Patent Abstract:
A method for the diagnosis of schizophrenia in an individual comprises: (i) measuring mRNA of either D3 dopamine receptor or α7 nicotinic acetylcholine receptor (α7 AChR), and of a control gene in peripheral blood lymphocytes (PBLs) of the individual and of at least one healthy control individual; (ii) calculating the ratio between the receptor mRNA and the control gene mRNA for each individual; and (iii) evaluating the ratio between the ratios obtained in (ii) for the tested individual and for the at least one healthy control individual, wherein (a) an increase in the D3 dopamine receptor mRNA or a decrease in the α7 AChR in the tested individual in comparison to healthy individuals, indicates that said tested individual has a higher likelihood of having schizophrenia than the healthy controls.

Full Description:
FIELD OF THE INVENTION 
     The present invention relates to methods for the diagnosis and follow-up of schizophrenia and other mental and neurodegenerative disorders, and kits for use in said methods. 
     BACKGROUND OF THE INVENTION 
     Schizophrenia is a neuropsychiatric disorder afflicting about one percent of the population. It is characterized by delusions, hallucinations, disorders in organizing thoughts logically, and emotional withdrawal. There is a well-known tendency for schizophrenia to run in families. 
     Although the exact pathogenesis of schizophrenia is still not known precisely, a common belief is that excessive activity at dopaminergic synapses in the brain plays a prominent role. To date, a definitive diagnosis of schizophrenia requires a 6-month duration of symptomotology, and relies on heterogeneous symptoms. Because there is neither an effective biological marker for identifying schizophrenia (Willner, 1997; Hietala and Syvalahti, 1996), nor an accurate and rapid diagnosis to ensure more optimal management at an early stage in the illness, there remains a vital need for a convenient assay for diagnosis and follow-up of schizophrenia. 
     Most of the drugs used to treat schizophrenia act to control the symptoms by neuroreceptor antagonism. Moreover, the dopaminergic basis of schizophrenia is strongly supported by the close correlation between clinical efficacy of antipsychotic medications and their potency to antagonize the binding of dopamine to its receptors (Creese et al., 1976). 
     Dopamine receptors are divided into two subclasses D1 and D2. The D1 subclass contains the D 1  and D 5  receptor subtypes, and the D2 subclass contains the D 2 , D 3  and D 4  subtypes (Levant, 1997). The dopamine hypothesis of schizophrenia relates specifically to the D2 subclass. Notably, most drugs effective in treating schizophrenia exhibit D2 receptor antagonistic activity, and administration of a selective D1-like antagonist has been reported to result in the worsening of symptoms (Karlsson et al., 1995). Among the receptors in the D2 subclass (D 2 , D 3  and D 4 ), the D 3  receptor is located principally in an area of the brain that could be very relevant to schizophrenia, the nucleus accumbens (Willner, 1997). Studies with positron-emission tomography and postmortem brain tissue have indicated increased levels of D2-like dopamine receptors in schizophrenics when compared with nonschizophrenic patients (Seeman and Niznik, 1990). Thus, the level of dopamine receptor could be employed as a marker for schizophrenia if it could be analyzed on an available tissue, preferably a peripheral one. 
     High affinity binding of dopaminergic ligands, as well as the presence of mRNA of several dopamine receptor subtypes (D 3 , D 4  and D 5 ) in human peripheral blood lymphocytes (PBLs) have been reported in recent years (Ricci et al., 1997, Takahashi et al., 1992). It should be noted, however, that neither D 2  nor D 1  dopamine receptor subtypes, which are the most abundant receptors in the brain and belong to the D2 and the D1 subclasses, respectively, have been detected in lymphocytes. Although the significance of dopamine receptors, as well as of other neurotransmitter receptors, in lymphocytes is still not clear, it has been suggested that they may reflect corresponding brain receptors. Several studies have demonstrated the increased binding of dopamine antagonists in lymphocytes of schizophrenic patients as compared with healthy individuals (Bondy et al., 1984; Bondy et al., 1985). In addition, a previous study carried out in the laboratory of the present inventors has demonstrated that spiperone (a D2 antagonist) binding in peripheral blood lymphocytes is higher in neuroleptic responders as compared with treatment-resistant schizophrenic patients (Grodzicki et al., 1990). However, the observed differences in binding studies were rather low and often not significant. The discrepancies obtained could have resulted from the crossreactivity of radioligands with different subtypes of the receptor and with other receptors (e.g. serotonergic), and from scattered levels of binding sites. Therefore, such binding assays in lymphocytes may not be suitable for a reliable assay for schizophrenia. 
     Such a correlation between the status of receptors in the brain and in PBLs has also been demonstrated in Alzheimer&#39;s disease, where muscarinic receptors are reduced in both brains and lymphocytes (Ferrero et al., 1991). A previous study by Nagai et al. (1996) demonstrated that patients with Parkinson&#39;s disease exhibit reduced levels of D 3  receptor mRNA in PBLs, as compared with healthy individuals. These latter findings provide another example of a disease that is associated with an insult in the central nervous system that is reflected in PBLs. This reduction has also been detected in medicated and non-medicated patients. 
     Central cholinergic systems were also shown to control basic functions of the brain. Acetylcholine mediates synaptic transmission in the vertebrate central nervous system through the activation of two major receptor subtypes, the muscarinic and nicotinic acetylcholine receptors (AChRs). The muscarinic receptors are G-coupled receptors, and the nicotinic receptors are ligand-gated ionic channels. Nicotinic AChRs are composed of five subunits organized around a central ion channel. Neuronal nicotinic AChRs are usually built as heteropentamers, composed of α(α2-α9), and β(β2-β4) subunits. α7, α8, and α9 can function as homomeric AChRs and are of special interest because they bind the curarinetric neurotoxin, α-bungarotoxin. (α-BTXβ). These receptors are characterized by a rapid rate of desensitization, and a high level of selectivity to calcium. 
     Several recent studies have suggested that nicotinic α7 AChR may be associated with some aspects of schizophrenia (Guan et al., 1999). Nicotine administration normalizes two psychophysiological deficits, typical for schizophrenia: disordered eye movements, and the P50 auditory evoked potential gating deficit (Olincy et al., 1998). The genes responsible for these two deficits are linked genetically to the chromosomal locus (15q14) of the α7-nicotinic receptor gene (Leonard et al., 2000). α7 AChR has been found to be expressed in the mammalian brain, especially throughout the hippocampus (Hellstrom-Lindahl et al., 1999), a brain region associated with schizophrenia. 
     Interestingly, the vast majority of schizophrenic patients are smoking. They appear to extract more nicotine than normal smokers, possibly due to different inhalation patterns (Olincy et al., 1997). This fact raised the possibility that nicotine might influence the levels of α7 receptor. However, searching for receptor differences between smokers and nonsmokers in the general population did not reveal any significant differences (Stassen et al., 2000). 
