METHOD AND KIT FOR EARLY DIAGNOSIS OF AUTOIMMUNITY AND LYMPHOMA IN CENTRAL NERVOUS SYSTEM

Abstract: The present invention relates to develop a series of B- and T-cell clonality assay kits. The data presented here has shown the importance of the B- and T-cell clonality assay kits in the early diagnosis and differential diagnosis for MS, PCNSL and other neurological diseases. In comparison to current diagnostic technologies (cytology, oligoclonal bands, CT, MRI, and surgical biopsy), the invented technology demonstrates its benefit as a technique which is high sensitive, specific, non-invasive, and economical, and which can be applied to different specimens (CSF cells, surgical biopsy and autopsy). Utilization of this technology to MS study has shown that the B-cell clonal expansion is present in majority of MS patients, which have undergone antigen-driven somatic mutation in the germinal centers of peripheral lymphoid tissues. This finding implicates an important pathogenic pathway for the development of demyelination in CNS of MS. Furthermore, this technology allows the establishment of clonal specific RNA library from pathogenic cells in the CNS of the patients, which is important for further understanding of the role of antigen(s) in the cause of B-cell clonal expansion, and towards to developing antigen specific therapeutic strategy.

Description: METHOD AND KIT FOR EARLY DIAGNOSIS OF AUTOIMMUNITY AND LYMPHOMA IN CENTRAL NERVOUS SYSTEM BACKGROUND OF THE INVENTION (a) Field of the Invention The invention relates to a series of B- and T- cell clonality kits for early diagnosis and dif ferential diagnosis of central nervous system (CNS ) autoimmunity and lymphoma in non- immunocompromised or immuno- def icient HIV-associated patients as well as the other neurological diseases . (b) Description of Prior Art Autoimmunity and lymphoid malignancy are the major neurological disorders in the CNS, including multiple sclerosis (MS) and primary CNS B-cell lymphoma in non-immunocompromised or immunodeficient HIV-associated patients. Currently protein chemistry (oligoclonal bands), magnetic resonance image (MRI) and computer tomograph (CT) are useful tools for helping diagnosis of the aforementioned neurological disorders. These methods, however, mainly provide indirect evidence or demonstrate lesions, which usually appear at the late stage of CNS autoimmunity or lymphoid malignancy. In addition, the prognosis for patients with lymphoid malignancy in CNS diagnosed by MRI or CT is poor with a median survival of five (5) months. Multiple Sclerosis MS is the most common CNS disease among young adults. More than a third of a million people in the U.S.A. live with MS and about 50,000 Canadians have MS (information from The National MS Society U.S.A. and National MS Society Canada) . The incidence of MS has risen significantly in the past two decades. MS is a chronic inflammatory disease, characterized by damage of the myelin sheaths and infiltration of mononuclear cell (T-cells, B-cells and macrophages) in white matter of the CNS. The symptom of MS The CNS includes the cerebrum, cerebellum, brain stem and spinal cord. The cerebrum is subdivided into frontal, parietal, occipital and temporal lobes. The symptom of MS is a very variable condition and the symptoms depend on which areas of the CNS have been affected. The systems commonly affected include: ♦ vision ♦ co-ordination ♦ strength ♦ sensation ♦ speech and swallowing ♦ bladder control ♦ sexuality ♦ cognitive function Examples: eye trouble, speech problems, paralysis, weakness (fatigue), shaking of hands, loss of coordination and bladder or bowel control, numbness or pricking feelings, staggering (loss of balance), dragging of feet. There is no specific pattern to MS and everyone with MS has a different set of symptoms, which vary from time to time and can change in severity and duration, even in the same person. MS is a chronic inflammatory disease, characterized by damage of the myelin sheaths in the CNS. It is believed that MS is an autoimmune disease, which results from immunity to myelin. However, there is no direct evidence to prove this hypothesis in MS studies. Myelin-specific T-cells, recognizing myelin basic protein (MBP) and proteolipid protein (PLP) , could be isolated from both MS patients and healthy individuals. The data on activation of T-cells are still rather fragmentary and are based largely on indirect methods for measuring T-cell receptor (TCR) affinity. Affinity maturation presumably applies to the typical memory T-cells generated in response to stimulating antigens. In addition, most of T-cells derived from postmortem MS brain plaque are antigen nonspecific (Steinman S, Proc Natl Acad Sci USA 93:2253-2256, 1996). The lack of myelin protein reactive T-cells from postmortem MS brain plaque tissue could be a result of apoptosis of antigen specific T-cells in the CNS. To date, there is no any specific methodology for the early diagnosis of MS based on myelin proteins-reactive T-cells. The finding of intra-blood brain barrier synthesis of immunoglobulins and oligoclonal bands by intrathecal B-cells is the most consistent immunological finding yet described in MS. However, the immunoglobulin from the cerebrospinal fluid (CSF) of the patients is comprised of both serum and intrathecal derived components. Therefore, it is important to study the nature of intrathecal B-cells, such as clonality, somatic hypermutation and antigen driven selection. The affinity maturation of B-cells, in contrast to T-cells, involves somatic hypermutation (Liu YJ, et al., Nature 342:929-931, 1989). This