CA2220245A1 - A novel method for early diagnosis of autoimmunity and lymphoma in central nervous system - Google Patents

Description

CA 0222024~ 1997-11-04 A NOVEL METHOD FOR EARLY DIAGNOSIS
OF AUTOIMMWNITY AND LYMPHOMA IN CENTRAL NERVOUS SYSTEM
BACKGROUND OF THE INVENTION
(a) Field of the Invention The invention relates to a method for early diagnosis of central nervous system (CNS) autoimmunity and lymphoma in non-immunocompromised or immuno-deficient HIV-associated patients as well as the systemic lymphoma CNS metastasis.
(b) Description of Prior Art Auto;mmlln;ty 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 auto;mmlln;ty 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.
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 ;mmlln;ty 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) CA 0222024~ 1997-11-04 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.
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 M~crobiol 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 and selection by which memory or antibody forming B-cells (plasma cells) with high-affinity antibodies 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.
Primary CNS B-cell lymphoma represents one of a large number of extranodal Non Hodgkin's Lymphoma (NHL), which are by definition usually restricted to a single organ at presentation. NHL-CNS appear to be CA 0222024~ 1997-11-04 similar to other NHL of extranodal sites lacking organized lymphoid tissue (e.g., bone, ovary, and testis) and have a tendency to relapse in the same or other extranodal sites, rather than developing as wide-spread nodal disease. 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.
It would be highly desirable to be provided with a method for an early diagnosis of multiple sclerosis and encephalomyelitis or lymphoid malignancy in the central nervous system of a patient with or without HIV infection.

SUMMARY OF THE INVENTION
One aim of the present invention is to provide a method for an earlier diagnosis of multiple sclerosis, brain lymphoma and other CNS diseases.
Another aim of the present invention is to provide a method 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 central nervous system sample, cerebrospinal fluid; brain and spinal cord biopsy specimens CA 0222024~ 1997-11-04 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.
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 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~0 and thereby neurological disorder in said patient.
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 differentiation of B cells in central nervous system. The results from R-R MS showed the high frequency of clonal B-cell evolution in the CNS. The utility of this method has been demonstrated with high sensitivity in patients with early multiple sclerosis; and other neurological diseases, as well as CNS lymphoid malignancy who have no MRI change or oligoclonal bands.

CA 0222024~ 1997-11-04 BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 illustrates an ethidium bromide stained 2~ agarose gel of framework region (FR) 3 RT-PCR
products from CSF cells;
Fig. 2 illustrates a predomin~nt complementarity determining region 3 (CDR3) nucleotide sequence of patients with MS or other neurological diseases; and HIV-positive PCNS non-Hodgkin's B-cell lymphoma; as well as Fig. 3 illustrates 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 15 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 cerebrospinal fluid (CSF); biopsy specimens of the patients with MS; CNS lymphoma and other neurological diseases will provide important evidence for diagnosis of autoimmllnity; malignancy and inflammation in the CNS. Based on this fundamental knowledge, a specific method has been developed and used to amplify VH, DH and JH region of VH; and VL and JL region of VL genes; identify clonal motifs and somatic hypermutations on the Ig VH genes of CSF
B-cells from eight (8) patients with relapsing 3 5 remitting multiple sclerosis (RR MS) (MRI with one to CA 0222024~ 1997-11-04 multiple lesions in CNS) and four (4) patients with early stage MS ~oligoclonal band negative or MRI
without lesion in CNS) and sixteen (16) patients with other neurological diseases (ONDs) who had oligoclonal band negative and with or without MRI changes.
Thirteen (13) of sixteen (16) patients with non-inflammatory ONDs, HIV-positive primary central nervous system (PCNS) non-Hodgkin's B-cell lymphoma;
motor neuron gangliosides (ALS); pseudotumer;
spinocerebellar degeneration spinal cord infarct;
myopathy; headache and neuropathy, and three (3) of sixteen (16) patients with inflammatory ONDs, acute disseminated encephalomyelitis (ADEM) and Herpes Zoster encephalitis (HZEM) The results are show in Fig 1.
