Human subjects
Families were recruited through the Reproductive Medicine Unit at KFSHRC based on the observation of abnormal embryo development during regular in vitro fertilization treatment of consanguineous couples. Eligible families and controls were enrolled after signing a KFSHRC IRB-approved written informed consent (RAC #2121053). Venous blood was collected in EDTA and, when possible, in Na-heparin tubes for DNA extraction and lymphoblastoid cell line establishment, respectively. All methods comply with the Helsinki Declaration.
Autozygome and linakge analysis
Determination of the entire set of autozygous intervals per genome (autozygome) was through genomewide SNP genotyping (Axiom SNP chip, Affymetrix) followed by mapping of runs of homozygosity as surrogates of autozygosity using AutoSNPa v4, as described before [10, 11]. Overlap in the autozygome of affected individuals was employed as a strategy to determine the critical disease locus. Statistical confirmation of the critical locus was achieved by linkage analysis using easyLINKAGE [12].
Whole exome sequencing
Exome capture was performed using TruSeq Exome Enrichment kit (Illumina) following the manufacturer’s protocol. Samples were prepared as an Illumina sequencing library, and in the second step, the sequencing libraries were enriched for the desired target using the Illumina Exome Enrichment protocol. The captured libraries were sequenced using Illumina HiSeq 2000 Sequencer. The reads are mapped against UCSC hg19 [13] by BWA ver.0.5.9rc1 [14], without unordered sequences and alternate haplotypes. The Picard-tools suite (ver.1.59) was then utilized to sort by mapping coordinates, and BEDtools (ver. 2.15.0) filtered out any reads not present in the targeted exonic regions. SNPs and Indels were detected by SAMTOOLS ver.0.1.18 [15] and annotated using ANNOVAR ver.Nov 2011 [16]. The candidacy of the resulting variants was based on their physical location within the autozygome of the affected individual, their population frequency and predicted effect on the protein as described before [9]. Data used in this paper come from a small and well-defined family. To protect the identity of individuals, these confidential data are not publicly available.
Western blot and phosphorylation analysis
Epstein Barr Virus (EBV) transformed cell lines were produced from three healthy donors (controls) and from three individuals who are homozygous for the TLE6 S510Y mutation (patients, see below). Western blot analysis was performed as described [17]. Briefly, cells were harvested by centrifugation and resuspended in lysis buffer (20 mM Tris pH 7.5, 350 mM NaCl, 0.05 % β-mercaptoethanol) supplemented with a protease inhibitor cocktail. After sonication and centrifugation, 30 μg of total cell lysates were analyzed by SDS-PAGE on 10 % acrylamide or on 8 % Phospho-tag acrylamide gels (Wako, TX, USA), followed by transfer of the proteins onto nitrocellulose membrane. After blocking in 5 % milk in TBS-Tween, the membranes were incubated with anti-TLE6, anti-KDHC3L/Ecat1 from (Abcam, Cambridge, MA, USA) or anti-OOEP, anti-Flag and anti-GAPDH from (Santa Cruz, CA, USA). After washing, secondary reactions were carried out with biotin conjugated secondary antibodies followed by anti-avidin-HRP conjugated antibody. Signals were visualized using an LAS 4000 mini (GE Healthcare, UK) and quantified using ImageQuant software (GE Healthcare, UK).
Phosphatase inhibitor treatment
A total of 30 μg of whole cell lysates from the two control individuals were incubated with 40 μM of calf intestine alkaline phosphatase (CIP) (Promega, Madison, WI, USA) and equimolar amount of PKI (5–24), PKA Inhibitor (Santa Cruz, CA, USA) in a reaction buffer (50 mM Tris–HCl, pH 9.3; 1 mM MgCl2; 0.1 mM ZnCl2; 1 mM spermidine). Extracts were then analyzed by western blot analysis as described before [17].
Production of TLE6 S510Y protein
Mutant TLE6 S510Y was produced using the Stratagene site-directed mutagenesis system (Stratagene, La Jolla, CA, USA) according to manufacturer’s instructions; using Flag tagged TLE6 plasmid (Origene, Rockville, MD, USA). Primers used to generate the mutation are sense: 5′-TCCTGAGCGTCAAGTTCT(A)CCCCTTTGGCCAGTGGTG-3′ and anti-sense: 5′-AGGACTCGCAGTTCAAGATGGGGAAACCGGTCACCAC-3. The base change was from C to A, shown between brackets. After verification of the mutation by sequencing, control transformed lymphocytes were transfected with wild-type and mutant plasmids using lipofectamine (Invitrogen, Waltham, MA, USA). Twenty-four hours post transfection, total cell lysates were prepared and analyzed by western blotting or immunoprecipitation.
Immunoprecipitation
Cell extracts were re-suspended in a buffer containing 50 mM Tris pH7.6, 150 mM or 500 mM NaCl, 1 mM EDTA, 1%Triton X-100, 1 mM PMSF supplemented with phosphatase and protease inhibitor cocktail (Sigma). A total of 60 μg of total protein was incubated with Flag-IgG sepharose beads (Sigma-Aldrich, St. Louis, MO, USA) or Protein A dynabeads (Invitrogen, Waltham, MA, USA) coupled to anti-OOEP antibody (Santa Cruz, CA, USA) or control IgG in a pull-down buffer (50 mM HEPES pH 7.5, 1 mM EDTA, 150 mM NaCl, 10 % glycerol, 0.1 % Tween 20, 0.5 mM DTT, 1 mM PMSF, 2 μg/mL leupeptin, and 2 μg/mL pepstatin A). Extracts were incubated with the beads for at least 2 h at 4 °C while mixing on a rotating wheel. After collection of the supernatants, the beads were washed with pull-down buffer and left as a 50 % slurry after a final wash. After elution, proteins were loaded on 10 % SDS-PAGE gel and immunoblotting was performed using the indicated antibodies.
In vitro phosphorylation
Wild-type and TLE6-S510Y proteins were expressed in HEK293 and immunoprecipitated using anti-Flag beads (Sigma-Aldrich, St. Louis, MO, USA). A total of 20 μg of purified proteins was added to a standard PKA mixture containing 20 mM Hepes (pH 7.5), 5 mM MgCl2, 1 mM unlabeled ATP, 1 mM 1,4-dithiothreitol, 100 mM NaCl, and 1 mM [γ32P]ATP (0.5 Ci/mmol- Perkin Elmer, Waltham, MA, USA), with different amounts of PKA (Promega, Madison, WI, USA) as indicated and incubated for 1 h at 37 °C. Reactions were stopped by adding 20 μL of 2X Laemmli sample buffer and proteins were separated by SDS-PAGE electrophoresis followed by autoradiography and immunoblotting.