Patent Publication Number: US-7709213-B2

Title: Method for diagnosing a pervasive developmental disorder

Description:
BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     The present invention relates in general to a method for diagnosing a pervasive developmental disorder, for example, autism. 
     2. Background Art 
     Since the first clinical descriptions of autism in the 1940s, there has been no definite etiology or pathological finding leading to a cure or reversal of the condition. Recent reports suggest that there may be a combination of environmental or perhaps in utero risk factors, possible autoimmune risk factors, and perhaps localized inflammation of central nervous system neuroglia pointing to a compartmentalized central nervous system inflammatory response in at least some patients with autism. 
     Although no specific cell count elevation or direct central nervous system antibodies have been found, some recent studies of cytokines in the central nervous system may implicate an isolated response separate from the peripheral immune system. 
     These studies have not addressed direct neuroglial or innate central nervous system immune responses. The reports of elevated cytokines support possible increased tumor necrosis factor alpha (TNF-α) levels, in that interleukin-6 and elevated levels of TNF-receptor I antibodies have been reported in the cerebrospinal fluid of autistic patients. The role of TNF-α as a neuromodulating agent has been described in brain development as well, and it may play a role in neurons and neuroglial cells modulating glutaminergic transmission. Excessive glutamate excitotoxic effects acting on NMDA receptors could occur in the presence of excess TNF-α. This occurrence can lead to effects on microglial activation as well as other cytokines, such as nuclear factor-kappa-beta (NF-Kβ), among others. Such changes have also been seen in models of inflammation inducing epileptic activity in which neuroglial inflammation has caused epileptic spikes. Because of the high frequency of epileptic spikes seen in some subgroups of autistic children, and given that similar inflammatory mechanisms may play a role in spike formation, this finding may be more than a coincidence. 
     Vargas D L, et al. Neuroglial activation and neuroinflammation in the brain of patients with autism, Ann Neural 2005; 57:304 and Zimmerman A, et al. Cerebrospinal fluid and serum markers of inflammation in autism, Pediatr Neurol 2005; 35:195-201, disclose measuring cytokine profiles. However, neither article measures TNF-α, much less comparing the levels of TNF-α in cerebrospinal fluid and serum at the same time. 
     However, to the best of Applicant&#39;s knowledge, there are currently no known biological early diagnostic markers to identify at risk patients for a pervasive developmental disorder or to monitor treatment targets. 
     SUMMARY OF THE INVENTION 
     It is an object of the present invention to provide a method for positively diagnosing a pervasive developmental disorder (PDD), which may allow early diagnosis in infancy or the first 2 years of life, and may allow early medical treatment to possibly alter the course of a disease, for example, autism. 
     It is another object of the present invention to use a TNF-α ratio as a key marker for abnormal central nervous system cytokine inflammation causing autistic disease. 
     Yet another object of the present invention is directed to monitoring and designing therapy for PDDs by showing an improvement in an elevated, abnormal ratio to a more normal TNF-α ratio. 
     Another object of the present invention is directed to diagnosing, for example, some catastrophic epilepsies utilizing the same ratio analysis. 
     The above objects are satisfied by a method for diagnosing a pervasive developmental disorder, such as autism, comprising obtaining a sample of cerebrospinal fluid (CSF) from a subject; obtaining a sample of serum from a subject; testing the cerebrospinal fluid of the subject for a concentration of TNF-α; testing the serum of the subject for a concentration of TNF-α; and positively diagnosing a pervasive developmental disorder when the concentration ratio of TNF-α in the cerebrospinal fluid of the subject to TNF-α in the serum of the subject exceeds approximately 2:1 over normal control concentrations. 
     In at least one embodiment, obtaining samples of cerebrospinal fluid and serum from a subject occur substantially simultaneously. 
     In at least one embodiment, positively diagnosing a PDD occurs when the concentration ratio of TNF-α (CSF) to TNF-α (serum) of a subject exceeds approximately at least one of 5:1, 10:1, 20:1, 50:1, 100:1, 250:1, or 500:1 over normal control concentrations. 
     In at least one embodiment, obtaining samples of CSF and serum from a subject occurs in a subject less than at least one of 84, 72, 60, 48, 36, 24, or 12 months old. 
     It will be understood that regardless of its ordinary meaning the term “normal control concentrations” will be defined as concentrations that are within a reasonable range for a subject (e.g., human patient) without a pervasive developmental disorder. 
     As used herein “substantially”, “generally”, “relatively”, “approximately”, and “about” are relative modifiers intended to indicate permissible variation from the characteristic so modified. It is not intended to be limited to the absolute value or characteristic which it modifies but rather approaching or approximating such a physical or functional characteristic. 