     Association between the α7 nicotinic receptor levels and Alzheimer&#39;s disease has also been investigated. Decrease in the expression of α7 AChR was observed in post mortem tissue from Alzheimer&#39;s disease patients, exhibiting a reduction of 36% in the hippocampus (Guan et al., 2000). Burghaus et. al. (2000) reported a decrease in protein amount of α7 AChR in Alzheimer&#39;s disease cortices. Wang et. al. (2000) described an interaction of α7 AChR and β-amyloid (1-42) as a mechanism involved in the pathophysiology of Alzheimer&#39;s disease. There have been some other conflicting reports demonstrating higher levels of the α7 AchR mRNA in the hippocampus (Hellstrom-Lindhal et al., 1999) as well as in lymphocytes (Hellstrom-Lindahl et al., 1997) of Alzheimer&#39;s disease patients, compared to healthy controls. 
     Freedman et al. (2000) reported that interneurons in the hippocampus and in other forebrain structures are decreased in number and function in subjects with schizophrenia. Decreased α7-nicotinic receptor immunoreactivity was found in the frontal cortex and in the nucleus reticularis thalami of schizophrenic patients (Freedman et al., 2000). Court et. al. (1999) described a reduction in the α-BTX binding, and no significant alterations in the nicotine binding in post mortem brains of schizophrenic patients. A significant decrease in the level of α7 AChR was also observed by Guan et. al. (1999) in the frontal cortex of schizophrenics when compared with controls, suggesting that α7 AChR may be involved in inhibitory neuronal pathways engaged in this disorder. 
     SUMMARY OF THE INVENTION 
     According to the present invention, we measured the mRNA levels of dopamine receptors and of α7 nicotinic acetylcholine receptor (AChR) in peripheral blood lymphocytes (PBLs) of schizophrenics and healthy individuals in order to find out if they can serve as peripheral markers for this disorder. Since the inhibitory D2 subclass of dopamine receptors is considered to be associated with neuropsychiatric disorders rather than the D1 subclass, we have focused only on the D 3  and D 4  subtypes, both belonging to the D2 subclass. We have then found a correlation between D 3  dopamine receptor on lymphocytes and schizophrenia, showing a significant elevation of above about 1.6, particularly 2-4, folds in mRNA level of D 3  but not of D 4 , in the schizophrenic patients. In addition, a significant decrease (&gt;20%, particularly 20-98%) of the α7 AChR mRNA levels in PBLs of schizophrenic patients was observed. The changes in the mRNA level of the D 3  dopamine receptor and of the α7 AChR in schizophrenic patients are not affected by different drug treatments. Moreover, non-medicated patients exhibit the same pattern, indicating that these changes are not a result of the medical treatment. 
     The present invention thus relates to the evaluation of the mRNA levels of D 3  dopamine receptor and/or of α7 AChR in PBLs of an individual as reliable peripheral markers for the identification and follow-up of schizophrenia, of other mental disorders, and of neurodegenerative disorders. 
     In one aspect, the invention relates to a method for the diagnosis and follow-up of a mental disorder or of a neurodegenerative disorder in an individual, comprising: 
     (i) measuring mRNA of D 3  dopamine receptor and/or of α7 AChR, and of a control gene in peripheral blood lymphocytes (PBLs) of said individual and of at least one healthy control individual; 
     (ii) calculating the ratio between the D 3  dopamine receptor mRNA and the control gene mRNA, and/or the ratio between α7 AChR mRNA and the control gene mRNA for each individual; and 
     (iii) evaluating the ratio between the ratios obtained in (ii) for the tested individual and for the at least one healthy control individual, wherein an increase in the ratio of the D 3  dopamine receptor mRNA and/or a decrease in the ratio of the α7 AChR mRNA in the tested individual in comparison to healthy individuals, indicate that said tested individual has a high likelihood of having said mental disorder or neurodegenerative disorder, wherein said increase in the D 3  dopamine receptor mRNA and/or decrease in the α7 AchR mRNA in the tested individual is correlated to said mental disorder or neurodegenerative disorder. 
     The mental disorder may be, for example, schizophrenia, maniac depression, Tourette syndrome or a similar disorder, and the neurodegenerative disorder may be, for example, Parkinson&#39;s disease, Alzheimer&#39;s disease or Huntington&#39;s disease. For each disease or disorder, the mRNA of the D 3  dopamine receptor, and/or of the α7 AChR, and of a control gene are measured in PBLs of tested individuals suffering from said disorder, and in PBLs of healthy control individuals, the ratio between the D 3  dopamine receptor mRNA and the control gene mRNA, and/or the ratio between α7 AChR mRNA and the control gene mRNA for each individual is calculated, and the correlation between said increase or decrease is evaluated for each disorder or disease in the same way as described herein in detail for schizophrenia. 
     In one embodiment, the invention relates to a method for the diagnosis and follow up of schizophrenia in an individual, comprising: 
     (i) measuring mRNA of D 3  dopamine receptor and/or of α7 AChR and of a control gene in PBLs of said individual and of at least one healthy control individual; 
     (ii) calculating the ratio between the D 3  dopamine receptor mRNA and the control gene mRNA and/or the ratio between α7 AChR mRNA and the control gene mRNA for each individual; and 
     (iii) evaluating the ratio between the ratios obtained in (ii) for the individual tested for schizophrenia and for the at least one healthy control individual, wherein an increase of above 1.6 fold, preferably 2-4, in the D 3  dopamine receptor mRNA and/or a decrease of more than 20%, preferably 20-98%, in the α7 AChR mRNA in the tested individual in comparison to healthy individuals, indicate that said tested individual has a high likelihood of having schizophrenia. 
     In order to carry out this assay, blood is obtained from individuals, PBLs are isolated therefrom, and total RNA is isolated from the lymphocytes by standard methods as well known in the art. The mRNA of the total RNA is then reverse-transcribed into cDNA that is used for PCR amplification using primers for the D 3  dopamine receptor, for the α7 AChR, and for a control house keeping gene such as β-actin, α-actin, NADH or tubulin. Measuring the D 4  dopamine receptor-mRNA can also serve as a control. Quantification of the PCR products by densitometry, PCR-ELISA, fluorescence techniques, or Southern blot, correlates to the mRNA levels of the D 3  dopamine receptor, α7 AChR, and of the control gene in the PBLs. For example, when the quantification of the PCR products is carried out by densitometry, the program, in a defined area, gives a number corresponding to the brightness intensity. 
     In one embodiment, the mRNA level of the D 3  dopamine receptor, and/or of the α7 AChR, and of a control gene of a tested individual, e.g. a schizophrenic individual, is compared with the mRNA level of the D 3  dopamine receptor, and/or of the α7 AChR, and of a control gene of a sole healthy individual, preferably of the same age and sex. In another embodiment, the comparison is made with a pool of PBLs of two or more healthy individuals. 
     In another aspect, the invention relates to a kit for use in the method of the invention. The kit comprises, for example, (i) means for isolating mRNA from PBLs; (ii) means for reverse transcription and for PCR; and (iii) means for detection of PCR products. The kit may also contain means for separating PBL from whole blood. 
     In one embodiment, the assay may be carried out by the use of DNA arrays or differential display. 
    