The method of the present application enables to identify the monoclonality of the cells in CSF and biopsy or autopsy specimens. Using this method, clonality of B-cells from CSF of patients and autopsy specimen (Fig. 2) has been demonstrated. Fig. 2 illustrates the DNA sequences of CDR3 region from CSF
of MS patients and ONDs as well as PCNS B-cell lymphoma. The sequences from each patients are grouped and subdivided into VH, N, D, N and JH
regions. The name of the germline D and JH genes having a maximum homology to the segments used in the VDJ joining are shown in parenthesis in the appropriate rows. Identity with the CDR3 sequence is indicated with a "-".
In view of the high frequency of clonal B cell evolution (expansion or differentiation) in the Relapsing and Remitting (R-R) MS and Early MS (EMS), here the invention proposes that the clonal motif of the B cells in the CNS ( CSF, brain and spinal cord biopsy or autopsy specimens) one of the important CA 0222024~ l997-ll-04 7 ~

laboratory criteria for diagnosis and differential diagnosis of MS and lymphoma.
Extraction of RNA from CSF
The cell pelletes of CSF from MS patients (n=12) and OND patients (n=16) were used as sources of RNA. Total RNA was extracted using RNAeasy~ kit (Qiagen Inc. Chatsworth, Ca). Table 1 summarizes the clinical and laboratory data for the patients under study.
Table l Sample Sex Age Disease CSF Head MRI Diagnosis PCR
of Duration product Onset (month) MS-1 F 48 <12 normal Multiple RR MShigh plaques density band MS-2 F 31 24 OB+ 2 spinal RR MShigh incr. IgG lesions density index band MS-3 F 24 1 OB Multiple RR MS high incr. IgG plaques density index band MS-4 F 48 12 OB Multiple RR MS high incr. IgG plaques density index band MS-5 M 41First Normal 1 spinal EMS high Attack lesion density band MS-6 F 29First OB Multiple EMS high Attack normal. plaques density IgG index band MS-7 F 41 24 OB Multiple RR MShigh normal IgG plaques no density index treatment band MS-8 F 29 12 OB Multiple RR MS high incr. IgG plaques density index band MS-9 F 21 First Normal Normal EMS density MS-10 F 22 First OB Normal EMS high Attack normal IgG density index band MS-11 F 43 1 OB 3 spinal RR MS high normal IgG lesions no density index treatment band CA 0222024~ l997-ll-04 MS-12 F 21 60 OB Multiple RR MS high incr. IgG plaques density index band NI F 68 First OB Headache high OND-1 normal IgG bands density index band NI F 66 First OB Spinal cord high OND-2 normal IgG infarct density index band NI Autopsy Multiple HIV- high OND-3 specimen plaques PCNS B_cell debnSity Lymphoma NI M 47 First OB Pseudotumer high OND-4 normal IgG density index band NI F 68 First OB Neuropathy Faint OND-5 normal IgG smear index NI M 39 First OB Migraine Faint oND-6 normal IgG smear index NI M 67 First OB ALS Faint oND-7 normal IgG smear lndex NI M 32 First OB ALS Faint OND-8 normal IgG smear index NI F 40 First OB Pseudotumer Faint OND-9 normal IgG smear index NI M 60 First OB Hydro- Faint