     References to “one embodiment”, “an embodiment”, or “in embodiments” mean that the feature being referred to is included in at least one embodiment of the invention. Moreover, separate references to “one embodiment”, “an embodiment”, or “in embodiments” do not necessarily refer to the same embodiment; however, neither are such embodiments mutually exclusive, unless so stated, and except as will be readily apparent to those skilled in the art. Thus, the invention can include any variety of combinations and/or integrations of the embodiments described herein. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     In accordance with the present invention, a method for diagnosing a pervasive developmental disorder (PDD), such as autism, is provided which comprises obtaining a sample of cerebrospinal fluid from a subject; obtaining a sample of serum from a subject; testing the cerebrospinal fluid (CSF) of the subject for a concentration of tumor necrosis factor alpha (TNF-α); and testing the serum of the subject for a concentration of TNF-α. In the case of an adolescent, e.g., a child 12 years or younger, a pervasive developmental disorder is positively diagnosed when the concentration ratio of TNF-α in the cerebrospinal fluid to TNF-α in the serum exceeds approximately 2:1 over normal control concentrations. 
     It will be understood that obtaining a sample of CSF from a subject, obtaining a sample of serum from a subject, testing the CSF of the subject for a concentration of TNF-α, and testing the serum of the subject for a concentration of TNF-α, are procedures and tests known by medical professionals having ordinary skill in the art. 
     In at least one embodiment of the present invention, obtaining samples of CSF and serum from the subject occur substantially simultaneously, for example, the samples are generally drawn at the same time. 
     In at least one embodiment of the present invention, positively diagnosing a PDD occurs when the concentration ratio of TNF-α in the CSF of the subject to TNF-α in the serum of the subject exceeds at least one of approximately 2:1, 5:1, 10:1, 20:1, 50:1, 100:1, 250:1, or 500:1 over normal control concentrations. 
     In at least one embodiment of the present invention, obtaining samples of CSF and serum from the subject occurs in a subject less than at least one of 84, 72, 60, 48, 36, 24, or 12 months old. Such early diagnosis may allow for reversal or at least enhanced therapy relative to conventional methods. 
     It will be understood that the present invention may also be helpful in diagnosing some catastrophic epilepsies. 
     In at least one embodiment, the present invention may be useful as a way to monitor and design therapy for at least one condition by showing an improvement in an elevated abnormal TNF-α (CSF) to TNF-α (serum) ratio to a normal ratio. 
     According to the present invention, the TNF-α (CSF) to TNF-α (serum) ratios are significantly elevated in autistic patients compared with patients in other known disease states and with control groups in the medical literature. There is no apparent correlation in the elevation of TNF-α levels between the CSF and serum of autistic patients, indicating that the effects on the levels within the CSF and serum are independent of the other. 
     This finding contrasts with the correlation suggested from review of previous studies, where changes in one TNF-α level generally corresponded with changes in the other TNF-α level, thus suggesting systemic inflammation, not isolated in the central nervous system. The finding according to the present invention suggests that, even ignoring the numerical difference between the levels of TNF-α in CSF and serum, there is a mechanism possibly unique to autism in which changes of TNF-α levels in the CSF may be independent of changes in the serum. Such a mechanism suggests a unique and isolated CNS response. Whether this response is genetic or environmentally induced is unclear, and whether it is due to an error in neurodevelopment or to immune system response is also unknown. 
     According to the present invention, there is an observed trend suggesting the possibility that a child&#39;s ratio of TNF-α in CSF and serum is a potential marker for the pathological process involved in autistic regression. This marker may reflect neuroglial and immune mechanisms found in the central nervous system different from typical infectious, autoimmune, or traumatic mechanisms previously described in the literature (in predominantly adult populations). The TNF-α ratio offers unique and specific therapeutic targets for treatment. This value of relative elevation may allow a biological marker to identify children at risk, and also allow for design of disease-altering treatment through lowering of TNF-α levels. Theoretically, if introduced early, such treatment could alter the course of the disease development and could allow tracking clinically. TNF-α levels in the CSF may be an early marker, allowing clinical diagnosis of autism, even before obvious clinical features become evident. 
     EXAMPLES 
     Example 1 
     Table 1 shows data for TNF-α in CSF and TNF-α in serum of patients (children) with autism. Four of the patients had not been previously treated for autism. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 TNF-α levels in autistic patients (pg/mL) 
               