process is induced in the specialized environment of the germinal centers and requires direct contact with antigens (immune complexes) displayed on follicular dendritic cells combined with a collaborative interaction with antigen-specific CD4+ T helper cells (MacLennan ICM, and Gray D Curr Top Microbiol Immunol 159:37-63, 1990; and Jacob J, Kelsoe G, Rajewsky K, and Weiss U Nature 354:389-392, 1991). Affinity mutation through somatic hypermutation of B-cell Ig V region genes is an efficient process of mutation. Through this process, memory or antibody forming B-cells (plasma cells) with high-affinity for its antigen are generated (Nossal GJV, Cell 68:1-2, 1992; Liu YJ, Johson GD, Gordon J, and MacLennan CM Immunol Today 13:17-21, 1992). In healthy brain, there are no antibody forming B-cells. In contrast, B-cell studies have revealed that the proportion of IgG-producing cells is greatly increased in the cerebrospinal fluid (CSF) from patients with MS. Our recent work have demonstrated that a dominant B-cell clonal expansion exhibit in the CSF of the majority MS patients at very early stage. Sequence analysis of VH genes of these clonally expanded B-cells suggests that these cells are hypermutated post germinal center memory or antibody- forming B-cells that had been positively selected through their antigen receptor. These findings provide the direct evidence of B-cell involvement in the pathogenesis of MS (Qin Y, Duquette P, Tabot P., Zhang Y., Pool R. , and Antel J. Journal of Clinical Investigation 102:1-6, 1998). The current technologies for diagnosis of MS The current technologies that are being used for diagnosis of MS are 1) Medical history, 2) Magnetic Resonance Imaging (MRI) , and 3) Lumbar puncture (CSF analyses) . Three basic parameters from above examinations have been used to confirm diagnosis of MS: 1) coming and going pattern of clinical neurological symptoms and signs caused by distinct CNS damage, 2) multifocal lesions in the CNS by MRI examination (12), and 3) IgG oligoclonal bands in the CSF. Primary center nervous system lymphoma Primary central nervous system lymphoma (PCNSL) compromised one (1) per cent of all intracranial neoplasms. A threefold increase in incidence has been shown since last two decades. PCNSL represents one of a large number of extranodal Non-Hodgkin' s Lymphoma (NHL) , mostly of B-cell origin, which are by definition usually restricted to a single organ at presentation. The increasing frequency, especially in the last decade, has to be correlated with the incidence of CNS lymphoma either in immune deficiency, or in non immune-compromised patients. The highest incidence occurs in patients with: -Congenital immunodeficiency syndrome -Acquired immunodeficiency syndrome (AIDS) -Organ transplantation recipient Differential Diagnosis Meningioma, Metastasis disease, Focal infectious lesions such as Toxoplasmosis multifocal progressive leukoencephalitis . The similarity between PCNSL and MS PCNSL and MS both occur in all age groups, from very young to the elderly. Similar to MS, the symptoms of early stage PCNSL is a very variable condition and the symptoms depend on which areas of the CNS have been invaded by lymphpoma cells. It appears to be similar to other NHL of extranodal sites, which lack organized lymphoid tissue (e.g., bone, ovary, and testis) . Lymphoma has a tendency to relapse in the same or different sites, rather than developing as widespread nodal disease. In this circumstance, it can easily be confused with other neurological conditions, such as MS and encephalomyelitis (DeAngelis LM, J Neurooncol . 9:177-81, 1990; Vaquero J, Martinez R, Rossi E, and Lopez R, J Neurosurg. 60:174-6, 1984; Young C, Gordon N, Safran HP, Schatz S, Stopa E, and King TC, Arch Pathol Lab Med . 120:275, 1996). In addition, both are capable of inducing demyelinating lesions and share the same feature of CSF B-cell clonal expansion. Currently technologies for diagnosis of PCNSL Magnetic resonance image (MRI) and computer topography (CT) are useful diagnostic tools. PCNSL mass is usually identified by CT or MRI scan (a densely enhancing area) , and surgical biopsy. These methods, however, mainly provide indirect evidence or passively demonstrate lesions in the CNS, which usually appear at the late stage of CNS demyelination or lymphoid malignancy. In addition, the prognosis for patients with lymphoid malignancy in CNS diagnosed by MRI or CT is poor, with a median survival of five months . The early stage of PCNSL can begin as an infiltrate process, in which no discrete mass is formed and the white matter lesion is often seen in the CNS. The lesions show the tendency of spontaneous disappearance and relapse in same or different area, being called 'ghost tumor' (Vaquero J, Martinez R, Rossi E, and Lopez R, J Neurosurg. 60:174-6, 1984). Figure 1 demonstrates an example of PCNSL indistinguishable from MS (Kuroda Y., Kawasaki T, Haraoka S, Fujiyama F, Kakigi R et al J " Neurol Sci . 