In February 2007, the Centers for Disease Control and Prevention (CDC) and the National Prion Disease Pathology Surveillance Center (NPDPSC) notified the Texas Department of State Health Services (DSHS) of a 32-year-old woman with an 18-month history of progressive neurological symptoms suggestive of CJD. (Table 1) Based on the medical record and her neurologist, her illness began in August 2005 with attention deficits and progressive memory loss. In June 2006, she demonstrated anisocoria and bizarre behavior, including talking incoherently to herself, and she was then referred to psychiatry. On a mini-mental state examination, she scored abnormally low in the measure of attention and calculation and she had reduced ability to repeat the names of three unrelated objects 14. Later in 2006 she was described as being in constant motion, having unfocused hand gestures, and continued difficulty with ambulation. She was reported as alert, but confused, sad, and having difficulty with her thought process. Physicians caring for the case patient discussed the possibility of several diagnoses such as viral encephalopathy, paranoid schizophrenia, and subacute sclerosing panencephalitis, yet the overall etiology remained unclear. By February 2007, the patient was unable to ambulate and became bed-bound. She continued to demonstrate bizarre behavior, inability to follow commands, and unintelligible speech. The patient expired in June 2007, 22 months after the onset of illness.

Over the course of her illness, she had EEGs, magnetic resonance imaging (MRI) studies, and cerebrospinal fluid (CSF) tests. The EEG study performed in July 2006 showed generalized slowing with bilateral periodic lateralized epileptiform discharges. A second EEG performed two to three weeks later was unsuccessful due to excessive movements of the patient. In April 2006, an MRI study was negative for intracranial abnormalities. Another MRI study was completed in February 2007 and it showed supratentorial parenchymal atrophy with no other acute intracranial findings. CSF studies performed in March 2007 were normal, including the amount of the 14-3-3 protein determined.

Because of the age of the patient and the potential for variant or iatrogenic CJD, in July 2007 an investigator from the DSHS (KMM) interviewed a family member to obtain additional information about the patient's travel history, past medical history, and the symptoms of the present illness. The patient had a history of travel outside the continental United States to Puerto Rico during 1995-96 where she had lived approximately one year. Her surgical history included two back surgeries for internal disc disruption and degenerative disc disease. An anterior lumbar discectomy with interbody fusion at L4-5 was performed in November 2000 utilizing cadaver donated bone and in August 2001 another fusion was performed at L5-S1 utilizing autologous bone. The donor of cadaver bone was pre-screened minimizing the possibility of iatrogenic transmission. There was no familial history of progressive neurological disease or dementia-like illness. The family member also confirmed the clinical history including the onset in August 2005 of progressive memory loss and, in February 2006, bizarre behavior that included the patient's sitting in a chair for hours making noises that progressively got louder.

Following preliminary autopsy results, the NPDPSC requested the DSHS re-interview the family to ask specifically about the patient's pattern of sleep. When questioned about insomnia, the family member recalled that the patient had experienced disturbed sleep at the time of her disease onset. The family member also reported that the patient's sleep pattern progressively deteriorated throughout her illness. Some nights, for example, the patient did not sleep. On other nights when she did appear to be sleeping, her sleep was intermittent. During nights that the patient did not sleep, she would roam the house at all hours, unable to calm down. By August of 2006, four hours was the maximum amount of sleep the patient would get in one stretch and at times she would go two to three days without sleep. Medications were prescribed to help her sleep but they were not beneficial.

Sequencing of the PrP gene open reading frame revealed methionine homozygosity at codon 129, with no pathogenic mutation.

The mediodorsal and pulvinar thalamic nuclei along with the inferior olives showed severe neuronal loss and astrogliosis but no spongiform degeneration (SD) (Figure 1A-D and Figure 2). Astrogliosis with possible neuronal loss and superficial non-specific spongiosis affected particularly the frontal cortices while typical fine SD was present in other cortical regions, including parietal and temporal cortex (Figure 1E and 1F). With the exception of the presence of some SD in the molecular layer of the hippocampal formation, the hippocampus, basal ganglia, and cerebellum were much less affected than the cerebral cortex. Torpedoes, fusiform swellings of the Purkinje cell axons, were detectable in the granule cell layer of the cerebellum. Except in the hippocampus, where only the molecular layer was even weakly stained, immunohistochemical evaluation for the prion protein (PrP) in the cerebral cortex demonstrated intense staining in a predominantly 'synaptic' pattern with occasional small clusters of coarse granules (Figure 1G). Meanwhile, the basal ganglia, thalamus, and cerebellum were just faintly stained.