OND-10 normal IgG cephalus smear index NI M 36 First OB Neuropathy Faint OND-11 incr. IgG smear index NI M 44 First OB Spino- Faint OND-12 normal IgG cerebellar smear index degeneratio n NI M 57 First OB Neuropathy Faint OND-13 normal IgG smear index IN F 64 First OB Multiple ADEM high OND-l normal IgG lesions density index band IN F 24 First OB ADEM high OND-2 normal IgG density index band IN F First Herpes high OND-3 Zoster density encephaliti band s CA 0222024~ 1997-11-04 g RR MS: Relapsing Remitting Multiple Sclerosis EMS: Early Multiple Sclerosis NI OND: Non-Inflammatory Other Neurological Disease IN OND: Inflammatory Other Neurological Disease ALS: Motor Neuron gangliosides ADEM: Acute Disseminated Encephalomyelitis Amplification VH, DH, and JH gene of CSF B-cells In accordance with one embodiment of the present invention, the first step used is RT-PCR to screen the Ig gene rearrangement of the CSF B-cells in patients with neurological diseases. Nested amplifications of 2 to 5 ~1 of cDNA with consensus primers were initially amplified in a final volume of 50 ul reaction buffer [50 mM Tris-HCL, PH 9.0 at 25~C:
20 mM ~NH4)2SO4; 3.0 mM MgCL2 ] containing 2 units of recombinant Taq Polymerase, 20 pmol of an upstream primer (FR2 5'-TGG[A/G]TCCG[C/A]CAG[G/C]C[T/C][T/C]
CNNG G-3' or Vcon: 5'-ACACGGCTGTGTATT-3'), designed using a sequence of framework region (FR) 2 or 3, and 20 pmol of downstream primer (LJH: 5'-ACCTGAGGAGACGGT-3'), designed to match the 3' ends of the six JH
segments. After 5 minutes of denaturation at 94~C, samples were amplified for 40 cycles, each cycle consisting of 50 seconds at 94~C, one minute at 55~C, and 40 seconds at 72~C. Reamplification of a 2 ul aliquot (1%) of the initial PCR product with 20 pmol primers Vcon and VLJH (5'-GGTGACCAGGGTCCCTTGGCCCCAG-3') was performed for 35 cycles [cycle conditions:denaturation step at 94~C for 50 sec; and an annealing/extension step at 59~C for 40 sec]. Aliquots of the final reaction product were analyzed by electrophoresis in a 1.5% agarose gel (Sigma) containing ethidium bromide. The following primer CA 0222024~ 1997-11-04 combinations in the once or nested PCR are also used in this invention:
upstream primers for analysis of VH chain:
VHLl 5'ATGGACTGGACCTGGAGG-3' VHL2 5'-ATGGACATACTTTGTACCAC-3' VHL3 5'-ATGGAGTTTGGGCTGAGC-3' VHL4 5'-ATGA~ACACCTGTGGTTCTT-3' VHL5 5'-ATGGGGTCAACCGCCATCCT-3' VHL6 5'-ATGTCTGTCTCCTTCCTCAT-3' VHl 5'-CAAGGCTTCTGGATACACCTTCACC-3' VH2 5'-CACCTTCTCTGGGTTGTCAGTCACC-3' VH3 5'-TGCAGCCTCTGGATTCACCTTCAGT-3' VH4 5'-CGCTGTCTCTGGTGGCTCCATCAGC-3' VH5 5'-TAAGGGTTCTGGATACAGCTTTACC-3' VH6 5'-TGCCATCTCCGGGGACAGTGTCTCT-3' FRl 5'-TCTGAGGTGCAGCTGGTGGAGTCTG-3' or FR2 5'-TGG[A/G]TCCG[C/A]CAG[G/C]C[T/C][T/C]
CNNGG-3' or Vcon 5'-ACACGGCTGTGTATT-3' with downstream primers:
JH 5'-CCCTGGACCAGTGGCAGAGGAGT-3' or C~ 5'-GAATTCTCACAGGAGACGAGGGG-3' C~ 5'-TTCTTCCTCTAGAAGGCGACCGGT-3' C~ 5'-AAGTAGTCCTTGACCAGGCAG-3' Ca 5'-GGGTCAGCTGGGTGCTGCTGG-3' C~ 5'-TGTAAGGGAGGTACGGTGGAGGCA-3' CA 0222024~ 1997-11-04 FOr the ana1YSiS Of VL Chain, the fO11OWing PrimerS