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
         FIGS. 1   a - 1   c  shows ethidium bromide staining of D 3 , D 4 , and β-actin PCR products obtained from mRNA of peripheral blood lymphocytes (PBLs) of schizophrenic (S) and control healthy (C) individuals. 
         FIG. 2  shows ethidium bromide staining of β-actin and α7 AChR PCR products obtained from mRNA of PBLs of schizophrenic (Sp) and control healthy (Hl) individuals. 
         FIG. 3  shows a comparison of α7 AChR/β-actin mRNA ratios in PBLs of healthy controls (HL), schizophrenic patients (S) and unmedicated patients (tested in their first hospitalization, FH. 
         FIG. 4  shows a comparison of α7 AChR/β-actin mRNA ratios in PBL of healthy smokers and non-smokers. 
     
    
    
     DESCRIPTION OF PREFERRED EMBODIMENTS 
     The invention will now be illustrated by the following non-limiting examples. 
     EXPERIMENTAL 
     Patients. Schizophrenic patients were recruited from Tyrat Hacarmel and Beer Yaacov Mental Health Centers, Israel, after providing written informed consent for participation in the study. The study has been approved by the Institutional Review Board for human studies in these two mental health centers. All patients were formally diagnosed according to the Diagnostic and Statistical Manual of Mental disorder-IV criteria and evaluated by using standard rating scales by a senior psychiatrist. Healthy individuals&#39; age and sex matched the patient group as much as possible. 
     Lymphocyte isolation. Blood (40-50 ml for D 3  dopamine receptor, or 20-30 ml for the α7 AChR) was drawn from the cubital vein into a heparinized plastic syringe, and then transferred into a sterile 50-ml plastic tube. Blood samples were diluted with an equal volume of phosphate-buffered saline (PBS), were placed onto FICOLL-PAQUE gradients, and then were centrifuged for 30 minutes at 400×g. FICOLL-PAQUE is sterile density gradient media which is based on FICOLL PM400 (polysucrose) and sodium diatrizoate. The lymphocyte layer was collected, and washed twice in PBS. The resulting pellet was immediately frozen at −80° C. until RNA preparation. 
     Reverse Transcription—PCR analysis: Total RNA was isolated from lymphocytes by the guanidinum-thiocyanate method, and the amount and quality of RNA were determined by spectrophotometry and gel electrophoresis (2% agarose for the D 3  dopamine receptor, or 1.5% for the α7 AChR; GibcoBRL). Two μg of total RNA were reverse transcribed into first-strand cDNA using poly-dT-priming and 20 units of Molony murine leukemia virus reverse transcriptase. Two μl cDNA product (80 ng RNA) was used for the PCR amplification at a final concentration of 1×PCR buffer (Perkin-Elmer), and 1 U of Taq DNA polymerase (Perkin-Elmer) in a 25 μl final volume. PCR was carried out in a DNA thermocycler (Minicycler MJ research, MA) for 23 cycles (β-actin), 38 cycles (D 3  and D 4  dopamine receptors), and 39 cycles (α7 AchR). Annealing temperatures for β-actin, for D 3  and for D 4  dopamine receptors was 60° C., while that for α7 AchR was 57° C. The amplification was found to be linear between 30 and 40 cycles for D 3  and D 4  dopamine receptors, as well as for the α7 AChR, and between 19 and 25 cycles for β-actin. 
     The PCR primers for D 3 -, D 4 -dopamine receptors, for α7 AchR, and for β-actin were designed to include at least one intron, to eliminate amplification of genomic DNA. Their sequences were as follows: 
     
       
         
               
               
             
               
               
             
               
               
             
               
               
             
               
               
             
               
               
             
               
               
             
               
               
             
               
               
             
               
               
             
               
               
             
               
               
             
               
               
             
               
               
             
               
               
             
               
               
             
           
               
                   
                 SEQ ID NO: 1 
               
             
          
           
               
                   
                 D3 dopamine receptor - 
               
               
                   
                 GGAGACGGAAAAGGATCCTCACTCG (nt 655-680); 
               
               
                   
                   
               
             
          
           
               
                   
                 SEQ ID NO: 2 
               
             
          
           
               
                   
                 TCAGCAAGACAGGATC TTGAGGAAGG (nt 1203-1177). 
               
               
                   
                   
               
             
          
           
               
                   
                 SEQ ID NO: 3 
               
             
          
           
               
                   
                 D4 dopamine receptor - 
               
               
                   
                 CGGGATCCCACCCCAGACTCCACC (nt 964-988); 
               
               
                   
                   
               
             
          
           
               
                   
                 SEQ ID NO: 4 
               
             
          
           
               
                   
                 CGGAATTCCGTTGCGGAACTCGGC (nt 1240-1216). 
               
               
                   
                   
               
             
          
           
               
                   
                 SEQ ID NO: 5 
               
             
          
           
               
                   
                 α7 AChR receptor- 
               
               
                   
                 AAGTTTGGGTCCTGGTCTTACG (nt 571-592); 
               
               
                   
                   
               
             
          
           
               
                   
                 SEQ ID NO: 6 
               
             
          
           
               
                   
                 GATCATGGTGCTGGCGAAGTA (nt 978-958). 
               
               
                   
                   
               
             
          
           
               
                   
                 SEQ ID NO: 7 
               
             
          
           
               
                   
                 β-actin- 
               
               
                   
                 TGAAGTGTGACGTGGACATCCG (nt 96-117); 
               
               
                   
                   
               
             
          
           
               
                   
                 SEQ ID NO: 8 
               
             
          
           
               
                   
                 GCTGTCACCTTCACCGTT CCAG (nt 543-522). 
               
             
          
         
       