High resolution and standard Western blot analyses of the abnormal and Protease K (PK)-resistant PrP (PrP^res) from 19 brain regions invariably disclosed the presence of PrP^res type 2, which, however, varied in amount according to the location (Figure 3A). Of the two main types of unglycosylated misfolded prion protein fractions used as biochemical surrogate markers in prion diseases, PrP^res type 2 protein is slightly lighter than the PrP^res type 1 as demonstrated by their electrophoretic mobility on Western blots. The highest concentration of PrP^res was observed in most cerebral cortical regions such as frontal, temporal (including entorhinal), and occipital cortices, but it was minimal in the hippocampus. Variations in amount were also detected within the same cortical region (i.e. superior, middle, and inferior frontal gyri: data not shown). Minimal amounts of PrP^res were demonstrated in the caudate nucleus and in the three thalamic nuclei examined: anterior ventral, mediodorsal, and pulvinar (Figure 3A). No PrP^res was detected in the cerebellum (Figure 3A). Detectability of PrP^res in the thalamic nuclei was enhanced either by increasing the concentration of the antibody 3F4 ten-fold or by precipitating PrP^res with sodium phosphotungstate. (Figure 3B and data not shown). The ratios of the three PrP^res glycoforms, (diglycosylated, monoglycosylated, and unglycosylated) in the cerebral cortex and in the pulvinar, the only thalamic nucleus where PrP^res could be assessed accurately, were 15:42:43 and 28:38:34 respectively.

Proteinase K (PK), sodium phosphotungstic acid (NaPTA), N-Lauroylsarcosine sodium salt (sarkosyl), and phenylmethylsulfonyl fluoride (PMSF) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Benzonase nuclease was purchased from Novagen (Gibbstown, NJ, USA). Reagents for enhanced chemiluminescence (ECL plus) and the horseradish peroxidase-conjugated antibody were produced by Amersham Biosciences (Piscataway, NJ, USA). The 3F4 monoclonal antibody (mAb) was used against PrP residues 106-110 24.

Human brain tissues were obtained at autopsy and stored at -80°C. Samples were taken from 19 different brain regions: superior, middle, and inferior gyri of the frontal and temporal cortices, the middle gyrus of the parietal cortex, visual and non-visual occipital cortices, entorhinal and hippocampal cortices, basal ganglia (caudate nucleus, putamen, globus pallidus), substantia nigra, thalamus (anterior ventral, mediodorsal and pulvinar nuclei), and cerebellum (hemispheres).

DNA was extracted from frozen brain tissues in all the cases, and genotypic analysis of PRNP coding region was performed as described 25.

Histopathology and PrP immunohistochemistry were performed as described 26. The sections were deparaffinized, rehydrated, and immersed in TBS-T. Endogenous peroxidase was blocked by Envision Flex Peroxidase Blocking Reagent (Dako) for ten minutes and washed. For 3F4 immunostaining only, sections were completely immersed in 1.5 mmol/L hydrochloric acid and microwaved for fifteen minutes. The slides were either incubated with GFAP 1:12000 (Sigma-Aldrich, St. Louis, MO) or 3F4 1:750 for one hour, washed, and incubated with Envision Flex/HRP polymer for 30 minutes (Dako). Envision Flex DAB (Dako) was used to visualize the immunoreactivity.

Brain homogenates (10% w/v) were prepared in lysis buffer with 100 mM TRIS-HCl (100 mM NaCl, 10 mM EDTA, 0.5% NP-40, 0.5% sodium deoxycholate, 100 mM Tris-HCl, pH 8.0) and centrifuged at 1000 × g for five minutes to collect the supernatant (S1). Homogenates were incubated with 5 Unit/ml (U/ml) PK [48 Units/mg specific activity at 37°C, with 1 U/ml equal to 20.8 μg/ml PK] at 37°C for one hour, and then stopped by the addition of 2 mM PMSF. Samples were mixed in an equal volume of 2 × sample buffer (6% SDS, 5% β-mercaptoethanol, 20% glycerol, 4 mM EDTA, 125 mM Tris-HCl, pH 6.8) and boiled for ten minutes.

Precipitation of PrP aggregates by NaPTA was conducted as described with minor modification 27. Briefly, 10% (w/v) homogenates from brain were prepared in PBS lacking Ca²⁺ and Mg²⁺. The samples were centrifuged at 1000 × g for 10 minutes at 4°C and 0.5 ml of supernatant was then mixed with an equal volume of 4% (w/v) sarkosyl prepared in PBS, pH 7.4, and incubated for 10 minutes at 37°C. Each sample was adjusted to final concentrations of 50 U/ml benzonase and 1 mmol/L MgCl₂ and incubated for 30 minutes at 37°C. Aliquots were adjusted with 81.3 μl of a stock solution containing 4% (w/v) NaPTA and 170 mmol/L MgCl₂ at a final concentration of 0.3% (w/v) NaPTA. Samples were incubated at 37°C for 30 minutes before centrifugation at 16,000 × g for 30 minutes. After isolation of the supernatant, the pellet was resuspended in 0.1% sarkosyl prepared in PBS, pH 7.4 for Western blotting.

Proteins were separated by both non-commercial, home-made 15% Tris-HCl, 20 cm-long SDS-PAGE gels and 15% Tris-HCl Criterion precast gels (Bio-Rad, Hercules, CA). Proteins were then transferred to PVDF membrane (Immobilon-P; Millipore) for two hours at 60 V. The 3F4 antibody was incubated for two hours at room temperature (1:40,000 and 1:4,000). After incubation with horseradish peroxidase-conjugated sheep anti-mouse IgG at 1:3000, the PrP bands were visualized on Kodak film (Eastman- Kodak, Rochester, NY) by the ECL Plus (GE Healthcare, Fairfield, CT) as described by the manufacturer. Densitometric analysis was performed with UN-SCAN-IT gel 5.1.