111 : 173 -9, 1992) : small foci of high-intensity were in the right frontal and the left occipital white matter on the T2 -weighted image in January 1990 (Fig. 1, upper left) . New lesions appeared in the left frontal white matter and a lesion in the right frontal white matter disappeared in February 1990 (Fig.l, upper right) . Patient was diagnosed as MS. The right frontal white matter lesions disappeared but diffuse cerebral atrophy was noted in May 1990 (Fig.l, lower left). Widespread, multicentric white matter lesions were seen in July 1990 (Fig. 1, lower right) . Figure 2 shows the autopsy findings. Diffuse infiltration of lymphoma cells of various sizes is found in the cerebral white matter lesions. Analysis of the immunoglobulin gene rearrangements provided evidences of PCNSL. It would be highly desirable to be provided with a method for an early diagnosis of MS and encephalomyelitis or lymphoid malignancy in the CNS of a patient with or without HIV infection. SUMMARY OF THE INVENTION One aim of the present invention is to provide a series of B- and T-cell clonality assay kits for an earlier diagnosis of multiple sclerosis, brain lymphoma and other CNS diseases. Another aim of the present invention is to provide a series of B- and T-cell clonality assay kits for diagnosis of demyelinating disease such as multiple sclerosis (MS) . Another aim of the present invention is to provide a sensitive technology for early diagnosis of primary CNS Non Hodgkin's B-cell lymphoma, as well as the systemic lymphoma CNS metastasis. Another aim of the present invention is to provide a specific technology for differential diagnosis between the clonal expansion or/and polyclonal inflammatory diseases. In accordance with the present invention there is provided a method for diagnosis of a neurological disorder in a patient, comprising the steps of: a) subjecting a nucleic acid sequence from a CNS sample, CSF; brain and spinal cord biopsy specimens or autopsy (necropsy) of said patient to analyze the clonality of the B-cells and T-cells by the amplification of VH, DH, and JH region (complementarity determining region 3, CDR3) of VH genes and VL, and JL region of VL immunoglobulin genes of B-cells, as well as genes of TCR; and b) analyzing sequences amplified in step a) by detecting amplified sequences of said gene regions wherein said amplified sequences are an indication of increased B-cell or T-cell expression and thereby neurological disorder in said patient. Still in accordance with the present invention there is provided a method for diagnosis of a neurological disorder in a patient, comprising the steps of : a) subjecting a nucleic acid sequence from a CNS of said patient to enzyme fragmentation of VH, DH, and JH (CDR3) or VL region genes of B-cells and genes of TCR; and b) analyzing fragments produced in step a) by detecting a signal on a southern blot, wherein said signal is an indication of increased B-cell or T-cell expression and thereby neurological disorder in said patient . In accordance with the present invention there is also provided a kit for determining B- and T-cell clonality for diagnosis of a neurological disorder in a patient. The kit comprises at least one upstream primer and at least one downstream primer derived from CDR3 genes of a B-cell immunoglobulin heavy chain, from variable region genes of a B-cell immunoglobulin light chain, or from a gene of a T-cell receptor γ. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 illustrates an example of PCNSL indistinguishable from MS; Fig. 2 illustrates the autopsy finding of diffuse infiltration of lymphoma cells in the cerebral white matter lesions; Fig. 3 illustrates the perivascular lymphocyte infiltration of the plaques from MS autopsy that were used for clonality assay; Fig. 4 illustrates the diffuse infiltration of malignant lymphocytes of the plaques from PCNSL autopsy that were used for clonality assay; Fig. 5 illustrates an ethidium bromide stained 2% agarose gel of CDR3 PCR products from CSF cells; Fig. 6 illustrates an ethidium bromide stained 2% agarose gel of CDR3 PCR products from 45 randomly selected cases (unknown diagnosis) ; Fig. 7 illustrates a predominant CDR3 nucleotide sequence of patients with MS or other neurological diseases; and HIV-positive PCNS non- Hodgkin's B-cell lymphoma; Fig. 8 illustrates an ethidium bromide stained 2% agarose gel of CDR3 RT-PCR products from plaques (lesions) of MS; and Figs. 9A to 9D illustrate the sequences of the VH gene from a dominant clone of CSF B-cells from patients with MS. DETAILED DESCRIPTION OF THE INVENTION In accordance with the present invention, there is provided a method for diagnosis and differential diagnosis of demyelinating disease, multiple sclerosis, lymphoid malignancy HIV-positive or negative Primary Central Nervous System (PCNS) Non- Hodgkin's B-cell lymphoma; the systemic lymphoma CNS metastasis; and other neurological diseases. Analysis of VH gene rearrangement; identifying the clonal motifs and analyzing antigen-driven selected mutations on the immunoglobulin (Ig) heavy chain or light chain variable region of dominant clone of B-cells from CSF; biopsy specimens of the patients with MS; CNS lymphoma and other neurological diseases will provide important evidence for diagnosis of autoimmunity; malignancy and inflammation in the CNS. Based on this fundamental knowledge, a series of specific B- and T-cell clonality assay kits have been developed and used to analyze the clonality and differentiation pathway of B- or T-cell in the CNS of the patients with neurological diseases. The criteria for establishing clonal relatedness were identity at the somatically formed VH, DH, JH genes and use of the same sets of VH, DH and JH genes. B and T-cell clonality assay kits Description Amplify genes of VH, DH and JH region (CDR3) for VH; and VL and JL region for VL of B-eelIs; and genes for various V and J segments of TCR. Analyze B- or T-cell clonality of PCR products on an ethidium bromide stained agarose gel . APPLICATIONS B- or T-cell clonality assay kits provide a high sensitive, specific, non-invasive technique for the earlier diagnosis and differential diagnosis of neurological diseases. It has been used to distinguish B-cell clonal