     
     Quantification of PCR products was performed by using a densitometer and a SCION IMAGE (Frederick, Md.) analysis, and/or PCR-ELISA. 
     PCR-ELISA: PCR was performed as described above except for the use of digoxigenin-labeled dNTPs. PCR products were incubated with biotinylated specific internal primers of the tested fragments that were immobilized in streptavidin-coated microtiter plates. The biotinylated internal primers served as capture probes. The bound digoxigenin-labeled PCR-products were then incubated with anti-digoxigenin-peroxidase conjugate that bound to the digoxigenin residues in the labeled PCR products. Peroxidase substrate solution was added, and the color developed was measured in a microtiter-plate reader. 
     Example 1 
     Table 1 summarizes the details (ages, sexes, and diagnoses) of schizophrenic patients and healthy controls from whom blood samples were obtained. RT-PCR was performed on total RNA preparations from these blood samples with primers specific for D 3  or D 4  dopamine receptor, and β-actin as a control. The specific PCR products were resolved on 2% agarose gels, and their sequences were verified. For each patient, a sex- and optimal age-matched healthy control was used, and the level of specific dopamine receptor mRNA was compared between sick and healthy patients. As depicted in  FIG. 1  (a and b) for several representative patients, the signals for D 3  receptor mRNA were significantly higher in schizophrenic patients than in healthy controls. This increase was found to be specific for the D 3  dopamine receptor, because no significant differences in the intensities of D 4  receptor bands were detected between schizophrenic patients and healthy controls ( FIGS. 1   b ,  1   c ). 
     Quantification of the intensities of the specific D 3  dopamine receptor bands was performed by densitometry. The results obtained for 13 patients are summarized in Table 2. Each schizophrenic patient was compared with a sex- and optimal age-matched healthy individual. For each of them, a ratio of the measured density value for D 3  receptor to the value for β-actin was determined. The ratio of these two values for a patient and a matched healthy control, respectively, represents the increased level (in folds) in D 3  specific mRNA in a given patient. As shown in Table 2, the increased levels obtained for the 13 patients range between 1.59 and 7.45 (mostly between 2-3). This increase in D 3  receptor mRNA in schizophrenic patients is significantly higher than the reported increases in binding levels and other recently suggested peripheral markers for schizophrenia (Avissar et al., 1997). Furthermore, the increase in D 3  receptor mRNA was not affected by different drug treatments. Although some of the patients received typical treatment and some atypical treatment (see Table 1), it can be noted that all patients exhibited a similar range of increase indicating that this was not a result of specific dopamine-receptor subtype blockade and up-regulation. Moreover, the present inventors found that this increase was not the consequence of a dopamine receptor antagonist treatment, because non-medicated patients (S12, S13) showed a similar increase in D 3  level (see Tables 1 and 2). 
     Another way to quantify the differences in a specific mRNA level was obtained from PCR-ELISA experiments (see Experimental part). Table 3 summarizes the results obtained from 6 patients. The increased mRNA levels observed are between 1.6 and 3.38 (average increase 2.30±0.63). It should be noted that there is a relatively good agreement between the quantitative values obtained by densitometry and by PCR-ELISA (see patients S1, S4 and S6 in Tables 2 and 3). 
     It should be added that the use of sex- and/or age-matched controls does not appear to be critical. The present inventors demonstrated that the differences in D 3  specific mRNA levels between schizophrenics and healthy individuals, determined by either densitometry or PCR-ELISA, were similar when compared with additional, not necessarily matched, controls (Table 4). This observation may be valuable in designing a practical assay wherein PBL from two or more healthy individuals may be pooled for use as a control. 
     In conclusion, these findings strongly suggest that D 3 -receptor mRNA levels in PBLs may serve as a convenient and reliable peripheral marker for schizophrenia, thus assist in early diagnosis (which is frequently unclear), and possible follow-up of the illness. 
     Example 2 
     Thirty four patients were included in this study, 14 men and 20 women, ranging from 18 to 67 year of age. Of these, 20 were hospitalized schizophrenic patients, and 14 unmedicated patients that were examined during their first hospitalization. 21 healthy controls were studied, 11 nonsmokers and 10 smokers, 8 male and 13 female ranging from 31 to 62 years of age. Table 5 summarizes the details (age, sex and diagnosis) of schizophrenic patients and healthy controls participating in this study. 
     RNA was prepared from blood samples and RT-PCR was performed on total RNA, using specific primers for the α7 AChR, and for β-actin as a control. The specific PCR products were resolved on 1.5% agarose gels. As depicted in  FIG. 2 , the signals for α7 AChR were significantly lower in 3 schizophrenic patients than in 3 healthy controls. Quantification of the intensities of the specific α7 AChR and β-actin bands was performed by densitometry. The results obtained in 28 experiments are summarized in Table 6a. For each individual, a ratio of the measured density value for the α7 AChR to the value for β-actin was determined. Each patient was tested 1-4 times. As seen in Table 6a, 10 of the 34 tested patients had no detectable band for α7 AChR. The α7 AChR/β-actin ratios for these patients was arbitrarily determined as &lt;0.1 (lower than the smallest calculated ratio in Table 6a). The α7 AChR/β-actin ratios for healthy controls (HL), schizophrenic patients (S) and unmedicated patients (first hospitalization, FH) obtained in all experiments are depicted in  FIG. 3 . The average values were 0.88±0.18, 0.36±0.30 and 0.34±0.26 for healthy controls, schizophrenic patients and unmedicated patients, respectively. 
     To determine the significance of the difference between healthy and schizophrenic patients, the α7 AChR/β-actin ratios obtained for 11 different healthy controls and for 14 different schizophrenic patients were compared by Sign test. In each experiment, the values obtained for the schizophrenic patients are significantly lower than for the corresponding healthy individuals (P&lt;0.004). 
     The reduction in the level of α7 AChR mRNA observed in schizophrenic patients was calculated by the following equation: 100−100[(α7 AChR/β-actin S)/(α7 AChR/β-actin Hav)]. First, the average α7 AChR/β-actin ratios of all healthy controls in a given experiment was calculated (Hav). The decrease (%) of the α7 AChR mRNA for each patient was obtained after subtracting the % of (α7 AChR/β-actin S)/(α7 AChR/β-actin Hav) ratio from 100%. The % of decrease for all experiments were calculated, and are depicted in the last column of Table 6a. As seen in this Table, there were only 8 determinations (representing 6 different patients), in which the percent decrease was lower than 20%. All the other determinations in patients resulted in significant decreases in the α7 AChR mRNA levels, ranging from 20% to 98% decrease. So far, the present inventors have not observed a correlation between the percent decrease of α7 AChR mRNA and the disease state. However, it is interesting to point out that one unmedicated patient (FH2) that was tested in his first hospitalization, and exhibited a very low % decrease in its α7 AChR mRNA (5.1%, representing an average of three independent determinations), turned to be non schizophrenic following detailed psychiatric evaluation. 
     The incidence of smoking in a mental illness, particularly in schizophrenia, is much higher than in the general population, 74-92% compared to 30-55%, respectively (Olincy et al., 1999). We have, therefore, tested whether smoking by itself has an effect on α7 AChR mRNA levels. Blood samples from healthy smokers that smoke a pack of cigarettes a day, and from healthy nonsmokers were analyzed for their α7 AChR mRNA levels. As depicted in Table 6b, there were no significant differences in the α7 AChR/β-actin ratios of smokers and nonsmokers. This suggests that the decrease in α7 AChR mRNA levels in schizophrenic patients is not a result of smoking. The α7 AChR/β-actin ratios for healthy smokers and nonsmokers are depicted in  FIG. 4 , demonstrating average ratios of 0.83±0.097 and 0.89±0.097, for smokers and nonsmokers, respectively. 
     The dopaminergic hypothesis of schizophrenia proposes that hyperactivity of dopamine transmission is responsible for the symptoms of this disorder. In the first example of the present invention we have demonstrated increased levels of D 3  dopamine receptor mRNA in PBLs of schizophrenic patients, when compared with the levels in healthy controls. In this example, the present inventors analyzed the mRNA levels of both D 3  dopamine receptor and of the α7 AChR in PBLs of a small group of patients. As expected, the levels of D 3  dopamine receptor mRNA were increased, whereas the levels of α7 AChR mRNA were decreased. As shown in Table 7, an increase of 55.52% in the levels of D 3  receptor mRNA and a decrease of 63.66% in the levels of α7 AChR mRNA were observed for patient SC8. The availability of two different biological markers (the mRNA level of D 3  dopamine receptor and α7 AChR) that can be both tested in PBLs makes the evaluation of schizoprenic patients by a peripheral and objective test, rather promising. Moreover, the fact that the mRNA level of these two receptor mRNAs changes in an opposite direction in schizophrenia, i.e., the gene expression of D 3  receptor increases, whereas the gene expression of α7 AChR decreases in PBLs of schizophrenic patients, that correlates with the changes in these receptors in the brain (as observed in post mortem schizophrenic patients), makes these assays experimentally convenient and reliable. 
     In conclusion, the decreased levels of mRNA of the α7 AChR in PBLs of schizophrenic patients, as presented herein, is consistent with earlier reports demonstrating a decrease in α7 AChR in post mortem brains of schizophrenic patients. Such correlation between decreased levels of α7 AChR mRNA in PBLs and the expression of schizophrenia justifies its application as a biological marker for this disease. 
     
       
         
               
             
               
               
               
               
               
             
               
             
               
               
               
               
             
           
               
                 TABLE 1 
               
               
                   
               
               
                 Characterization of patients (Example 1). 
               