are USed in thiS inVentiOn:
the PrimerS fOr VK Chain UPStream PrimerS
D/W 5'-ATGGACACGAGGGCCCCCACTCAG-3' 18/19 5'-ATGGA~ACCCCAGCGCAGCT-3' L4 5'-ATGGACATGAGGGTCCCCGCTCAGCTC-3' RP 5'-ATGGGGTCCCAGGTTCACCTC-3' BL41 5'-ATGAGGCTCCCTGCTCAGCTCCTG-3' EB15 5'-ATGGTGTTGCAGACCCAGGT-3' With dOWnStream Primer CK3 ~ S5'-GCTCTTTGTGACGGGC
the PrimerS fOr V~ Chain UPStream PrimerS:
LII 5'-ATGGCCTGGGCTCTGCTGCTC-3' LIII 5'-ATGGCATGGATCCCTCTCTT-3' BL2 5'-ATGACCTGCTCCCCTCTCCT-3' 4A 5'-ATGGCCTGGACTCCTCTCTTTCTG-3' L~EV 5'-ATGGCCTGGGCTCCACTACT-3' With dOWnStream Primer C~ 5'-TATGAACATTCTGTAGGGGCCAC-3' SeqUenCing VH, DH, and JH (CDR3 regiOn) geneS Of the CSF B_Ce11S
The SeCOnd SteP in aCCOrdanCe With One embOdiment Of the PreSent inVentiOn iS SeqUenCing Of PCR PrOdUCtS fOr fUrther identifiCatiOn Of C1Ona1itY

CA 0222024~ l997-ll-04 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. (Sanger F, et al., Proc Natl Acad Sci USA 74:5463, 1977). 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 was considered as being derived from the dominantly clonal B-cells. Our criteria for establishing such clonal relatedness are identity at the somatically formed VH, DH, and JH and use of the same sets of VH, DH, and JH.
Based on the complete sequence, oligonucleotides specific for each patient-derived domin~nt VH, DH and JH region were designed.
Amplification and sequencing the VH gene expressed by ~mi ~t CSF B-cell clone In accordance with one embodiment of the invention, the third step used is to investigate the role of the antigen-driven selection in the ~omin~nt clonal expansion of the CSF B-cells. Five (5) ~l of cDNA was amplified with 6 VH family or family leader primers and patient-specific CDR3 primers or VLJH
primer in a reaction volume of 50 ul. The final concentrations of reagents in the solution were the same as described above. After five minutes of denaturation at 94~C, the samples were amplified for 40 cycles, each cycle consisting of 1 minute at 94~C, CA 0222024~ 1997-11-04 1 minute at 59~C and 1 minute at 72~C, and with final extension at 720C for 10 minutes. 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 using the FASTA
program and the GenBank~ Database (Genetics Computer Group of the University of Wisconsin), 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
Screening the Ig VH gene rearrangement of CSF B cells from MSi B-cell lymphoma and OND
In order to screen Ig gene rearrangement of the CSF B-cells, an analysis of the VDJ rearrangement of IgH was undertaken. The first strain cDNA from eight patients with R-R MS, four early MS and thirteen patients with NI-ONDs, headache; spinal cord infarct;
HIV-positive Primary Central Nervous System (PCNS) Non-Hodgkin's B-cell lymphoma; ALS; pseudotumer;
spinocerebellar degeneration; myopathy; and neuropathy, and three patients with IN-ONDs, ADEM;
HZEM was amplified by RT-PCR technique (Table 1).