expanded autoimmune diseases, such as MS, and neoplasm of primary central nervous system non-Hodgkin v s lymphoma and B-cell polyclonal expanded inflammatory diseases, such as encephalomyelitis and other neurological diseases. PROTOCOL The B- and T-cell clonality assay kits use the modification of procedure based on amplifying genes of VH, DH and JH region (CDR3) for VH; VL and JL region for VL of B-cells; and genes for various V and J segments of TCR using PCR technique. The procedure involves the instruction of the sample preparation, extraction of the DNA and a set of the primers for amplifying the genes of framework regions (FRs) and J regions of heavy and light chains of immunoglobulin and TCR. The clonality of B- and T-cells can be analyzed following an ethidium bromide staining of agarose gel of PCR products. CONTENTS For fresh cells or frozen tissues: Cell wash buffer Digestion buffer Proteinase K dNTPs A set of primers Distilled water 1.5 ml microfuge tubes For paraffin embedded specimen Xylene Graded alcohol Digestion buffer Proteinase K DNTPs A set of primers Distilled water 1.5 ml microfuge tube Set of primers For CDR3 genes of B-cell immunoglobulin heavy chain, the following primers are suggested: Upstream primers : FR2 5 ' -TGG [A/G] TCCG [C/A] CAG [G/C] C [T/C] [T/C] CNNGG-3 ' (SEQ ID N0:1) FR3 5 ' -ACACGGCTGTGTATT-3 ' (SEQ ID NO : 2 ) Downstream primers : LJH: 5 ' -ACCTGAGGAGACGGT-3 ' (SEQ ID NO : 3 ) VLJH 5' -GGTGACCAGGGTCCCTTGGCCCCAG-3 ' (SEQ ID N0:4) For variable region genes of B-cell immunoglobulin light chain, the following primers are suggested: VK Upstream primers : Vκl 5 ' GACATCC (A/G) G (T/A) TGACCCAGTCTCC (A/T) TC-3 ' (SEQ ID NO: 5) Vκ2 5 ' CAG (T/A) CTCCACTCTCCCTG (C/T) CCGTCA-3 ' (SEQ ID NO: 6) Vκ3 5 ' TCTCCAG (G/C) CACCCTGTCT) T/G) TGTCTC-3 ' (SEQ ID NO: 7) Vκ4 5 'AGACTCCCTGGCTGTGTCTCTGGGC-3 ' (SEQ ID NO : 8 ) Vκ5 5 'CAGTCTCCAGCATTCATGTCAGCGA-3 ' (SEQ ID NO: 9) Vκ6 5 ' TTTCT (G/C) TCTGTGACTCCAGGA (G/A) GGAGAA-3 ' (SEQ ID NO:10) Downstream primer: Jκl ,2,4, 5 'ACTCACGTTTGAT (T/C) TCCA (G/C) CTTGGTCC-3 ' (SEQ ID N0:11) Jκ3 5 ' GTACTTACGTTTGATATCCACTTTGGTCC-3 ' (SEQ ID NO: 12) Jκ5 5 'GCTTACGTTTAATCTCCAGTCGTGTCC-3 ' (SEQ ID NO: 13) Vλ Upstream primers : LII 5' -ATGGCCTGGGCTCTGCTGCTC-3 ' (SEQ ID NO: 14) LIII 5 ' -ATGGCATGGATCCCTCTCTT-3 ' (SEQ ID NO: 15) BL2 5 ' -ATGACCTGCTCCCCTCTCCT-3 ' (SEQ ID NO: 16) 4A 5' -ATGGCCTGGACTCCTCTCTTTCTG-3 ' (SEQ ID NO: 17) LBV 5 ' -ATGGCCTGGGCTCCACTACT-3 ' (SEQ ID NO: 18) Downstream primers : Cλ 5 ' -TATGAACATTCTGTAGGGGCCAC-3 ' (SEQ ID NO: 19) For genes of T-cell receptor-γ, the following primers are suggested: Upstream primers : Vγ2 5 ' CTTCCTGCAGATGACTCCTACAACTCCAAGGTTG-3 ' (SEQ ID NO: 20) Vγ3 5 ' CTTCCTGCAGATGACGTCTCCACCGCAAGGGCTG-3 ' (SEQ ID NO: 21) Vγ4 5 ' CTTCCTGCAGATGACTCCTACACCTCCAGCGTTG-3 ' (SEQ ID NO: 22) Vγ5 5 ' TTCCTGCAGATGACGTCTCCAACTCAAAGGATG-3 ' (SEQ ID NO:23) Vγ8 5 ' CTTCCRFCAGATGACTCCTACAACTCCAGGGTTG-3 ' (SEQ ID NO:24) Vγ9 5 ' GG (A/G/C/T) ACTGCAGGAAAGGAATCTGGCATTCCG-3 (SEQ ID NO:25) VγlO 5'CTCTGCAGAATCCGCAGCTCGACGCAGCA-3 ' (SEQ ID NO: 26) Vγll 5 'CACTGCAGGCTCAAGATTGCTCAGGTGGG-3 ' (SEQ ID NO: 27) Vγl2 5 ΆCTCTGCAGCCTCTTGGGCACTGCTCTAAA-3 ' (SEQ ID NO: 28) Downstream primers : JGT12 5 'AAGTGTTGTTCCACTGCCAAA-3 ' (SEQ ID NO: 29) JGT3 5'AGTTACTATGAGC(T/C)TAGTCCC-3 ' (SEQ ID NO:30) JGT4 5'TGTAATGATAAGCTTTGTTCC-3 ' (SEQ ID NO:31) Immunoglobulin heavy chain variable region genes assay kit Description Amplify genes on Ig VH of B-cells. Study the differentiation pathway of B-cell. APPLICATIONS Immunoglobulin heavy chain region genes assay kit has been used to amplify the entire Ig VH genes of B-cells from the neurological disease. After subsequently sequencing PCR products, the differentiation pathway of pathogenic B-cells in the CNS and their origin can be analyzed by studying the antigen-driven somatic mutation on the genes Ig VH region. CONTENTS For fresh cells or frozen tissues: Cell wash buffer Digestion buffer Proteinase K dNTPs A set of primers Distilled water 1.5 ml microfuge tubes For paraffin embedded specimen Xylene Graded alcohol Digestion buffer Proteinase K dNTPs A set of primers Distilled water 1.5 ml microfuge tubes Set of primers Upstream primers : VH1 5'CCTCAGTGAAGGT(C/T)TCCTGCAAGGC-3 ' (SEQ ID NO:32) VH2 5 'GTCCTGCGCTGGTGAAA(G/C)CCACACA-3 ' (SEQ ID NO:33) VH3 5'GGGGTCCCTGAGACTCTCCTGTGCAG-3 ' (SEQ ID N0:34) VH4 5'GACCCTGTCCCTCACCTGC(A/G)CTGTC-3 ' (SEQ ID NO-.35) VH5 5'AAAAAGCCCGGGGAGTCTCTGA(A/G)GA-3 ' (SEQ ID NO:36) VH6 5 'ACCTGTGCCATCTCCGGGGACAGTG-3 ' (SEQ ID NO: 37) Downstream primers : JH1-5 5'GGTGACCAGGGT(A/G/C)CC(A/G)TGGCCCCAG-3 ' (SEQ ID NO: 38) JH6 5 'GGTGACCGTGGTCCCTTGCCCCCAG-3 ' (SEQ ID NO:39) Primer of oligonucleotides specific for each patients-derived dominant VDJ region is designed for analyzing the antigen-driven selected somatic hypermutation on the Ig VH genes derived from dominant clonal B-cells. The B-cell clonality kits have been developed for Ig VH gene amplification and identification of the clonality of B-cells from CSF of the patients with multiple sclerosis and other neurological diseases. These kits can also be used for analysis of surgical, biopsy and autopsy specimens of MS plaques and PCNSL. Patients The CSF cell samples (from 2-8 ml) were obtained from each of twelve (12) MS patients and fifteen (15) patients with other neurological diseases (Table 1) as well as 47 random selected cases (unknown diagnosis) . Seven of 12 MS patients were considered as at early stage of disease, CSF was collected from initial onset of clinical symptom. MS diagnosis was confirmed on the second-episode. MS patients including 11 females and 1 