WO1998036091A1

WO1998036091A1 - Method of determining susceptibility to late-onset alzheimer's disease and dementia with lewy bodies

Info

Publication number: WO1998036091A1
Application number: PCT/GB1998/000467
Authority: WO
Inventors: Christopher Miles Morris
Original assignee: Medical Research Council
Priority date: 1997-02-14
Filing date: 1998-02-13
Publication date: 1998-08-20
Also published as: AU6223098A

Abstract

The present invention relates to a method of determining susceptibility to late-onset Alzheimer's disease and dementia with Lewy bodies in an APOE-e4 negative patient comprising determining the HLA-DR polymorphism type of the patient.

Description

Method of Determining Susceptibility to late-onset Alzheimer's Disease and Dementia with Lewy Bodies

The present invention relates to genetic testing. Specifically, the present invention relates to a method of determining a person's susceptibility to Alzheimer's disease and dementia with Lewy bodies based on detecting the presence of particular HLA-DR antigen polymorphisms on chromosome 6 of the human genome. More particularly, the invention concerns the detection of polymorphisms in the HLA-DR subregion of Class II major histocompatibility complex (MHC) loci, to determine susceptibility to late- onset Alzheimer's disease (AD) and dementia with Lewy bodies (DLB) in patients that are negative for the APOE-e4 allele.

Alzheimer's disease is a devastating disorder characterised by a progressively worsening and debilitating dementia. Whilst some intellectual dysfunction is a natural result of the aging process, the dementia caused by Alzheimer's disease is by no means normal.

Alzheimer's disease affects millions of individuals of all races and ethnic backgrounds and is the most common disease underlying senile dementia. The number of sufferers is expected to expand markedly as the proportion of the aged in the population increases (Plum, 1979) . After onset of the disease, life expectancy ranges from only five up to twenty years . The course of the disease is different for each individual and often infection or malnutrition is the ultimate cause of death.

Whilst neurodegeneration associated with Alzheimer's disease probably accounts for 50-60% of cases of dementia in elderly patients, neuropathologic autopsy studies have reported Lewy bodies in the brainstem and cortex of approximately 15-25% of elderly demented patients, constituting the largest pathologic sub group after pure AD (McKeith et al., 1996).

The high frequency, poor prognosis and heavy emotional, economic and medical costs of these diseases have prompted a search for genetic markers to facilitate the prediction of those individuals who are at risk, their early diagnosis and thus their potential treatment.

Onset of the disease before age 60 (early-onset Alzheimer's disease) is infrequent and caused either by mutations in the presenilin-1 or 2 genes on chromosome 14 and 1, respectively or by a mutation in the amyloid protein precursor gene on chromosome 21.

In late-onset AD, initial evidence that implicated a locus on chromosome 19 was confirmed by the finding of an association between AD and the apolipoprotein E locus (APOE) on this chromosome in 1993 (Strittmatter et al., 1993). In addition, an intronic polymorphism in the presenilin-1 gene may be associated with this disorder (Wragg et al., 1996).

The pathology of the disease involves characteristic brain lesions consisting of β-amyloid-containing neuritic plaques (NP) and neurofibrillary tangles (NFT) (Khachaturian, 1985; Braak and Braak, 1994). Because reactive glial cells surround NP and NFT it is thought that they may play an important role in the pathogenesis of AD. These cells are found throughout the brain even in the early stages of the disease . Products that are expressed specifically or predominantly by reactive glial cells include APOE, HLA-DR antigens, complement proteins and cytokines. The levels of the latter three protein types are increased in the brains of sufferers of AD (Boyles et al., 1985; Dickson and Rogers, 1992; Barron, 1995) .

Lewy bodies are intracytoplasmic, spherical, eosinophilic neuronal inclusion bodies occurring particularly in the brainstem, sub-cortical nuclei, limbic cortex (cingulate, entorhinal, amygdala) and neocortex. Cortical lewy bodies are readily visualised with antiubiquitin immunocytochemical detection. ..Differing interpretations of the relative significance of Lewy body (LB) and AD pathology have led to various terminologies being applied to these cases including LB variant of AD, AD with Parkinson's disease changes, dementia associated with cortical LB, diffuse LB disease and senile dementia of LB type. As used herein, the term dementia with Lewy bodies (DLB) encompasses these terms.

