistry

FFPE samples were embedded in paraffin after fixation with 10% formaldehyde. 4 μm slices were cut from FFPE samples, mount on coated slides, and dried for 3 h at 42 °C. After deparaffinization, rehydration, and wash, antigen activation was performed by heating in boiled citrate buffer solution (pH 6.0). Samples were incubated for 1 h at 20 °C in a 4% Donkey Serum/1% BSA/Tris-buffered saline with Tween 20 (TBS-T) solution followed by 4 °C overnight incubation with primary antibodies in 2% Donkey Serum/TBS-T. After five times washes with TBS-T, samples were exposed to secondary antibodies for 1 h at 20 °C. For GJA4 staining, samples were incubated for 1 h in biotinylated antibody, followed by incubation in streptavidin secondary antibody in 2% Donkey serum/TBS-T for 1 h at 20 °C. Three more washes with TBS-T were followed by incubation with the TOTO®-3 nuclear stain. Images were recorded on the ZEISS Axio Imager M1 (Carl Zeiss, Oberkochen, Germany). Following antibodies were used: GJA4 (Abcam ab181701, 1:500), CD31 (R&D Systems AF3628, 1:200), αSMA (Abcam ab21027, 1:200), Ki-67 (Dako, M7240, 1:200), Donkey anti-Rabbit IgG, biotin-SP (Merck Millipore AP182B, 1:500), Streptavidin, Alexa Fluor 488 (Thermo Fisher Scientific S11223, 1:200), Donkey anti-Goat IgG, Alexa Fluor 546 (Invitrogen A-11056, 1:200), TOTO®-3 (Thermo Fisher Scientific T3604, 1:500).

Preparation of connexin complementary RNA (cRNA) and injection into xenopus oocytes

Whole-cell voltage clamp recording with Xenopus oocytes were performed as described [17]. The experiments were approved by the Animal Experiments Committee and Gene Modification Experiments Safety Committee at Osaka University (approval numbers FBS-14-002-1 and 04294). Complementary DNAs (cDNAs) encoding GJA4 WT and GJA4 c.121G > T (p.Gly41Cys) were amplified using PCR and cloned into pGEM-HeFx plasmids. The plasmids were linearized using restriction enzymes and then used as a template for in vitro synthesis of cRNA (mMESSAGE mMACHINE T7 Transcription Kit, Invitrogen, Waltham, MA) according to the manufacturer’s protocol. Oocytes were collected from Xenopus laevis. An adult Xenopus female was anesthetized with ethyl-3-aminobenzoate methanesulfonate, and the ovarian lobes were collected using surgical knife and forceps. The eggs were treated with collagenase solution (20 mg/ml collagenase I (Sigma-Aldrich, Burlington, VT) and 20 mg/ml hyaluronidase (Sigma-Aldrich) in OR2 buffer (82.5 mM NaCl, 2 mM KCl, 1 mM MgCl2, and 5 mM HEPES [pH 7.5, adjusted with NaOH]) at 18 °C for 2 h. Stage V and VI oocytes were collected manually and used for cRNA injection. Then 1 ng of GJA4 WT or GJA4- c.121G > T (p.Gly41Cys) cRNA was injected with 10 ng of antisense oligonucleotide DNA for Xenopus cx38 into Xenopus oocytes. Water was co-injected with the antisense oligonucleotide as a negative control. Oocytes injected with cRNA were incubated at 18 °C overnight in ND96 buffer (93.5 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 2 mM MgCl2, and 5 mM HEPES; adjusted to pH 7.5 using NaOH). Then, single oocytes were used for hemichannel current recording. For recording transjunctional currents, the vitelline membrane was removed manually using forceps in a hypertonic solution (200 mM aspartic acid, 1 mM MgCl2, 10 mM EGTA, 20 mM KCl, and 10 mM HEPES [pH 7.5]) and the oocytes were manually paired with the vegetal poles together prior to incubation.

Hemichannel current recording

Hemichannel current was measured using the whole-cell voltage clamp technique. Current and voltage electrodes were prepared with a micropipette puller P-1000 (Sutter Instrument, Novato, CA) to obtain a resistance of 0.5–1.0 MΩ. The pipette was filled with solution containing 3 M KCl, 10 mM EGTA, and 10 mM HEPES (pH 7.4). Voltage clamp experiments were performed using the iTEV90 multielectrode clamp amplifier (HEKA, Reutlingen, Germany). To obtain the hemichannel current, the cells were initially clamped at − 40 mV and then subjected to 2 s voltage steps from − 30 to + 50 mV in 10 mV increments.

Transjunctional (gap junction) current recording

Transjunctional currents were measured using the dual whole-cell voltage clamp technique with two iTEV90 multielectrode clamp amplifiers. To measure transjunctional current, both cells were initially clamped at − 40 mV. One cell was then subjected to 3 s voltage steps from − 140 to + 60 mV in 10 mV increments. Currents detected in the second oocyte were recorded, and junctional conductance was calculated using the current value at the end of the steady state. Conductance was obtained using the equation, Gj = Ij/(V1-V0), where Ij is the current value of the second oocyte. V0 and V1 are the voltages of the first and second oocyte, whose currents were obtained as Ij at each voltage step, Vj = V1−V0. The obtained Gj value was then normalized and plotted against the Vj values.

