The proband had a normal childhood with good school performance. At age 13 years, in conjunction with a fever and flu-like symptoms, he experienced a generalized seizure lasting four minutes upon awakening from sleep, followed by a second seizure on awakening eleven days later. An EEG revealed bilateral slow waves and generalized spike activity which became especially marked with photic stimulation, and he was treated with diphenylhydantoin, then phenobarbital, which caused excessive drowsiness and finally primidone. The patient remained well until a sledding accident at age 14, which resulted in a ruptured spleen, requiring an emergency splenectomy. In the succeeding months, generalized grand mal seizures became increasingly frequent, and he began having myoclonic jerks and staring spells. Multiple hospital admissions followed for almost continuous seizure activity and myoclonus, which became intractable, in spite of multiple drug regimes. Serum copper and ceruloplasmin were normal. A brain CT scan at age 17 was normal. By the age of 18 he was functioning well below his chronological age intellectually, with poor memory, poor fund of knowledge and slow mentation. He was dysarthric with slow hand movements, upper extremity myoclonus and staring spells. The EEG revealed bilateral slowing and spike activity. An electroretinogram, technetium brain scan and lumbar puncture were normal.
A work-up at age 19 revealed deficient GCase and Gaucher cells were found in the bone marrow and liver, as well as the previously removed spleen. His renal function and hearing were normal. The patient underwent a series of plasmapheresis treatments to reduce blood glucocerebroside accumulation, which decreased the frequency of generalized seizures, but the myoclonic and staring spells continued. An evaluation at age 20 revealed impaired memory, difficulty naming common objects and a poor fund of knowledge. Visual pursuit was very slow. There was asterixis of the upper extremities and very frequent myoclonic movements, more severe in the arms than legs. Muscle tone and strength were normal, and deep tendon reflexes were absent at the ankles, but preserved elsewhere. He was clumsy and dysmetric. By age 21, he became wheelchair-bound. Intravenous apomorphine dramatically reduced his myoclonus and photoconvulsive responses. He was continued on Sinemet for 10 months with improvement in physical activity. He was then switched to bromocriptine but this was discontinued at age 25 because of adverse behavioral effects and increasing unresponsiveness to the drug. Lithium was tried for six months but was also ineffective.
On examination at age 26 years, there was perseveration and interruption of speech by myoclonic jerking, and he was transiently inattentive during flurries of myoclonic seizures. There was marked impairment of horizontal saccadic eye movements, and muscle tone was mildly increased. A vitamin B12 level was normal. He was treated for a urinary tract infection. Urinalyses done on prior and subsequent admissions were negative for protein. The following year a gastrostomy was performed because the patient refused to eat and was losing weight. Prior to his death at age 28, he was diagnosed with bacterial endocarditis. A full autopsy was performed demonstrating hepatomegaly, accessory splenic tissue, osteoporosis and Gaucher cells in the bone marrow and lymph nodes. Neuropathology revealed meningeal fibrosis, with mild focal gliosis of the left cingulate gyrus and anterior basal ganglia and degeneration of the lateral columns of the spinal cord, but no Gaucher cells. His kidneys were unremarkable.
Following his diagnosis, GCase levels were measured on his parents and three siblings. His brother was also deficient in GCase activity and carried the identical GBA genotype. A thorough examination at age 26 was normal, and a technetium liver-spleen scan showed the spleen to be at the upper limit of normal. He has remained healthy without palpable hepatosplenomegaly, bone pain, episodes of bleeding, seizures or cognitive impairment. Now 60 years of age, he has his own business and has worked in physically demanding occupations. He has not received enzyme replacement therapy.
The proband’s mother, a carrier now in her 80’s, also has no cognitive or neurologic impairment. Her GBA genotype was c.535G>C (p.Asp140His) + c.10936G>A (p.Glu326Lys)/WT.
Lymphocytes, lymphoblasts or fibroblasts were collected from the siblings, their mother, 13 other patients with GD and myoclonic epilepsy and 40 controls under NIH Institute Review Board approved clinical protocols.
RNA was isolated from fibroblasts from the siblings, patients with GD and normal controls using the Qiagen RNeasy Mini Kit (QIAGEN, Valencia, CA) and cDNA was generated using the High Capacity RNA-to-cDNA Mastermix (Applied Biosystems, Forest City, CA). SCARB2 cDNA was synthesized using the following two primer sets: exon 1-1F: 5’-GTCTTCGACGCCTCTGCGGC-3’; exon 12-1R: 5’-CAACTCATGGGTATTGCC-3’and exon 12-8F: 5’-GGTAGCTTCATCCAATATATC-3’; exon 12-2R 5’-GTGAACCAACTGTATAAGCTAC-3’, and sequenced on an Applied Biosystems 3130Xl Sequencing Analyzer using the primers listed in Supp. Table S1A
. Genomic sequencing of SCARB2
was performed on genomic DNA from patients and controls using the primers in Supp. Table S1B
. The Gaucher genotypes were established by sequencing GBA
, as previously described [Stone et al., 2000
]. All mutations are described using the nomenclature specified at www.hgvs.org/mutnomen
cDNA nucleotides are numbered designating the adenine of the first ATG translation initiation codon as nucleotide 1 (GenBank reference sequence NM_000157.2). Amino acid designations are based on the primary GBA
translation product, including the 39-residue signal peptide. For SCARB2
, coding DNA nucleotides are numbered based on the adenine of the first ATG translation initiation codon as nucleotide 1 (GenBank reference sequence NM 005506.3). The reference sequence NP 005497.1 was used for amino acid numbering.