            
           
           
               
               
               
               
               
            
               
                 Patient 
                 Treated 
                 CSF 
                 Serum 
                 CSF-serum ratio 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 1 
                 No 
                 124 
                 1.6 
                 77.5 
               
               
                 2 
                 No 
                 140 
                 2.0 
                 70.0 
               
               
                 3 
                 No 
                 181 
                 8.2 
                 22.1 
               
               
                 4 
                 No 
                 16 
                 2.1 
                 7.6 
               
               
                 5 
                 Yes 
                 12 
                 1.7 
                 7.1 
               
               
                 6 
                 Yes 
                 13 
                 1.3 
                 10.0 
               
               
                 7 
                 Yes 
                 11 
                 1.3 
                 8.5 
               
               
                 8 
                 Yes 
                 4 
                 2.3 
                 1.7 
               
               
                 Averages 
                 Not Treated 
                 115.25 
                 3.47 
                 33.17 
               
               
                   
                 Treated 
                 10.00 
                 1.65 
                 6.06 
               
               
                   
                 Overall 
                 62.63 
                 2.56 
                 24.44 
               
               
                   
               
            
           
         
       
     
     TNF-α is elevated in the cerebrospinal fluid of children with autism and regression, even in cases previously treated with known immunosuppressive agents. The fact that these children showed consistently higher ratios of CSF to serum TNF-α suggests a compartmentalized local reaction in the central nervous system. This finding differs from previous literature as discussed below. 
     Based upon a review of other known conditions that affect the central nervous system, such as stroke, infection, multiple sclerosis, systemic lupus, traumatic brain injury, and human immunodeficiency virus or acquired immunodeficiency syndrome (HIV/AIDS) infection, the cerebrospinal fluid and serum levels of TNF-α were approximately 1:1 in both test and control groups, as shown in Tables 2-3. Of 37 reviewed articles addressing tumor necrosis factor in humans, 25 measured TNF-α levels in the cerebrospinal fluid or serum, and 6 of these studies allowed for direct comparison of the two between test groups and control groups, wherein N represents the number of patients. 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 TNF-α levels in other conditions (pg/mL) 
               
            
           
           
               
               
               
               
               
               
            
               
                 Authors 
                 Diagnosis 
                 CSF 
                 N 
                 Serum 
                 N 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 Attallah &amp; Ibrahim, 2004 
                 Meningitis 
                 123.3 
                 21 
                 88.6 
                 21 
               
               
                 Baraczka et al., 2004 
                 MS 
                 2.4 
                 50 
                 1.32 
                 50 
               
               
                 Baraczka et al., 2004 
                 Systemic Lupus 
                 1.8 
                 50 
                 2.63 
                 50 
               