Cell culture

Human umbilical vein endothelial cells (HUVECs) were purchased from Takara Bio Inc. (Kusatsu, Japan). The cells were cultured in EGM-2 medium (Lonza, Basel, Switzerland) supplemented with EGM-2 BulletKit (Lonza), 100 U/ml penicillin, 100 μg/ml streptomycin (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), and 10 μM Rho-kinase inhibitor Y27632 (Selleck Chemicals, Houston, TX), which promotes the proliferation of endothelial cells [18].

Retrovirus production

The experiments were approved by the Committee on Genetically Modified Organisms at UT (approval number 37-5). Full-length GJA4 WT, GJA4 c.121G > T (p.Gly41Cys), GJA4 WT-FLAG (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys), or GJA4 c.121G > T (p.Gly41Cys)-FLAG were cloned into pMXs vector. GFPNLS was also cloned into pMXs vector for vector control. pMXs vectors were then co-transfected with packaging plasmids (pCMV-gag-pol-PA and pCMV-VSVg) into 293AAV cells (Invitrogen) using Lipofectamine 2000 (Invitrogen). Retroviral supernatants were collected 48 h after transfection.

Retroviral transduction

For retroviral transduction, HUVECs were seeded at 8,000 cells/cm2. The next day, retroviral supernatant of human GJA4 WT, GJA4 c.121G > T (p.Gly41Cys), GJA4 WT-FLAG, GJA4 c.121G > T (p.Gly41Cys)-FLAG or human GFPNLS (vector control) was obtained and mixed with complete EGM-2 medium in the presence of 4 μg/ml polybrene (Nacalai Tesque Inc., Kyoto, Japan). The medium was changed on days 1, 2, and 4.

RNA extraction, reverse transcription, and quantitative PCR (qPCR)

Total RNA was extracted with the Quick-RNA Miniprep Plus Kit (ZYMO RESEARCH, Irvine, CA) and reverse transcribed with PrimeScript RT Master Mix (Takara Bio). qPCR was performed using the THUNDERBIRD SYBR qPCR Mix (TOYOBO) and the StepOnePlus Real-Time PCR System (Applied Biosystems). The sequence of the primers used are as follows: forward primer (CCGTGGTGGGTAAGATCTGG), reverse primer (GCCTGGTCATAGCAGACGTT).

Cell biology experiments

For the assessment of cell shape, cells were observed and imaged on day 5. For the assessment of cellular viability, the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay was performed with the MTT Cell Proliferation Assay Kit (Cayman Chemical), according to the manufacturer’s instructions using Synergy LX (BioTek Instruments, Inc, Winooski, VT) on day 4. For assessing the ability to form a normal capillary network, we performed the tube formation assay. On day 4, 96-well culture plates were coated with 50 μl/well Matrigel (Corning) and incubated for 30 min at 37 °C. After harvesting HUVECs cultured on 6-well plates, 20,000 cells were seeded on coated plates in EGM-2 medium and cultured in a CO2 incubator. Thirty minutes before the end of the incubation period, cells were treated with 2 μg/ml calcein AM (FUJIFILM Wako Pure Chemical Corporation) and incubated at 37 °C, 5% (v/v) CO2. Tube formation observed at the 6 h time point was imaged. The degree of tube formation was assessed by measuring total mesh area and the number of master junctions using ImageJ (NIH). For experiments assessing the ability of CBX to rescue the phenotypes of HUVECs overexpressing GJA4 p.Gly41Cys, HUVECs were treated with 20 μM CBX (in EGM-2 media) from day 1 to the start of each assay (the start of the MTT assay and the time of seeding on Matrigel for the tube formation assay). Images were taken on an Olympus IX73 inverted fluorescence and bright field microscope using the cellSens software.

Immunocytochemistry

For immunocytochemistry, cells were fixed using 4% paraformaldehyde, permeabilized using 0.5% Triton-X100, and blocked using 5% normal goat serum. Then, the cells were incubated with the FLAG antibody (FUJIFILM Wako Pure Chemical Corporation 014–22383 1:500) overnight at 4 °C, washed three times, and incubated with goat anti-mouse IgG, Alexa Fluor Plus 555 secondary antibody (Invitrogen A32727, 1:200), and DAPI (Dojindo D523, Kumamoto, Japan, 1:1,000) for 1 h at 20 °C. After further washing, Phosphate-buffered saline (PBS) was added to cells, and images were taken on the Olympus IX73 inverted fluorescence and bright field microscope using the cellSens software.

Statistical analyses

Comparisons between two groups were performed with a two-tailed t test. P value of < 0.05 was considered significant. Analyses were performed using JMP Pro version 15.0.0 (SAS Institute, Inc., Cary, NC).

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