male with clinically or laboratory- supported definite MS diagnosis with a mean age of 34 11 years. The average duration of disease was 2 ± 0.4 years. The mean expanded disability status score (EDSS) was 3.2 ± 1.3. All MS patients were categorized as having relapsing-remitting disease. Oligoclonal bands of all CSF samples were analyzed for presence of oligoclonal immunoglobulin bands by isoelectric focussing or agarose electrophoresis . MRI scan demonstrated that the lesion (s) varying from no lesion to multi lesions and areas were affected from spinal cord to brain individually (Table 1) . The control patients were divided into inflammatory (I) (3 of 15 cases) : 2 patients with subacute illnesses diagnosed as acute disseminated encephalomyelitis and 1 with a remote history of Herpes Zoster encephalitis. Twelve (12) out of fifteen (15) were patients with noninflammatory ONDs, including amyotrophic lateral sclerosis (ALS) ; pseudotumer ; spinocerebellar degeneration; spinal cord infarct; myopathy; headache and neuropathy. The patients with other neurological diseases (ONDs) who had oligoclonal band negative (15 of 16 cases) (Table 1) . DNA samples were also collected from paraffin embedded specimens from plaques of four (4) patients with MS (Fig 3) and two (2) patients with PCNSL (Fig 4) . Extraction of RNA or DNA from CSF The cell pellets of CSF; surgical biopsy and autopsy specimens were used as sources of RNA or DNA. Protocol 1: Preparation of samples The precise method used depends upon the type of sample. A. The cerebrospinal fluid cells 1. Collect the cells from 2 to 8 ml cerebrospinal fluids by centrifugation in a 15 ml tube at 1,200 rpm for 10 min at 4°C. 2. Collect fluid for other lab examine and wash cell pellet in 5 ml wash buffer by centrifugation as above. 3. Discard the supernatant completely and store cell pellet at -80°C. B The frozen specimens Collect 3 to 5 sections (10 μM thick) from each specimen into 1.5 ml microfuge tube and store the sections at -80°C. The paraffin embedded specimens 1. Collect 3 to 5 sections (10 μM thick) from each specimen into 1.5 ml mirofuge tube. 2. Dewax the sample with 1 ml xylene and shake for 10 min. Collect the sample by centrifugation at 13,000 rpm for 7 min. 3. Discard the supernatant and rehydrate the sample with 1 ml 100% graded alcohol for 10 min. Collect the sample by centrifugation as above. 4 Repeat steps 2 and 3 for 3 times. 5 Wash the sample with 1 ml 100% graded alcohol for 2 min and centrifugation as above . Discard the alcohol and dry the sample at 60°C for 15 min. Protocol 2 Extraction of RNA Total RNA of the frozen CSF cell pellets and Specimens are extracted using RNAeasyTM Kit (Qiagen Inc. Chatsworth, Ca) . Protocol 3 Extraction of DNA Prepare samples as described in Protocol 1 above . Add the digestion buffer to each sample: 20 μl for CSF cell pellet; 200 μl for frozen specimen; 200 to 400 μl for paraffin embedded specimen. 3. Add proteinase K stock (20 mg/ml) into each sample in a final concentration of 2 mg/ml . 4. Incubate samples at 55°C: 2 hours for sample of CSF cells; 5 hours for sample of frozen specimens ; overnight (16 hours) for samples of paraffin embedded specimens. 5. Incubate samples at 95°C for 8 min. Table 1 summarizes the clinical and laboratory data for the patients under study. TABLE 1 Summary of clinical and Lab features of patients included in the CSF analysis A. R-R MS samples Clinical MRI CSF Sex Age of Disease OB Cell Number Onset Duration (years) •_ 1. F 48 3 + - 9X10 4 2. F 31 4 + + 8X10 4 3. F 24 1 + + 4X10 4 4. F 48 2 + + 3X10 4 5. F 22 6 + + 8X10 4 6. F 28 1 1 SL - 1 .6X10 5 7. F 18 1 + + 4X10 4 8. F 29 1 1 SL + 1X10 5 9. F 36 1 + + 6X10 4 10. F 21 1 + + 2X10 4 11. M 41 4 + + 1 .6X10 5 12. F 43 1 NL _ 2 .5X10 5 B . OND Samples Clinical CSF Sex Age Diagnosis OB Cell Number Onset I-OND 1. F 64 ADEM 2X10 4 2. F 24 ADEM 5X10 4 3. F 86 Remote HZEM UN NI-OND 1. F 68 Headache 3X10 4 2. F 66 Spinal cord 2X10 4 infarct 3. 47 Pseudotumor 5X10 4 4. F 68 Neuropathy 6X10 4 5 M 34 Migraine 3X10 4 6. M 67 ALS UN 7. M 32 ALS 5X10 3 8. F 40 Pseudotumor 2.5X10 4 9. M 60 Hydrocephalus 4X10 4 10. M 36 Neuropathy + 2X10 4 11. M 44 Spinocerebeller - 1X10 5 Degeneration 12. M 57 Neuropathy 2X10 4 OB : oligoclonal bands ; Cell number : the total numbers of white blood cells in the specimen (2 -8 ml) ; + multiple plaques ; SL : spinal lesion; NL : normal ; ADEM : acute disseminated encephalomyelitis ; ALS : amyotrophic lateral sclerosis ; HZEM : Herpes Zoster encephalitis . UN : unknown . Amplification Vh, DH, and JH genes of B-cells derived from CSF and brain lesions In accordance with one embodiment of the present invention, the f irst step is to use B-cell clonality assay kit for screening the CDR3 region of Ig gene rearrangement derived from B-cells in the CNS of patients with neurological diseases. The method of the present invention is simple, convenient, specific, highly sensitive and needs only a small sample of CSF with mild or no side effects and biopsy and autopsy specimens. It provides a clear-cut information to support a diagnosis or a differential diagnosis. The method has provided us an important tool to study the pathogenesis of B-cells and T-cells in CNS. The results from B-cells derived from CSF and the plaques of MS brain using said kits showed the high frequency of clonal B-cell evolution in the CNS these patients (Figs. 3 and 4) . The utility of this B- and T-cell clonality assay kits has been demonstrated with high sensitivity in patients with early multiple sclerosis; and other neurological diseases, as well as CNS lymphoid malignancy (Figs. 5 and 6) who have no MRI change or oligoclonal bands (Table 1) . PCR reaction conditions The PCR was performed in a final volume of 50 μl reaction buffer that contain 2.5 units of recombinant Taq Polymerase, 50 mM/L Tris-HCL, PH 9.0 at 25°C: 20 mM/L (NH4) 2 S0 4 ; 3.0 mM/L MgCl 2 , 0.1 mM/L each of deoxyadenosine triphosphate, deoxycytidine triphosphate, deoxyguanosine triphosphate, and thymidine triphosphate, 25 pmol of each primer, and 50 to 200 ng of DNA. For amplification of the Ig genes, a semi- nested PCR was performed: The first round, each sample was amplified with upstream primers FR2 or FR3 , respectivelly, and downstream primer LJH, for 40 cycles of 50 seconds at o o o 94 C, one min at 55 C, and 40 seconds at 72 C. The second round, 2 μl aliquot (1%) of the initial PCR product was reamplified with primers FR3 and VLJH, for 30 cycles of 94 C for 50 sec; 59 C for 40 sec and 72°C for 1 min. Aliquots of the final reaction product were analyzed by electrophoresis in a 1.5 to 2% agarose gel (Sigma) containing ethidium bromide . The second step in accordance with one embodiment of the present invention is sequencing of PCR products for further identification of clonality of the CSF B-cells. The PCR products were purified by electrophoresis and recovered from the gel by treatment with gelase (CalbioChem, San Diego, CA. ) . The recovered DNA was ligated into the pGEM™ T vector (Promega, La Jolla, CA.), which was used to transfect Escherichia coli DH5a. Six (6) to eight (8) white colonies were picked up at random and grown overnight in 3 ml of LB (Luria-Bertani) medium. The double- stranded DNA template from the colonies was sequenced by the method of Sanger et al . The CDR3 gene sequences of the CSF B-cells were analyzed using the FASTA program, the sequences of expressed D segments were compared with those of the published germline D and DIR segments. The most common sequence from each patient was considered as being derived from the dominantly clonal B-cells. Based on the complete sequence, oligo-nucleotide primer specific for each patient -derived dominant VH, DH and JH region were designed. Antigen-driven selection in the differentiation of the dominantly clonally expanded B-cells In accordance with one embodiment of the invention, the third step is to use Immunoglobulin heavy chain variable region genes assay kit for investigating the role of the antigen-driven selection in the dominant clonal expansion of the CSF B-cells. A semi -nested PCR was performed: The first round, five 250 ng of DNA was amplified with 6 VH family primers and LJH for 40 cycles of 94°C for 1 min, 55°C for 1 min and 72°C for 1 min. The second round, 2 to 5 μl aliquot of the initial PCR product was reamplified with primers 6 VH family primers and the primer of oligonucleotides specific from each patients-derived dominant, for 35 cycles of 94 C for 1 min; 59 C for 1 min and 72 °C for 1 min. Aliquots of the final reaction product were analyzed by electrophoresis in a 1% agarose gel (Sigma) containing ethidium bromide. The amplified VH product was sequenced as described above. All sequences were confirmed by sequencing in both orientations . Assignment of mutations Mutations, identified by comparing each sequence with germline sequences, were defined on the basis of nucleotide changes in the VH segment, with any variability at the joining sites of the VH, D, and JH gene segments not being classified as mutations, since they might result either from the insertion or mutation of N regions. Two nucleotide exchanges in a single codon were scored as one replacement mutation. Results and discussion Clonality In order to analyze B-cell clonality of CSF cells and the cells from surgical biopsy or autopsy specimens, CDR3 (VDJ region) of immunoglobulin genes from these specimens was screened using invented technology. Following electrophoresis, the clonality and quantity of PCR products were estimated by ethidium bromide staining of the agarose gel and the results are shown in Fig 5 and 6. PCR products from all MS and I-OND patients, 3 NI-OND as well as 8 of 45 randomly selected (unknown diagnosis) controls showed high density bands suggesting a possible limited clonality pattern. PCR products from 9 NI-OND cases (cases 4 to 12) and 37 of 45 unknown diagnosis cases, showed only a faint smear or invisible bands, reflecting size differences in the VH, DH, and JH regions in a polyclonal population of B-cells. Monoclonal PCR band also demonstrated from the plaques from 3 of 4 MS patients and 2 PCNSL patients (Fig. 7) . Fig. 7 shows the dominant CDR3 sequences of CSF B- cells. Dominant sequences from each patients are grouped and subdivided into VH, N, D, N, and JH regions. Names of the germline D and JH genes with maximum homology to the segments used in the VDJ joining are shown in parentheses in the appropriate rows . Clonality of CSF B-cells from patients with MS and OND as well as plaques which showed a possible or probable clonal band of PCR products on ethidium- bromide stained agarose gels, was determined by sequencing PCR products of CDR3 genes. CSF B-cells from 10 of 12 MS patients, including the both cases of first clinical onset or multi-episodes as well as MS plaques had dominated clonal or dual clonal expansion. Four (4) of sixteen (16) controls were found to have dominant B-cell clones (Fig. 8) . The case from inflammatory ONDs group showed that CSF B-cell clonal expansion has history of systemic cancer. Three cases from non- inflammatory ONDs: case 1 has been diagnosed as a primary central nervous non-Hodgkin' s lymphoma, in this study, that serves