Following electrophoresis, the quantity of PCR
products were estimated by ethidium bromide staining of the agarose gel. Compare to patients with NI-ONDs (NI-OND cases 5 to 13), the increase of Ig gene rearrangement was detected from CSF B cells of the all CA 0222024~ l997-ll-04 patients with RR MS (RR MS cases 1 to 8) and early MS
(EMS cases 9 to 12) and IN-ONDs (IN-OND cases 1 to 3), in a pattern of a high density band. In the controls with NI-OND, the increase of Ig gene arrangement was observed in four of thirteen cases (NI-OND cases 1 to 4). The RNA from Nine (9) NI-ONDs (NI-OND cases 5 to 13), only a faint smear, reflecting size difference in the VH, DH, and JH region in a polyclonal population of the CSF B cells, was seen (Fig.1). The screen of the VH gene rearrangement from CSF B cells provide us first information of the involvement of the B-cell immune response in the neurological disorders. They could be inflammatory antibody-forming or memory B
cells, and/or low-grade or high-grade extranodal B-cell lymphoma. Analyzing the pattern of PCR productsband could provide an estimate of clonal or polyclonal B-cell evolution (expansion or differentiation) in the CNS of the patients. To distinguish between inflammatory B cells and B-cell lymphoma, the nucleotide sequence of clonal motifs of increased CSF
B cells was next investigated.
Nucleotide sequence of clonal motif of CSF B-cells The PCR fragments which showed high density bands were cloned into the pGEM~ T vector and at least six (6) to eight (8) colonies were sequenced for each patient. The VH, DH, and JH region sequences from six (6) of eight (8) R-R MS and all four (4) EMS cases showed the dominant monoclonal ~cases 1, 2, 3, 5, 6, 7, 8, 9 and 10) or two clonal (cases 4) sequence/s per case. The same phenomenon was observed in the CSF B
cells from NI-ONDs (NI-OND cases 1, 2 and 3) and one of three IN-ONDs (IN-OND case 1~ who had the increase of VH, DH, and JH gene rearrangement. In identical clones from CSF B cells of each case, the VH-N-D-N-JH
regions were identical both in length and sequence CA 0222024~ 1997-11-04 (Fig. 2). The domin~nt clones from R-R MS cases 1, 2, 3, 4 and EMS cases 5 and 10 appeared to contain 2 DH
gene segments: DM1 in the inverse orientation, rearranged to A1 in the forward orientation (R-R MS
case 1); DN1 in the forward orientation, rearranged to LR4 in the inverse orientation (R-R MS case 2); LR3 in the inverse orientation, combined with XP3 in the forward orientation (R-R MS case 3); DN1 combined with A1, both in the forward orientation (R-R MS case 4);
DN1 in the forward orientation, rearranged to XP'1 in the inverse orientation (EMS case 5); and XP'1 rearranged to LR4, in the forward orientation (EMS
10). R-R MS cases 7, 8 and EMS 6, 9 used one DH gene segment: XP3 in the forward orientation (EMS case 6);
LRl in the forward orientation (R-R MS case 7)); Xp'1 in the forward orientation (R-R MS case 8); and XP'1 in the forward orientation (EMS case 9). Two B-cell clones equally dominantly selected in the CSF was found in R-R MS cases 4. From non-inflammatory OND, the dominant clones are LR4 combined with LR2 in the forward orientation used by NI-OND case 1; XP'1 combined with DA5, in forward orientation (NI-OND case 2) and XP4 in the forward orientation used by NI-OND
case 3. XP'1 in the forward orientation used by IN-OND
case 1. The different sequences of VH, DH, and JH were found in the PCR products with high density band from CSF B cells of two patients with R-R MS (R-R MS cases 11 and 12); one patient with NI-OND (NI-OND case 4) and two patients with IN-OND (IN-OND cases 2 and 3), showed clear differences, both in length and sequence.
The high frequency of B-cell clonal expansion in the CSF of relapsing-remitting and early MS might be indicative of a monoclonal disease process with a low malignant potential, similarly to low-grade CA 0222024~ 1997-11-04 mucosa-associated lymphoid tissue (MALT) B-cell lymphoma.
The dominant CDR3 region nucleotide sequences from each case were synthesized as patient-clone-specific primers. Each patient-derived primer specifically amplified only the VH gene from the corresponding CSF sample.