               
                   
               
             
             
               
                 Schizophrenic Patients: 
               
             
          
           
               
                 Number 
                 Age 
                 Sex 
                 Diagnosis 
                 Comments 
               
               
                   
               
               
                 S1 
                 21 
                 M 
                 Chronic negative 
                   
               
               
                   
                   
                   
                 schizophrenia 
               
               
                 S2 
                 27 
                 M 
                 Chronic negative 
               
               
                   
                   
                   
                 schizophrenia 
               
               
                 S3 
                 25 
                 M 
                 Chronic negative 
               
               
                   
                   
                   
                 schizophrenia 
               
               
                 S4 
                 27 
                 F 
                 Positive psychosis 
               
               
                 S5 
                 49 
                 F 
                 Acute schizophrenia 
               
               
                 S6 
                 57 
                 F 
                 Residual Schizophrenia 
               
               
                 S7 
                 41 
                 M 
                 Undifferentiated schizophrenia 
               
               
                 S8 
                 54 
                 M 
                 Paranoid schizophrenia 
               
               
                 S9 
                 47 
                 M 
                 Undifferentiated schizophrenia 
               
               
                 S10 
                 42 
                 M 
                 Undifferentiated schizophrenia 
               
               
                 S11 
                 42 
                 M 
                 Paranoid schizophrenia 
               
               
                 S12 
                 21 
                 M 
                 Undifferentiated schizophrenia 
                 Non-medicated 
               
               
                 S13 
                 40 
                 F 
                 Paranoid schizophrenia 
                 Non-medicated 
               
               
                   
               
             
          
           
               
                 Healthy Controls: 
               
             
          
           
               
                   
                 Number 
                 Age 
                 Sex 
               
               
                   
                   
               
               
                   
                 C1 
                 45 
                 F 
               
               
                   
                 C2 
                 37 
                 F 
               
               
                   
                 C3 
                 37 
                 M 
               
               
                   
                 C4 
                 62 
                 F 
               
               
                   
                 C5 
                 22 
                 M 
               
               
                   
                 C6 
                 44 
                 M 
               
               
                   
                 C7 
                 31 
                 M 
               
               
                   
                 C8 
                 32 
                 M 
               
               
                   
                 C9 
                 49 
                 F 
               
               
                   
                 C10 
                 27 
                 M 
               
               
                   
                 C11 
                 36 
                 F 
               
               
                   
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
               
             
               
               
               
               
               
               
               
               
               
             
               
             
               
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 2 
               
               
                   
               
               
                 Densitometric evaluation of D 3  and D 4  mRNA levels in patients as 
               
               
                 compared with their levels in healthy individuals. (Example 1) 
               
               
                   
               
             
             
               
                 A. 
               
             
          
           
               
                 Schizophrenic 
                   
                   
               
               
                 patients: 
                 Controls: 
                 D 3  fold 
               
             
          
           
               
                   
                 β-actin 
                 D 3   
                   
                   
                 β-actin 
                 D 3   
                   
                 increase 
               
               
                 Number 
                 Arb. units 
                 Arb. units 
                 D 3 /β-actin 
                 Number 
                 Arb. units 
                 Arb. units 
                 D 3 /β-actin 
                 (S/C) 
               
               
                   
               
               
                 S1 
                 67 
                 51 
                 0.671 
                 C3 
                 86 
                 23 
                 0.267 
                 2.513 
               
               
                 S2 
                 87 
                 56 
                 0.643 
                 C3 
                 86 
                 23 
                 0.267 
                 2.408 
               
               
                 S3 
                 82 
                 60 
                 0.731 
                 C3 
                 86 
                 23 
                 0.267 
                 2.737 
               
               
                 S4 
                 98 
                 100 
                 1.02 
                 C2 
                 95 
                 61 
                 0.642 
                 1.588 
               
               
                 S5 
                 55 
                 143 
                 2.600 
                 C2 
                 58 
                 59 
                 1.017 
                 2.556 
               
               
                 S6 
                 75 
                 153 
                 2.040 
                 C4 
                 79 
                 70 
                 0.886 
                 2.302 
               
               
                 S7 
                 19 
                 171 
                 9.000 
                 C10 
                 85 
                 163 
                 1.917 
                 4.694 
               
               
                 S8 
                 138 
                 426 
                 3.087 
                 C8 
                 425 
                 176 
                 0.414 
                 7.456 
               
               
                 S9 
                 89 
                 60 
                 0.674 
                 C7 
                 121 
                 28 
                 0.231 
                 2.917 
               
               
                 S10 
                 107 
                 71 
                 0.663 
                 C7 
                 121 
                 28 
                 0.231 
                 2.870 
               
               
                 S11 
                 303 
                 277 
                 0.914 
                 C3 
                 271 
                 29 
                 0.107 
                 6.644 
               
               
                 S12 
                 319 
                 227 
                 0.711 
                 C7 
                 273 
                 86 
                 0.315 
                 2.257 
               
               
                 S13 
                 237 
                 354 
                 1.493 
                 C9 
                 199 
                 130 
                 0.653 
                 2.870 
               
               
                   
               
             
          
           
               
                 B. 
               
             
          
           
               
                   
                 Controls: 
                   
               
             
          
           
               
                 Schizophrenic patients: 
                   
                 β-actin 
                 D 4   
                   
                 D 4  fold 
               
             
          
           
               
                   
                 β-actin 
                 D 4   
                   
                   
                 Arb. 
                 Arb. 
                 D 4 /β- 
                 increase 
               
               
                 Number 
                 Arb. units 
                 Arb. units 
                 D 4 /β-actin 
                 Number 
                 units 
                 units 
                 actin 
                 (S/C) 
               
               
                   
               
               
                 S1 
                 74 
                 70 
                 0.945 
                 C3 
                 67 
                 70 
                 1.044 
                 0.905 
               
               
                 S2 
                 71 
                 72 
                 1.010 
                 C3 
                 67 
                 70 
                 1.044 
                 0.967 
               
               
                 S3 
                 72 
                 74 
                 1.027 
                 C3 
                 67 
                 70 
                 1.044 
                 0.983 
               
               
                 S4 
                 67 
                 71 
                 1.059 
                 C2 
                 82 
                 73 
                 0.890 
                 1.189 
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
               
             
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Evaluation by PCR-ELISA of D3 mRNA levels in patients as compared 
               
               
                 with their levels in healthy individuals. (Example 1) 
               
             
          
           
               
                 Schizophrenic 
                   
                   
               
               
                 patients: 
                 Controls: 
                 D 3  fold 
               
             
          
           
               
                   
                 β-actin 
                   
                   
                   
                 β-actin 
                 D 3   
                   
                 increase 
               
               
                 Number 
                 (O.D) 
                 D 3  (O.D) 
                 D 3 /β-actin 
                 Number 
                 (O.D) 
                 (O.D) 
                 D 3 /β-actin 
                 (S/C) 
               
               
                   
               
             
          
           
               
                 S1 
                 0.556 
                 0.868 
                 1.56 
                 C8 
                 0.918 
                 0.552 
                 0.601 
                 2.595 
               
               
                 S2 
                 0.808 
                 2.225 
                 2.75 
                 C9 
                 0.405 
                 0.33 
                 0.814 
                 3.378 
               
               
                 S3 
                 0.224 
                 0.253 
                 1.13 
                 C8 
                 0.533 
                 0.272 
                 0.510 
                 2.215 
               
               
                 S4 
                 0.629 
                 0.394 
                 0.626 
                 C8 
                 0.876 
                 0.316 
                 0.360 
                 1.738 
               
               
                 S5 
                 0.340 
                 0.823 
                 2.420 
                 C2 
                 0.365 
                 0.533 
                 1.46 
                 1.657 
               
               
                 S6 
                 0.339 
                 0.899 
                 2.652 
                 C3 
                 0.368 
                 0.444 
                 1.206 
                 2.199 
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 4 
               
             
             
               
                   
               
               
                 Evaluation of D 3  mRNA levels in patients as compared with their 
               
               
                 levels in several healthy individuals (Example 1) 
               
             
          
           
               
                   
                 Schizophrenia 
                   
                 Controls: 
                   
               
             
          
           
               
                   
                 patients: 
                   
                 D 3 /β- 
                   
               
             
          
           
               
                 Number 
                   
                 Number 
                 actin 
                 Ratio 
               
               
                   
               
             
          
           
               
                 S8 
                 3.087 
                 C8 
                 0.414 
                 7.456 
               
               
                   
                   
                 C9 
                 0.498 
                 6.198 
               
               
                 S5 
                 2.600 
                 C2 
                 1.017 
                 2.6 
               
               
                   
                   
                 C3 
                 1.145 
                 2.27 
               
               
                   