as a control of monoclonal B-cells, • case 2 suffered from headache band for more than 6 months, using current diagnostic technologies no diagnosis was made, was found B-cell clonal expansion occurring in the CSF; case 3 with initially diagnosed as spinal cord infarct, was found B-cell clonal expansion in the CSF. Similar symptom has been reported in the patients who had systemic lymphoma CNS metastasis. Using our novel technology, 8 in 45 randomly selected cases (unknown diagnosis) were identified as having high-density clonal band. Three (3) of these eight (8) cases has been recently confirmed the diagnosis of MS and one (1) of them considered as a possible MS. Other one(l) case with B- cell clonal expansion in the CSF, the diagnosis of Guillian-Barre Syndrome was entertained. MRI showed the multiple hyperintense white matter lesions located in the centrum semi-ovale, corona radiale and the periventricular areas and suggested a microvascular disease . Among the MS cases analyzed, 3 of the 10 MS patients did not have oligoclonal bands detected by CSF electrophoresis. Conversely, 2 MS cases with polyclonal sequences did have CSF oligoclonal bands. In this regard none of the I-OND or NI-OND cases with dominant B-cell expansion had detectable oligoclonal bands (Table 1, Figs. 5 to 9) . This indicates that analysis of CSF oligoclonal band does not define the extent of B-cell clonality. Furthermore, B-cell clonal expansion was found from an individual, who had no detectable lesion in his MRI scan. This new technology can screen B-cell clonal expansion at early stage before CNS lesion appears. When the technique was applied to paraffin embedded autopsy specimens of the plaques from MS and HIV-related primary central nervous system non- Hodgkin's B-cell lymphoma, PCR products illustrated high density of clonal band and sequence analysis demonstrate a dominant B-cell clonal expansion. The polyclonal and monoclonal expansion was detected from the surgical biopsy specimens of encephalomyelitis (Fig. 7) . Using this kits, the B-cell clonal expansion can be identified at very early stage of MS as well as in the late stage of autopsy MS plaques. The invented technology permits examination of a wider array of patients then can be performed on surgical biopsy (invasive diagnosis) and autopsy tissue, and is particularly suited for patients at relatively early stages of disease. Somatic hypermutation The high frequency of dominant clonal expansion of CSF B-cells expressed by MS patients raises the questions whether theses cells are transformed or under the pressure of chronic antigen-driven selection. Positive selection of clones bearing somatic mutations, resulting in an increase in affinity of the surface receptor for antigen, has been shown to be the primary mechanism underlying the process of affinity maturation during a specific immune response. Somatic hypermutation of Ig V region genes is generally believed to occur in the germinal centers of lymphoid tissue. To elucidate these issues, the sequences of VH of CSF B-cells derived from 4 MS patients were analyzed. Figs. 9A to 9D show the DNA and the deduced amino acid sequences of the VH segments of the CSF B-cells from MS patients. The immunoglobulin (Ig) VH gene rearrangement in cases 1, 2, 3 and 10 used gene segments of V71-2; 4dl54; 4d68; and 4d76, which belong to the VH IV families (Table 2) . Table 2 Utilization Of VH Gene Segments Case VH Gene Closest VH Fetal Member Family Germline gene Identity% 1 VHIV V71-2 58P2 94 2 VHIV 4dl54 58P2 94 3 VHIV 4d68 58P2 94 10 VHIV 4d75 58P2 93 The differences in nucleotide and predicted amino acid sequences when compared with the closest known germline VH genes are summarized in Table 3. Gene rearrangement mutations in the framework region (FR) of functional V regions are usually counterselected to preserve the structure of the antibody V domain. This results in replacement to silence (R/S) ratios below those expected on the basis of random mutagenesis. The combined R:S ratios for the FRs and CDRs domains, derived from the sum of all mutated codons in the VH sequences of the CSF B-cells assignable to the germline gene segment, are shown in Table 3. Table 3 Differences in Nucleotide Sequences of Dominant Clone from CSF Of four (4) Cases Compared With Closest Known Germline Genes No. of nucleotide differences R:S Case FR1 CDRl FR 2 CDR2 FR3 Total Total CDRs FRs in FRs in CDRs 1 1 2 2 5 6 7 9 6.0 0.5 2 1 3 2 6 4 9 7 3.0 0.5 3 4 0 0 5 9 5 13 4.0 1.0 10 5 3 1 6 6 9 12 2.0 0.8 For cases 1, 2, 3 and 10, taken together, the average R:S ratio in the framework regions was 0.7 (expected~2.9) . The average R:S ratio in CDRl and CDR2 was 4.0. A higher CDR R:S mutation ratio reflects the positive selective pressure of an antigen on those gene products which come into close contact with antigen, while a lower FR R:S mutation ratio reflects the negative pressure for mutant selection applied to structural components that need to be conserved. This pattern is consistent with the notion of an antigen- driven selection of antibodies with high-affinity antigen-binding sites. Our data provide direct evidence that intrathecal clonally expanded B-cells from the CSF of MS are hypermutated postgerminal center antibody- forming or memory lymphocytes that have undergone antigen selection. Further study is to identify whether specific antigen/s, anti-apoptosis gene(s), virus infection, and/or other molecular events, which have been defined in the PCNSL might be related to the B-cell clonal expansion in the CNS of MS. While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims.