Pathological studies of deceased AD and DLB patients provide an insight into the biochemical nature of the disease. Although such studies may well lead to the eventual discovery of a treatment or cure for AD and DLB, at present no such cure is available. Particularly in its early stages, the symptoms of AD and DLB are shared by many other forms of senile dementia. Therefore, whereas the pathological findings of neurofibrillary tangles in AD and Lewy bodies in DLB are unequivocal, in the living patient differential diagnosis is largely by clinical exclusion from other forms of dementia. The debilitating nature of the diseases in their later stages means that early prognosis would greatly ease the great emotional trauma and financial burden that these diseases presents to both the patient and family.

There is thus a great need for a diagnostic test that could determine an individual's susceptibility to developing late- onset Alzheimer's disease and dementia with Lewy bodies and/or could refute or substantiate a diagnosis of late- onset Alzheimer's Disease and dementia with Lewy bodies.

Description of the invention

According to the present invention there is provided a method of determining susceptibility to, or of supporting a diagnosis of late-onset Alzheimer's disease and dementia with Lewy bodies in an APOE-e4 negative patient comprising determining the HLA-DR polymorphism type of the patient.

In man, the class II (D) region of the MHC contains three subregions, HLA-DP, DQ and DR. In each individual, there may be a number of genes, depending on the haplotype of the cell. There is only one α gene (DRA) with which the product of the functional β genes (DRB) associate. The products of the various HLA-DRB genes give rise to a number of serologically-detectable differences between individuals, that arise from distinct antigenic determinants present in the polypeptide chain.

An increased frequency of particular HLA alleles has been noted in affected individuals in both population and family studies, in connection with various diseases. The strongest association is found in narcoleptic patients, all of which have the specificity HLA-DR2. An increased association has also been noted for the HLA-DR genes, for insulin-dependent diabetes mellitus and rheumatoid arthritis.

It has been found that an association exists between the expression in a patient of certain HLA-DR antigen types and the susceptibility of that patient to late-onset AD and dementia with Lewy bodies. Thus, according to the present invention, there is provided a method of determining the susceptibility to AD in an APOE-e4 negative patient comprising determining whether the patient expresses an HLA- DR antigen type selected from the group comprising HLA-DR 1, 2, 3, 4, and 6..

It has been found that the effects of HLA-DR antigens can be masked by the^' presence of the dominant APOE-e4 allele in the genome of the patient. Hence, in the present invention the presence of APOE-e4 is eliminated when determining susceptibility to this disease through HLA-DR typing. Thus, according to the present invention there is provided a method of determining susceptibility to late-onset Alzheimer's disease and dementia with Lewy bodies comprising determining both the APOE-e4 genotype and the HLA-DR antigen type of the patient. The invention further provides use of HLA-DR typing in a method of determining susceptibility to late-onset Alzheimer's disease and dementia with Lewy bodies in an APOE-e4 negative patient.

It is shown herein that when AP0E-e4 positive cases are discounted, HLA-DR1, 2 and 3 are associated with an increased susceptibility and HLA-DR4 and 6 a decreased susceptibility to late-onset AD. When both HLA-DR alleles present in an APOE-e4 negative patient are taken into account, the combined effect of the separate HLA-DR1, 2 and 3 alleles gives as much as a 10-fold greater risk of development of the disease over the risk carried by patients who ^• do not carry these alleles. The combined effect of HLA-DR4 and 6 gives as much as a 10-fold decrease in this risk.

By APOE-e4 negative patients is meant those patients that do not possess an APOE-e4 allele. The APOE genotype has been established as the single most important genetic determinant of susceptibility to sporadic and late-onset familial AD yet identified.

Approximately 65% of the population are APOE-e4 negative, and as many as 25-50% of cases of late-onset AD occur in the absence of APOE-e4. The typing of HLA-DR therefore provides for the first time a clinically useful diagnostic test to support or refute a diagnosis of AD. The typing of HLA-DR may also provide the basis of a screening test to identify persons most at risk from developing the disease.

APOE has an important role in blood plasma as a transporter of cholesterol and as a modulator of atherogenic lipoprotein metabolism. It exists in three common allelic forms: APOE- e2, APOE-e3 and AP0E-e4 (Morris et al . , 1996). These three alleles encode forms of the apolipoprotein which differ by amino acid substitutions at one or both of two sites, imparting distinctive physical and biochemical properties to each isoform. It has been reported that a total of 80% of familial and 64% of sporadic late-onset cases have at least one APOE-e4 compared to only 30% of control subjects. This finding has implicated APOE-e4 as an important factor in the aetiology of more than half of all AD (Corder et al . , 1993) .