                   
                 C4 
                 0.886 
                 2.934 
               
               
                 S6 
                 2.040 
                 C2 
                 1.017 
                 2.005 
               
               
                   
                   
                 C3 
                 1.145 
                 1.78 
               
               
                   
                   
                 C4 
                 0.886 
                 2.302 
               
               
                 S4 
                 1.02 
                 C2 
                 0.642 
                 1.588 
               
               
                   
                   
                 C8 
                 0.656 
                 1.554 
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
             
               
             
               
               
               
               
             
               
             
               
               
               
               
             
               
             
               
               
               
               
             
               
             
               
               
               
               
             
           
               
                 TABLE 5 
               
             
             
               
                   
               
               
                 Characteristics of patients and healthy donors (Example 2) 
               
             
          
           
               
                 Sample 
                 Age 
                 Gender 
                 Diagnosis 
               
               
                   
               
             
          
           
               
                 Healthy controls (nonsmokers) 
               
             
          
           
               
                 HL16 
                 49 
                 F 
                   
               
               
                 HL15 
                 32 
                 M 
               
               
                 HL14 
                 31 
                 M 
               
               
                 HL10 
                 37 
                 M 
               
               
                 HL5 
                 40 
                 M 
               
               
                 HL9 
                 39 
                 F 
               
               
                 HL18 
                 36 
                 F 
               
               
                 HL11 
                 62 
                 F 
               
               
                 HL6 
                 35 
                 M 
               
               
                 HL19 
                 32 
                 M 
               
               
                 HL20 
                 35 
                 F 
               
             
          
           
               
                 Healthy smokers 
               
             
          
           
               
                 SM1 
                 52 
                 F 
                   
               
               
                 SM2 
                 35 
                 M 
               
               
                 SM3 
                 35 
                 F 
               
               
                 SM4 
                 41 
                 M 
               
               
                 SM6 
                 44 
                 F 
               
               
                 SM7 
                 37 
                 F 
               
               
                 SM8 
                 45 
                 F 
               
               
                 SM9 
                 54 
                 F 
               
               
                 SM10 
                 55 
                 F 
               
               
                 SM11 
                 50 
                 F 
               
             
          
           
               
                 Schizophrenic patients 
               
             
          
           
               
                 SP1 
                 21 
                 M 
                 Schizophrenia-residual type 
               
               
                 SP2 
                 27 
                 M 
                 Schizophrenia-paranoid type 
               
               
                 SP3 
                 25 
                 M 
                 Schizophrenia-undifferentiated type 
               
               
                 SP7 
                 49 
                 F 
                 Schizophrenia-paranoid type 
               
               
                 SP8 
                 57 
                 F 
                 Schizophrenia-residual type 
               
               
                 SP9 
                 31 
                 F 
                 Schizophrenia-paranoid type 
               
               
                 SP12 
                 56 
                 F 
                 Schizophrenia-undifferentiated type 
               
               
                 SP13 
                 54 
                 M 
                 Schizophrenia-paranoid type 
               
               
                 SP15 
                 31 
                 F 
                 Schizophrenia-paranoid type 
               
               
                 SP19 
                 54 
                 F 
                 Schizophrenia-paranoid type 
               
               
                 SP20 
                 45 
                 F 
                 Schizophrenia 
               
               
                 BY8 
                 39 
                 M 
                 Schizophrenia-residual type 
               
               
                 BY9 
                 62 
                 F 
                 Disorganized Schizophrenia 
               
               
                 BY10 
                 67 
                 F 
                 Disorganized Schizophrenia 
               
               
                 BY11 
                 50 
                 M 
                 Schizophrenia 
               
               
                 SC6 
                 51 
                 M 
                 Schizophrenia 
               
               
                 SC7 
                 24 
                 F 
                 Schizophrenia 
               
               
                 SC8 
                 64 
                 F 
                 Schizophrenia 
               
               
                 SC10 
                 63 
                 F 
                 Schizophrenia 
               
               
                 SP5 
                 65 
                 F 
                 Schizophrenia-undifferentiated type 
               
             
          
           
               
                 Schizophrenic patients (First hospitalization) 
               
             
          
           
               
                 BY14 
                 40 
                 F 
                 Schizophrenia-paranoid type 
               
               
                 FH2 
                 30 
                 F 
               
               
                 FH3 
                 27 
                 M 
                 Personality disorder 
               
               
                 FH4 
                 40 
                 M 
                 Psychotic episode 
               
               
                 FH5 
                 18 
                 M 
                 Schizophrenia 
               
               
                 FH6 
                 21 
                 M 
                 Personality disorder 
               
               
                 FH7 
                 26 
                 M 
                 Acute psychotic disorder 
               
               
                 FH9 
                 26 
                 F 
                 Schizophrenia-moderately ill 
               
               
                 FH10 
                 23 
                 F 
                 Schizophrenia-markedly ill 
               
               
                 FH11 
                 20 
                 F 
                 Schizophrenia-moderately ill 
               
               
                 FH12 
                 35 
                 M 
                 Acute psychotic disorder 
               
               
                 FH13 
                 38 
                 M 
                 Schizophrenia-moderately ill 
               
               
                 FH14 
                 20 
                 F 
                 Acute psychotic disorder 
               
               
                 FH15 
                 48 
                 F 
                 Acute psychotic disorder 
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 6a 
               
             
             
               
                   
               
               
                 Evaluation of α7/β-actin mRNA levels of schizophrenic patients compared to healthy 
               
               
                 controls (Example 2) 
               
             
          
           
               
                   
                 Schizophrenic patients 
                 Healthy controls 
                   
               
             
          
           
               
                 Exp. 
                   
                 β-actin 
                 α7 
                   
                 Sample 
                 β-actin 
                 α7 
                   
                   
               
               
                 No. 
                 Sample No. 
                 (arb. Units) 
                 (arb. Units) 
                 α7/β-actin 
                 No. 
                 (arb. Units) 
                 (arb. Units 
                 α7/β-actin 
                 Decrease % 
               
               
                   
               
             
          
           
               
                 1 
                 SP1 
                 206.49 
                 75.77 
                 .37 
                 HL16 
                 228.25 
                 223.65 
                 .98 
                 59.99 
               
               
                   
                 SP2 
                 220.25 
                 109.33 
                 .496 
                 HL15 
                 235.74 
                 230.62 
                 .99 
                 45.87 
               
               
                   
                 SP3 
                 215.3 
                 91.46 
                 .42 
                 HL14 
                 206.45 
                 163.76 
                 .79 
                 53.68 
               
               
                 2 
                 SP7 
                 155.53 
                 134.14 
                 .86 
                 HL16 
                 166.3 
                 152.18 
                 .92 
                 17.35 
               
               
                   
                 SP8 
                 168.35 
                 140.64 
                 .84 
                 HL5 
                 131.47 
                 154.08 
                 1.17 
                 19.95 
               
               
                   
                 SP9 
                 135.95 
                 112.06 
                 .82 
                   
                   
                   
                   
                 21.01 
               
               
                 3 
                 SP3 
                 133.48 
                 118.31 
                 .89 
                 HL16 
                 104.04 
                 138.75 
                 1.33 
                 23.08 
               
               
                   
                 SP12 
                 143.23 
                 123.66 
                 .86 
                 HL15 
                 130.7 
                 118.01 
                 .90 
                 25.07 
               
               
                   
                   
                   
                   
                   
                 HL14 
                 106.07 
                 129.44 
                 1.22 
               
               
                 4 
                 SP19 
                 173.83 
                 93.35 
                 .54 
                 HL14 
                 175.14 
                 138.94 
                 .79 
                 41.15 
               