VH family and somatic mutation The entire sequences of VH genes from CSF
B-cells of five cases with RR MS were further amplified with each patient-specific-CDR3 primer and 5' VH family specific leader region primers. Fig. 3 shows the nucleotide and deduced amino acid sequences of the VH segments of the CSF B-cells. Homology of sequences are identified with "-". Lowercase letters are used to identify substitution which do not affect the amino acid sequence (silent mutation) and the uppercase letters are used to identify mutations which affect the amino acid sequence.
The immunoglobulin (Ig) VH gene rearrangement in cases l; 2, 3 and 4 used VH gene segments of the VHIV
families V71-2; 4dl54; 4d68; and 4d76 which derived from fetal line 58P2, while that in case 5 used VH
gene segments of the VHIV and VHI, respectively. The differences in nucleotide and predicted amino acid sequences when compared with the closest known germline VH genes are summarized in table 2. The VH
gene sequence in case 1 showed the highest degree of similarity (94%) with the germline sequence V71-2 gene, which is more than 98% identical with the "fetal" gene, 58P2; it contained 16 substitutions, 7 CA 0222024~ l997-ll-04 occurring in the CDRs and all resulting in amino acid substitutions. The ratios of replacement (R) to silence (S) mutations in the CDRs and FRs were 7 and O.5, respectively. The VH gene sequence of case 2 was 94% identical with the germline 4dl54, which is more than 94% identical with the "fetal" 58P2 gene; 16 substitutions were found in the VH gene. The R:S
ratios in the CDRs and FRs were 3.5 and O.7. The VH
gene sequence in case 3 displayed the highest degree of similarity to the germline 4d68 (94%), the counterpart of the "fetal" 58P2 gene; it contained 18 substitutions and the R:S ratio in the CDRs and FRs was 4 and 1.6, respectively. The VH gene sequence in case 4 was 93% identical with the germline sequence 4d76, which is more than 94% identical with the "fetal" 58P2 gene. The VH gene sequence in case 5 was 85% identical with the germline sequence, hvlflO~
which is 93% identical to the "fetal" 20P3 gene, it contained 34 substitutions, distributed throughout the sequence. The R:S ratios were 14 and 2.1 in the CDRs and FRs, respectively.
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 2. Taken together cases 1, 2, 3, 4 and 5, the average R:S ratio CA 0222024~ 1997-11-04 in the framework regions was 1.27, as expected for a part of the antibody molecular structure essential for the overall maintenance of protein structure. However, the average R:S ratio in CDR1 and CDR2 was 7.1. This pattern is consistent with the notion of an antigen-driven selection of antibodies with high-affinity antigen-binding sites.

Table 2 10 Utilization Of VH Gene Segments Case VH Gene Closest VH Fetal Member Family Germline gene Identity %

VHIV v71-2 58P2 94 2 VHIV 4dl54 58P2 94 3 VHIV 4d68 58P2 94 4 VHIV 4d75 58P2 93 VHI hvlflO 20P3 85 Table 3 Differences In Nucleotide Se~l~ncec Of Dominant Clone From CSF Of Four (4) Cases Compared With Closest Known Germline Genes No. of nucleotide differences R S
Case FR1 CDRl FR2 CDR2 FR3 Total Total CDRs FRs in CDRs in FRs 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 4 5 3 1 6 6 9 12 2.0 0.8 7 3 1 12 11 15 19 8.0 1.3 CA 0222024~ 1997-11-04 Analysis of the pattern of somatic mutation 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 2. Taken together cases 1, 2, 3, 4 and 5, the average R:S ratio in the framework regions was 1.27, as expected for a part of the antibody molecular structure essential for the overall maintenance of protein structure. However, the average R:S ratio in CDR1 and CDR2 was 7.1. This pattern is consistent with the notion of an antigen-driven selection of antibodies with high-affinity antigen-binding sites.
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 (10)

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.