               
                   
                 Erythernatosus 
               
               
                 Monno et al., 1999 
                 HIV 1   
                 6.0 
                 13 
                 22 
                 11 
               
               
                 Monno et al., 1999 
                 HIV 2   
                 11.3 
                 9 
                 50.1 
                 8 
               
               
                 Monno et al., 1999 
                 HIV 3   
                 17.8 
                 25 
                 38.3 
                 22 
               
               
                 Shiozaki et al., 2005 
                 Traumatic Brain 
                 16.6 
                 18 
                 5.1 
                 18 
               
               
                   
                 Injury 
               
               
                 Sjögren et al., 2004 
                 Dementia 
                 0.6 
                 15 
                 3.4 
                 19 
               
               
                 Vila et al., 2000 
                 Stroke 
                 22.8 
                 33 
                 21.1 
                 83 
               
            
           
           
               
               
               
            
               
                 Weighted means 
                 19.16 
                 19.33 
               
               
                   
               
               
                   1 w/ progressive multifocal leukoencephalopathy 
               
               
                   2 w/ HIV-1 leukoencephalopathy 
               
               
                   3 w/ opportunistic infections 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 TNF-α levels in control conditions (pg/mL) 
               
            
           
           
               
               
               
               
               
               
            
               
                 Authors 
                 Diagnosis 
                 CSF 
                 N 
                 Serum 
                 N 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 Attallah &amp; Ibrahim, 2004 
                 Control 
                 63.42 
                 19 
                 65.3 
                 19 
               
               
                 Baraczka et al., 2004 
                 Control 
                 0.42 
                 50 
                 1.66 
                 50 
               
               
                 Monno et al., 1999 
                 Control 
                 4.2 
                 8 
                 36 
                 11 
               
               
                 Shiozaki et al., 2005 
                 Control 
                 18.4 
                 17 
                 5 
                 17 
               
               
                 Sjögren et al., 2004 
                 Control 
                 0 
                 5 
                 normal 
                 — 
               
               
                 Vila et al., 2000 
                 Control 
                 11.1 
                 48 
                 15.1 
                 148  
               
            
           
           
               
               
               
            
               
                 Weighted Means 
                 14.18 
                 16.49 
               
               
                   
               
            
           
         
       
     
     TNF-α CSF-to-serum ratios ranged from 0.18 to 3.25 (mean=1.04, S.D.=1.01) for test groups and 0.12 to 3.68 (mean=1.15, S.D.=1.46) for control groups. There were no indications that these values differed significantly from the expected 1:1 ratio (t=0.266, P=0.795), nor were there indications that the CSF-to-serum ratios differed significantly between the test groups and the control groups (t=−0.168, P=0.869). 
     Weighted analyses of these studies suggested that there were no statistically significant differences overall in TNF-α levels between test groups and control groups in serum (t=1.638, P=0.102) or cerebrospinal fluid (t=1.758, P=0.080), nor between serum and CSF in test groups (t=0.066, P=0.947) or control groups (t=1.117, P=0.265). 
     A ranked correlation analysis suggested relations between TNF-α levels in the cerebrospinal fluid and the serum of patients in the test groups (r=0.717, P=0.030) but not the control groups (r=0.500, P=0.391). The largest ratio between TNF-α levels in CSF and serum was found in a study of patients with traumatic brain injury, in which both the test group (traumatic brain injury with extracranial injury) and the control group (traumatic brain injury without extracranial injury) showed elevated ratios. 
     As shown in Table 4, the overall TNF-α CSF-to-serum ratios were 0.99 for patients of Table 2 and 0.86 for controls of Table 3. In contrast, the overall TNF-α CSF-to-serum ratios for the autistic patients of Table 1 were significantly higher. 
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 Comparison of TNF-α in CSF and serum levels and ratios 
               
            
           
           
               
               
               
               
            
               
                   