               
                   
                 SP15 
                 159.95 
                 undetectable 
                 &lt;0.1 
                 HL19 
                 175.91 
                 164.57 
                 .94 
                 &gt;89.05 
               
               
                   
                   
                   
                   
                   
                 HL16 
                 164.39 
                 165.8 
                 1.01 
               
               
                 5 
                 BY8 
                 108.54 
                 80.63 
                 .74 
                 HL16 
                 114.12 
                 131.7 
                 1.15 
                 35.63 
               
               
                   
                 BY10 
                 97.75 
                 undetectable 
                 &lt;0.1 
                   
                   
                   
                   
                 &gt;91.31 
               
               
                   
                 BY11 
                 101.5 
                 undetectable 
                 &lt;0.1 
                   
                   
                   
                   
                 &gt;91.31 
               
               
                 6 
                 BY14 
                 182.8 
                 114 
                 .62 
                 HL18 
                 181.07 
                 128.85 
                 .69 
                 10.27 
               
               
                 7 
                 FH2 
                 221.62 
                 188.4 
                 .85 
                 HL18 
                 241.88 
                 210.59 
                 .87 
                 3.97 
               
               
                   
                 SP19 
                 228.01 
                 138.64 
                 .61 
                 HL15 
                 236.86 
                 194.03 
                 .82 
                 31.31 
               
               
                   
                 SP20 
                 171.5 
                 undetectable 
                 &lt;0.1 
                 HL11 
                 235.08 
                 227.08 
                 .97 
                 &gt;88.72 
               
               
                   
                 SP3 
                 202.13 
                 undetectable 
                 &lt;0.1 
                   
                   
                   
                   
                 &gt;88.72 
               
               
                   
                 BY10 
                 240.7 
                 179.56 
                 .75 
                   
                   
                   
                   
                 15.73 
               
               
                 8 
                 SC7 
                 170.88 
                 144.4 
                 .85 
                 HL6 
                 148.91 
                 144.1 
                 .97 
                 12.68 
               
               
                   
                 SC10 
                 124.7 
                 95.67 
                 .77 
                   
                   
                   
                   
                 20.72 
               
               
                   
                 FH6 
                 162.22 
                 undetectable 
                 &lt;0.1 
                   
                   
                   
                   
                 &gt;90.30 
               
               
                   
                 SC6 
                 158.41 
                 undetectable 
                 &lt;0.1 
                   
                   
                   
                   
                 &gt;90.30 
               
               
                 9 
                 SC7 
                 201.25 
                 127.85 
                 .64 
                 HL6 
                 187.14 
                 157.69 
                 .84 
                 24.61 
               
               
                   
                 SC10 
                 215.77 
                 86.5 
                 .40 
                   
                   
                   
                   
                 52.42 
               
               
                   
                 FH6 
                 201.53 
                 81.66 
                 .41 
                   
                   
                   
                   
                 51.91 
               
               
                   
                 FH7 
                 165.53 
                 68.99 
                 .42 
                   
                   
                   
                   
                 50.54 
               
               
                   
                 SC6 
                 180.86 
                 undetectable 
                 &lt;0.1 
                   
                   
                   
                   
                 &gt;88.10 
               
               
                   
                 SC8 
                 209.27 
                 undetectable 
                 &lt;0.1 
                   
                   
                   
                   
                 &gt;88.10 
               
               
                 10 
                 FH2 
                 227.06 
                 199.01 
                 .88 
                 HL11 
                 229.63 
                 170.67 
                 0.74 
                 −9.80 
               
               
                   
                   
                   
                   
                   
                 HL14 
                 239.21 
                 204.11 
                 0.85 
               
               
                 11 
                 SP3 
                 190.77 
                 51.58 
                 .27 
                 HL9 
                 190.34 
                 123.79 
                 .65 
                 58.43 
               
               
                 12 
                 SP1 
                 166.62 
                 90.67 
                 .54 
                 HL5 
                 155.5 
                 100.14 
                 .64 
                 41.36 
               
               
                   
                 SP2 
                 167.84 
                 90 
                 .54 
                 HL9 
                 164.2 
                 103.37 
                 .63 
                 42.22 
               
               
                   
                 SP3 
                 157.41 
                 undetectable 
                 &lt;0.1 
                 HL9 
                 167.36 
                 102.55 
                 .61 
                 &gt;85.62 
               
               
                   
                 SP20 
                 145.1 
                 undetectable 
                 &lt;0.1 
                 HL14 
                 167.28 
                 150.2 
                 .90 
                 &gt;85.62 
               
               
                 13 
                 SP15 
                 126.61 
                 undetectable 
                 &lt;0.1 
                 HL10 
                 154.99 
                 85.57 
                 0.55 
                 &gt;81.31 
               
               
                   
                   
                   
                   
                   
                 HL9 
                 145.48 
                 74.96 
                 0.52 
               
               
                 14 
                 SP3 
                 186.89 
                 undetectable 
                 &lt;0.1 
                 HL9 
                 177.55 
                 137.62 
                 0.78 
                 &gt;86.85 
               
               
                   
                 SP15 
                 166.82 
                 undetectable 
                 &lt;0.1 
                 HL10 
                 170.43 
                 139.54 
                 0.82 
                 &gt;86.85 
               
               
                   
                 SP19 
                 181.28 
                 undetectable 
                 &lt;0.1 
                 HL15 
                 170.05 
                 100.22 
                 0.59 
                 &gt;86.85 
               
               
                 15 
                 SP9 
                 166.45 
                 undetectable 
                 &lt;0.1 
                 HL15 
                 168.02 
                 90.39 
                 0.54 
                 &gt;80.59 
               
               
                   
                 SP19 
                 139.61 
                 undetectable 
                 &lt;0.1 
                 HL16 
                 168.81 
                 91.01 
                 0.54 
                 &gt;80.59 
               
               
                   
                   
                   
                   
                   
                 HL10 
                 166.02 
                 81.96 
                 0.49 
               
               
                   
                   
                   
                   
                   
                 HL18 
                 168.06 
                 82.67 
                 0.49 
               
               
                 16 
                 SP19 
                 220.14 
                 undetectable 
                 &lt;0.1 
                 HL14 
                 217.55 
                 131.95 
                 0.61 
                 &gt;85.30 
               
               
                   
                 SP20 
                 197.13 
                 undetectable 
                 &lt;0.1 
                 HL5 
                 203.7 
                 153.4 
                 0.75 
                 &gt;85.30 
               
               
                 17 
                 SP15 
                 179.26 
                 undetectable 
                 &lt;0.1 
                 HL9 
                 209.93 
                 146.15 
                 0.70 
                 &gt;85.72 
               
               
                 18 
                 FH2 
                 61.84 
                 26.08 
                 0.52 
                 HL18 
                 76.29 
                 46.87 
                 0.61 
                 21.94 
               
               
                   
                 FH3 
                 54.38 
                 4.93 
                 0.11 
                 HL11 
                 72.83 
                 52.98 
                 0.73 
                 83.00 
               
               
                 19 
                 FH3 
                 63.37 
                 6.54 
                 0.10 
                 HL5 
                 29.82 
                 14.75 
                 0.49 
                 74.90 
               
               
                   
                 SP9 
                 52.77 
                 undetectable 
                 &lt;0.1 
                   
                   
                   
                   
                 &gt;79.60 
               
               
                   
                 SP5 
                 32.03 
                 undetectable 
                 &lt;0.1 
                   
                   
                   
                   
                 &gt;79.60 
               
               
                   
                 SP12 
                 45.16 
                 undetectable 
                 &lt;0.1 
                   