Plasmids, site-directed mutagenesis and transfection
A pCMV6-Neo vector expressing wild type LIMP-2 protein was purchased from Origene Inc. (Rockville, MD). The LIMP-2 mutation c.1412A>G was generated by PCR-based site-directed mutagenesis using the Quickchange mutagenesis kit according to the manufacturer’s guidelines (Stratagene, La Jolla, CA). Fibroblasts from the proband’s sibling (P2) were stably transfected with the mutated and wild type SCARB2 using FuGENE 6 according to the guidelines of the manufacturer (Roche, Indianapolis, IN), and were selected with Neomycin (300ng/ml) for 3 weeks. Selected colonies were grown and utilized for enzyme assays and Western blot analyses.
Quantitative real time PCR analysis
RNA expression was quantified using the StepOnePlus Real-Time PCR System (Applied Biosystems) in a 20ul volume containing 10 ul of TaqMan Universal PCR master mix, 1 ul of SCARB2 or GBA probe (with primer), 1 ul β-Actin probe and primer and 25ng cDNA. One cycle at 50°C for 2 min and one at 95°C for 10 min were followed by 40 cycles at 95°C for 15 sec and 60°C for 1 min. Three replicates of each sample were assayed.
GCase activity assay
GCase activity was assayed on fibroblasts from the siblings, mother and controls using 4-methylumbelliferyl-β-D-glucopyranoside substrate (Sigma, St. Louis, MO) as previously described [Raghavan et al., 1980
]. Three replicates of each sample were performed.
SDS-PAGE and Western blotting
Fibroblasts, grown in monolayers to 90% confluence, were harvested and sonicated at 4°C in lysate buffer (60 mM KH2PO4, 0.1% Triton-X and protease inhibitor, pH 5.9). After quantification with BCA (Thermo scientific, Rockford, IL) the lysate was separated by SDS-PAGE, and transferred to iBlot PVDF nitrocellulose membranes (Invitrogen, Carlsbad CA). Blots were blocked in phosphate-buffered saline (PBS) containing 0.1% Tween-20 (Sigma) and 5% fat-free milk for 1 h at room temperature. The blocked membrane was incubated in blocking buffer containing primary antibody LIMP–2, 1:1000, (Santa Cruz Biologics, Santa Cruz, CA,) and glucocerebrosidase 1:15000,(custom-made antibody, R386) overnight at 4 °C, followed by three 10 min washes, and was incubated in blocking buffer containing horseradish peroxidase (HRP)-conjugated secondary antibody 1:3000 (KPL, Gaithersburg, MD) for 1 h at room temperature. HRP immunoblots were developed using enhanced chemiluminescence (ECL Plus, GE Healthcare).
Immunofluorescence staining and confocal microscopy
Fibroblasts were grown in Lab-Tek 4 chamber slides (Fisher Scientific, Pittsburgh, PA) to 60% confluency, fixed in 3% paraformaldehyde, permeablized with 0.1 % Triton-X for 10 min and blocked in PBS containing 0.1% saponin, 100 µM glycine, 0.1% BSA and 2% donkey serum. They were incubated with mouse monoclonal anti-LAMP-2 (1:100)
(Developmental Studies Hybridoma bank, University of Iowa), rabbit polyclonal anti-GCase antibody R386 (1:500), or mouse monoclonal calnexin (1:100, Abcam) for 2 h at room temperature. The cells were washed, and incubated with secondary donkey anti-mouse or anti-rabbit antibodies conjugated to ALEXA-488 or ALEXA-555 (Invitrogen), washed again, and mounted in VectaShield with DAPI (Vector Laboratories, Burlingame, CA). Cells were imaged with a Zeiss LSM510 META confocal laser-scanning microscope (Carl Zeiss, Microimaging Inc, Germany) using an Argon (458, 477, 488, 514 nm) 30 mW laser, a HeNe (543 nm) 1 mW laser, and a laser diode (405 nm). Images were acquired using a Plan Apochromat 63X/1.4 oil DIC objective. All the images were taken at the same laser setting. Zeiss LSM510 META quantification software was used to analyze the images. Briefly, the co-localization coefficients of a total of 16 cells from 6 different fields were calculated. The mean and SD of these values are represented in . The experiment was performed three independent times.
Figure 2 Localization of GCase in patient and control fibroblasts. A) GCase (red) and LAMP-2 (green) stain in cells from a control (WT). B) The non-affected sibling P2 and C) the affected proband P1. D) Relative colocalization of GCase and LAMP-2 based on the (more ...)
Analysis of extra-cellular secretion
Approximately the same quantity of fibroblasts from the siblings and controls were seeded in T75 culture flasks. The next day, media was replaced with an equal volume of serum free OPTI-MEM medium. After 24 hours, the conditioned medium (CM) was passed through a 0.22 uM filter and concentrated at 4000 rpm at 4 C for 45 min in iCON concentrators (9K pore size) (Pierce) to a volume of 250 uL. 30 ul of lysate from the CM was loaded on a 4–12% bis-tris acrylamide gel, blotted and probed with antibody to GCase and LIMP-2. To analyze different glycosylated forms of GCase, the medium was treated with N-Glycanase, Glyko PNGase F (GKE-5003, Prozyme, CA) for 6 h at 37°C.Treated and non-treated samples were analyzed by SDS-PAGE.