                 CSF Levels 
                 Serum Levels 
                   
               
               
                   
                 (pg/mL) 
                 (pg/mL) 
                 Ratios 
               
               
                   
                   
               
            
           
           
               
               
               
               
            
               
                 Autism (Table 1) 
                   
                   
                   
               
               
                 Not Treated 
                 115.25 
                 3.47 
                 33.17 
               
               
                 Treated 
                 10.00 
                 1.65 
                 6.06 
               
               
                 Overall 
                 62.63 
                 2.56 
                 24.44 
               
               
                 Other Studies 
               
               
                 Test Groups (Table 2) 
                 19.16 
                 19.33 
                 0.99 
               
               
                 Control Groups (Table 3) 
                 14.18 
                 16.49 
                 0.86 
               
               
                   
               
            
           
         
       
     
     Example 2 
     TNF-α Study 
     I. Patients 
     Ten male pediatric patients meeting DSM-IV-TR criteria for autism with a history of regression in language and eye contact between 15 and 24 months of age were given the option of having a lumbar puncture performed under a general anesthetic while undergoing such sedation for neuroimaging studies such as a diagnostic scheduled magnetic resonance imaging study of their brains. All families gave written and verbal consent for the procedures. In addition, one male patient (age 2.8 years) with Lennox-Gastant syndrome and speech delay had a lumbar puncture as part of his epilepsy evaluation and had only spinal fluid levels of TNF-α measured, providing a spontaneous single alternative case to compare whether the CSF TNF-α would be elevated in a young severely epileptic child with language delay as well. 
     Of the 10 patients, 4 had been previously treated for presumed autoimmune issues clinically with prednisone, thalidomide, or carnosine and turmeric supplements. These particular patients also had treatment with valproic acid for abnormal sleep electroencephalographic patterns with frequent sleep-activated central-temporal spikes but not a continuous spike-wave in sleep pattern or Landau-Kleffner diagnosis. Patient characteristics are given in Table 5. 
                     TABLE 5                  Characteristics of autistic patients in TNF-α study                                                     Age of                           Age,   Regression,   24-hr EEG Baseline at   Prior Immunosuppressive   Current       Patient   Sex   years   months   Initial Visit   Therapy   Medications                                                 1   M   9.5   18   Abnormal sleep temporal-central   Pulse dose prednisone and   Memantine,                       spike-wave R &gt; L.   thalidomide in past; turmeric at   valproic acid,                           time of CSF study   risperdal, focalin       2   M   6.0   24   Centrotemporal spike-wave   Pulse dose prednisone, IVIG   Valproic acid,                       activity in sleep R &gt; L.   in past; turmeric at time of   memantine,                           CSF study   carnitine       3   M   4.5   15   Sleep right temporal spikes   None   Valproic acid       4   M   4.5   15   Sleep right and left temporal   None   Valproic acid                       spikes       5   M   8.3   18   Secondary generalized   Pulse dose prednisone in past;   Valproic acid,                       polyspikes from temporal lobes   turmeric at time of CSF   memantine,                       in sleep   study   adderall       6   M   6.7   15   Generalized polyspikes in sleep   Brief prednisone in past;   Valproic acid,                           turmeric at time of CSF study   memantine, carnitine       7   M   2.5   15   Bilateral temporal and secondary   None   Valproic acid                       generalized spikes in sleep       8   M   4.5   18   Mild left temporal slowing in sleep   None   None       9   M   2.5   15   Normal   None   None       10   M   9.7   24   Normal   None   None               CSF = Cerebrospinal fluid       EEG = Electroencephalogram       IVIG = Intravenous immunoglobulin       R &gt; L = Greater activity over the right versus left side            
II. Methods
 