                   
                   
                   
                 &gt;79.60 
               
               
                   
                 SP13 
                 20.1 
                 undetectable 
                 &lt;0.1 
                   
                   
                   
                   
                 &gt;79.60 
               
               
                   
                 FH4 
                 60.35 
                 undetectable 
                 &lt;0.1 
                   
                   
                   
                   
                 &gt;79.60 
               
               
                 20 
                 FH7 
                 43.61 
                 10 
                 0.23 
                 SM7 
                 95.28 
                 55.89 
                 0.59 
                 65.26 
               
               
                   
                 FH9 
                 99.78 
                 14.48 
                 0.15 
                 SM8 
                 105.05 
                 72.68 
                 0.69 
                 78.01 
               
               
                   
                 FH5 
                 81.75 
                 undetectable 
                 &lt;0.1 
                 SM9 
                 84.78 
                 49.92 
                 0.59 
                 &gt;84.85 
               
               
                   
                   
                   
                   
                   
                 SM10 
                 89 
                 68.77 
                 0.77 
               
               
                 21 
                 FH9 
                 92.99 
                 50.54 
                 0.54 
                 SM9 
                 81.75 
                 69.1 
                 0.85 
                 35.70 
               
               
                   
                 FH7 
                 94.76 
                 undetectable 
                 &lt;0.1 
                   
                   
                   
                   
                 &gt;88.24 
               
               
                   
                 SC6 
                 79.03 
                 undetectable 
                 &lt;0.1 
                   
                   
                   
                   
                 &gt;88.24 
               
               
                 22 
                 FH9 
                 63.85 
                 32.77 
                 0.51 
                 SM9 
                 63.48 
                 50.93 
                 0.80 
                 36.03 
               
               
                   
                 FH7 
                 53.35 
                 undetectable 
                 &lt;0.1 
                   
                   
                   
                   
                 &gt;87.5 
               
               
                 23 
                 SC8 
                 66.84 
                 6.25 
                 0.09 
                 C3 
                 84.21 
                 17.32 
                 0.21 
                 54.54 
               
               
                   
                 SC6 
                 39.87 
                 undetectable 
                 &lt;0.1 
                   
                   
                   
                   
                 &gt;52.39 
               
               
                 24 
                 FH9 
                 92.3 
                 47.22 
                 0.51 
                 H2 
                 127.12 
                 111.29 
                 0.88 
                 46.85 
               
               
                   
                 FH10 
                 66.85 
                 undetectable 
                 &lt;0.1 
                 HL6 
                 127.53 
                 133.84 
                 1.05 
                 &gt;89.64 
               
               
                 25 
                 FH11 
                 95.22 
                 76.43 
                 0.80 
                 C3 
                 99.08 
                 103.75 
                 1.05 
                 23.35 
               
               
                   
                 FH12 
                 101.4 
                 92.47 
                 0.91 
                   
                   
                   
                   
                 12.91 
               
               
                   
                 FH13 
                 93.01 
                 82.36 
                 0.89 
                   
                   
                   
                   
                 15.44 
               
               
                   
                 FH14 
                 72.02 
                 34.75 
                 0.48 
                   
                   
                   
                   
                 53.92 
               
               
                   
                 FH15 
                 101.95 
                 26.55 
                 0.26 
                   
                   
                   
                   
                 75.13 
               
               
                 26 
                 FH15 
                 108.85 
                 38.52 
                 0.35 
                 C3 
                 120.6 
                 119.12 
                 0.99 
                 64.17 
               
               
                   
                 FH14 
                 95.75 
                 undetectable 
                 &lt;0.1 
                   
                   
                   
                   
                 &gt;89.9 
               
               
                 27 
                 SC8 
                 67.04 
                 0.75 
                 0.01 
                 C3 
                 71.26 
                 64.85 
                 0.91 
                 98.78 
               
               
                   
                 FH14 
                 47.29 
                 3.21 
                 0.07 
                   
                   
                   
                   
                 92.54 
               
               
                   
                 FH15 
                 74.17 
                 29.25 
                 0.39 
                   
                   
                   
                   
                 56.67 
               
               
                 28 
                 BY9 
                 212.5 
                 94.95 
                 0.45 
                 HL18 
                 187.8 
                 114.39 
                 0.61 
                 35.71 
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 6b 
               
             
             
               
                   
               
               
                 Evaluation of α7/β-actin mRNA levels of smokers compared to non smokers 
               
               
                 (Example 2) 
               
             
          
           
               
                   
                 Smokers 
                 Non smokers 
                   
               
               
                   
                 (healthy controls) 
                 (healthy controls) 
               
             
          
           
               
                 Exp 
                 Sample 
                 β-actin 
                 α7 
                   
                 Sample 
                 β-actin 
                 α7 
                 α7/β- 
                   
               
               
                 No. 
                 No. 
                 (arb. Units) 
                 (arb. Units) 
                 α7/β-actin 
                 No. 
                 (arb. Units) 
                 (arb. Units 
                 actin 
                 Decrease % 
               
               
                   
               
             
          
           
               
                 1 
                 SM1 
                 245.51 
                 228.35 
                 .93 
                 HL11 
                 229.63 
                 170.67 
                 0.74 
                 −16.52 
               
               
                   
                 SM3 
                 235.09 
                 187.18 
                 .80 
                 HL14 
                 239.21 
                 204.11 
                 0.85 
                 .26 
               
               
                   
                 SM4 
                 228.96 
                 192.34 
                 .84 
                   
                   
                   
                   
                 −5.24 
               
               
                 2 
                 SM6 
                 148.88 
                 125.74 
                 .84 
                 HL6 
                 148.91 
                 144.1 
                 .97 
                 12.72 
               
               
                   
                 SM7 
                 170.86 
                 125.14 
                 .73 
                   
                   
                   
                   
                 24.31 
               
               
                   
                 SM8 
                 170.29 
                 149.14 
                 .88 
                   
                   
                   
                   
                 9.50 
               
               
                 3 
                 SM7 
                 216.7 
                 223.81 
                 1.03 
                 HL6 
                 187.14 
                 157.69 
                 .84 
                 −22.57 
               
               
                   
                 SM8 
                 165.87 
                 117.62 
                 .71 
                   
                   
                   
                   
                 15.85 
               
               
                 4 
                 SM1 
                 240.84 
                 202.56 
                 0.84 
                 HL18 
                 241.88 
                 210.59 
                 .87 
                 4.99 
               
               
                   
                   
                   
                   
                   
                 HL15 
                 236.86 
                 194.03 
                 .82 
               
               
                   
                   
                   
                   
                   
                 HL11 
                 235.08 
                 227.08 
                 .97 
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 7 
               
             
             
               
                   
               
               
                 Comparison of α7 and D 3  mRNA levels 
               
             
          
           
               
                   
                 β-actin 
                 α7 
                   
                 Decrease 
                 β-actin 
                 D3 
                   
                 Increase 
               
             
          
           
               
                 Sample 
                 (arb. Units) 
                 (arb. Units) 
                 α7/β-actin 
                 % 
                 Fold 
                 (arb. Units) 
                 (arb. Units) 
                 D3/β-actin 
                 % 
                 Fold 
               
               
                   
               
             
          
           
               
                 C3 
                 83.36 
                 67.28 
                 0.81 
                   
                   
                 83.36 
                 0.65 
                 0.007 
                   
                   
               
               
                 SC8 
                 67.31 
                 19.86 
                 0.30 
                 63.66 
                 2.7 
                 67.31 
                 1.18 
                 0.02 
                 55.52 
                 2.86 
               
               
                   
               
             
          
         
       
     
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