Claims: WHAT IS CLAIMED IS: 1. A method for diagnosis of a neurological disorder in a patient, comprising the steps of: a) subjecting a nucleic acid sequence from a central nervous system sample, cerebrospinal fluid; brain and spinal cord biopsy specimens or autopsy (necropsy) of said patient to amplification of VH, DH, and JH region of VH genes and VL, and JL region of VL genes; and b) analyzing sequences amplified in step a) by detecting amplified sequences of said gene regions wherein said amplified sequences are an indication of increased B-cell expression and thereby neurological disorder in said patient. 2. A method for diagnosis of a neurological disorder in a patient, comprising the steps of: a) subjecting a nucleic acid sequence from a central nervous system sample of said patient to enzyme fragmentation of VH, DH, and JH or VL gene regions ; and b) analyzing fragments produced in step a) by detecting a signal on a southern blot, wherein said signal is an indication of increased B-cell expression and thereby neurological disorder in said patient. 3. The method of claim 1, wherein the nucleic acid sequence of step a) is subjected to PCR amplification. 4. The method of claim 1, 2 or 3, further comprising before step a) the step of isolating the nucleic acid sequence from the central nervous system sample . 5. The method of claim 2, wherein in step b) the fragments are gel-separated prior to the southern blot. 6. The method of claim 1, wherein said sequences detected in step b) are further subjected to the following steps: c) sequencing said sequences; and d) evaluating clone expansion of said sequences by comparing different sequences obtained in step c) , wherein homology of different sequences is an indication of clone expansion, which is an indication of increased B-cell expression and thereby of a neurological disorder. 7. The method of claim 2 , wherein said produced fragments analyzed in step b) are further subjected to the following steps: c) sequencing said fragments; and d) evaluating clone expansion of said sequences by comparing different sequences obtained in step c) , wherein homology of different sequences is an indication of clone expansion, which is an indication of increased B-cell expression and thereby of a neurological disorder. 8. The method of claim 3 , wherein the nucleic acid sequence of step a) is amplified by PCR with FR2 upstream primer and LJH downstream primer. 9. The method of claim 8, wherein nucleic acid amplified is further amplified by PCR with Vcon and VLJH primers . 10. The method of claim 1 or 2 , wherein the central nervous system sample is cerebrospinal fluid (CSF) or brain tissue of said patient. 11. The method of claim 3, wherein the nucleic acid sequence of step a) is amplified by PCR using an upstream primer and a downstream primer derived from CDR3 genes of a B-cell immunoglobulin heavy chain, from variable region genes of a B-cell immunoglobulin light chain, or from a gene of a T-cell receptor γ. 12. The method of claim 11, wherein the upstream primer derived from CDR3 genes of a B-cell immunoglobulin heavy chain is selected from the group consisting of SEQ ID NO:l and SEQ ID NO : 2 , and wherein the downstream primer derived from CDR3 genes of a B- cell immunoglobulin heavy chain is selected from the group consisting of SEQ ID NO: 3 and SEQ ID NO:4. 13. The method of claim 11, wherein the upstream primer derived from variable region genes of a B-cell immunoglobulin light chain is selected from the group consisiting of SEQ ID NO: 5, SEQ ID NO : 6 , SEQ ID NO : 7 , SEQ ID NO: 8, SEQ ID NO : 9 , SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18 and wherein the downstream primer derived from variable region genes of a B-cell immunoglobulin light chain is selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 19. 14. The method of claim 11, wherein the upstream primer derived from a gene of a T-cell receptor γ is selected from the group consisiting of SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28 and wherein the downstream primer derived from a gene of a T-cell receptor γ is selected from the group consisting of SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 31. 15. The method of claim 11, wherein the upstream primer is selected from the group consisting of SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, and SEQ ID NO: 37, and wherein the downstream primer is selected from the group consisting of SEQ ID NO: 38 and SEQ ID NO: 39. 16. A kit for determining B- and T-cell clonality for diagnosis of a neurological disorder in a patient, said kit comprising at least one upstream primer and at least one downstream primer derived from CDR3 genes of a B-cell immunoglobulin heavy chain, from variable region genes of a B-cell immunoglobulin light chain, or from a gene of a T-cell receptor γ. 17. The kit of claim 16, wherein wherein the upstream primer derived from CDR3 genes of a B-cell immunoglobulin heavy chain is selected from the group consisting of SEQ ID NO : 1 and SEQ ID NO: 2, and wherein the downstream primer derived from CDR3 genes of a B- cell immunoglobulin heavy chain is selected from the group consisting of SEQ ID NO : 3 and SEQ ID NO: 4. 18. The kit of claim 16, wherein the upstream primer derived from variable region genes of a B-cell immunoglobulin light chain is selected from the group consisiting of SEQ ID NO : 5 , SEQ ID NO: 6, SEQ ID NO : 7 , SEQ ID NO: 8, SEQ ID NO : 9 , SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, and wherein the downstream primer derived from variable region genes of a B-cell immunoglobulin light chain is selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 19. 19. The kit of claim 16, wherein the upstream primer derived from a gene of a T-cell receptor γ is selected from the group consisiting of SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, and SEQ ID NO: 28, and wherein the downstream primer derived from a gene of a T-cell receptor γ is selected from the group consisting of SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 31. 20. The kit of claim 16, wherein the upstream primer is selected from the group consisting of SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, and SEQ ID NO: 37, and wherein the downstream primer is selected from the group consisting of SEQ ID NO: 38 and SEQ ID NO: 39.

Inventors: QIN YUFEN, QIN, YUFEN
Applicants: QIN YUFEN, QIN, YUFEN
Application year: 1998
Main classification: C12Q1
IPC codes: C12Q1