     The procedure was elective and was done to rule out a degenerative process. The patients had routine clinical CSF studies performed including cell count for red and white blood cells, total protein, and glucose levels. The calculation of production of immunoglobulin synthesis and oligoclonal bands, myelin basic protein, and glutamate levels were also performed. In addition, levels of TNF-α were simultaneously drawn from serum and CSF. The CSF samples were frozen and analyzed by Inter Science Institute (Inglewood, Calif.) using a quantitative sandwich enzyme immunoassay technique with a monoclonal antibody specific for TNF-α. The TNF-α levels in CSF and serum of patients were analyzed and ratios were calculated. The ratios were analyzed with a paired-samples r test, and relation trends were examined with correlation analyses. 
     III. Results 
     All patients had normal red and white cell counts in their CSF. They also had normal protein, glucose, and glutamate levels. The CSF showed no elevation of myelin basic protein, an absence of oligoclonal bands, and no evidence of immunoglobulin synthesis. TNF-α levels are reported in Table 6. 
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 TNF-α levels in serum and cerebrospinal fluid 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Autoimmune 
                 CSF 
                 Serum 
                 CSF/Serum 
               
               
                 Patient 
                 Treatment 
                 (pg/mL) 
                 (pg/mL) 
                 Ratio 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 1 
                 No 
                 124 
                 1.6 
                 77.5 
               
               
                 2 
                 No 
                 140 
                 2.0 
                 70.0 
               
               
                 3 
                 No 
                 181 
                 8.2 
                 22.1 
               
               
                 4 
                 No 
                 16 
                 2.1 
                 7.6 
               
               
                 5 
                 No 
                 155 
                 2.7 
                 57.4 
               
               
                 6 
                 No 
                 385 
                 1.4 
                 275 
               
               
                 7 
                 Yes 
                 12 
                 1.7 
                 7.1 
               
               
                 8 
                 Yes 
                 13 
                 1.3 
                 10.0 
               
               
                 9 
                 Yes 
                 11 
                 1.3 
                 8.5 
               
               
                 10 
                 Yes 
                 4 
                 2.3 
                 1.7 
               
               
                   
                 Mean 
                 104.1 
                 2.5 
                 53.7 
               
               
                   
                 S.D. 
                 121.3 
                 2.3 
               
               
                   
               
            
           
         
       
     
     All but one patient had normal (&lt;8.2 pg/mL) serum levels of TNF-α, ranging from 1.3 to 8.2 pg/mL. The CSF levels of TNF-α ranged from 4 to 385 pg/mL and were higher particularly in the patients who were naïve to prior immunomodulatory therapies. An established reference range for TNF-α levels in CSF is not available. 
     The overall ratio of the average CSF-to-serum levels for TNF-α was 53.7, with ratios for individual patients ranging from 1.7 to 275. Paired sample analysis suggests a significant difference between serum and cerebrospinal fluid (t=2.384, P=0.049), and independent t tests indicate significantly higher TNF-α levels in the cerebrospinal fluid of untreated patients in comparison with treated patients (t=2.986, P=0.024) (patients 1-6 and 7-10, respectively). 
     The CSF-to-serum ratios for the untreated and treated patients were 55.6 and 6.3, respectively, and the TNF-α levels in the serum were found to not be significantly different between the two groups. Overall, there is no correlation evident in TNF-α levels between samples taken from the cerebrospinal fluid and serum (r=0.311, P=0.453). 
     Elevation of cerebrospinal fluid levels of TNF-α was significantly higher than concurrent serum levels of TNF-α in all of the patients studied. The ratio averaged 53.7:1. This ratio is significantly higher than the elevations reported for other pathological states for which cerebrospinal fluid and serum TNF-α levels have been simultaneously measured. 
     The single patient with Lennox-Gastaut syndrome had cerebrospinal fluid levels of TNF-α of 2 pg/mL, clearly below the levels seen in the autistic patients. 
     The foregoing description merely explains and illustrates the invention and the invention is not limited thereto except insofar as the appended claims are so limited, as those skilled in the art who have the disclosure before them will be able to make modifications without departing the scope of the invention.