The biochemical basis of the APOE-e4 association is unknown. It has been suggested that the various APOE isoforms may differentially affect amyloid decomposition, tangle formation, neuronal plasticity, cholinergic function and other biological aspects of the pathophysiology of AD.

HLA-DR and APOE typing of a patient are carried out ex vivo. APOE typing is preferably carried out as described in Benjamin et al., 1995. Assessment of HLA-DR polymorphism type may be either through the use of specific antibodies directed against the antigenic determinants of the HLA-DRB polypeptides or by analysis of the genotype of the patient. Preferably, typing is by genetic analysis of the HLA-DRB locus .

In order to ascertain the genotype of a patient, a sample of the DNA of that patient must be available. This sample may be obtained from any tissue of the body and does not have to be a brain biopsy, with which obvious problems are associated in the live patient. Commonly-used tissues for biopsy are the blood, buccal epithelium, skin or hair.

Molecular DNA typing of the HLA-DR gene locus may be carried out by detection and assignation of the DNA polymorphisms in the HLA-DRB gene through the use of various techniques that will be well known to those of skill in the art. There are three preferred methods. These are first the detection of restriction fragment length polymorphisms (RFLPs) ; second, Southern blotting of PCR-amplified DNA using specific probes; and third, direct sequencing of PCR products. The latter method, which although more laborious is more stringent, is generally the preferred method of the present invention.

One embodiment of the invention involves the detection of RFLPs. RFLPs are changes in a specific DNA (termed a polymorphism if the differences between human individuals occur more frequently than every 10⁷ bases) that may be traced using restriction enzymes. When a polymorphism occurs in a consensus sequence that is recognised by a particular restriction enzyme, so that this sequence is no longer recognised, the DNA fragments produced by restriction enzyme digestion will be of different sizes. The various possible fragment sizes from a given region therefore depend on the precise sequence of the DNA in the region. This variation in the fragment sizes is termed a restriction fragment length polymorphism (RFLP) , and can be visualised by separating the DNA according to its size on an agarose gel.

The individual fragments may be visualised by annealing to a labelled oligonucleotide probe that is specific for the sequence of the fragment of interest. In the case of the present invention, the sequence of interest is the HLA-DRB gene locus. More preferably, the sequence of interest is exon 2 of the HLA-DR gene locus. Various methods of labelling the probe will be known by those of skill in the art and will most commonly involve the use of radioactivity or fluorescent or enzymatic tags.

According to the present invention, the more preferred method of detection of polymorphisms is through the amplification of a DNA fragment which is then analysed using probes specific for the particular polymorphism of interest. Alternatively the amplified DNA fragment may be sequenced directly. Preferably, the DNA fragment is amplified using the polymerase chain reaction (PCR) .

The amplified DNA fragment may comprise any portion of the HLA-DRB gene that is sufficiently diverse in sequence to allow it to be distinguished from other HLA-DRB types. The region of choice from which the fragment should be isolated is most preferably the second exon of HLA-DRB. This exon is highly polymorphic and is the most widely divergent sequence in each of the HLA-DRB types (Middleton et al., 1993).

A diagnostic length of DNA may be amplified by PCR using primers raised to conserved DNA sequence in the HLA-DRB gene. By a diagnostic length is meant a fragment of sufficient length to allow discernment of the characterising polymorphisms of each HLA-DR antigen type. Thus, the fragment must be of sufficient length to allow an oligonucleotide primer to hybridise specifically with this sequence. As will be apparent to those of skill in the art, this fragment of DNA is of at least 50 bases, preferably 100 bases, and most preferably more than 400 bases in length.

The primers used to amplify the DNA fragment may be designed by anyone of skill in the art. Most preferably the primers DRB AMP-A and DRB AMP-B (Kimura and Sasazuki, 1992) are used to amplify a short fragment of the HLA-DRB gene surrounding the second exon. Preferably the reaction conditions for PCR are as described in Kimura and Sasazuki, 1992.

The PCR product can be purified and immobilised for hybridization by methods commonly used in the art. The fragment may be purified by submarine gel electrophoresis and immobilized on membranes (Boehringer) as described in Middleton et al . , 1993 and Kimura and Sasazuki, 1992.

For analysis by Southern blotting, the purified and immobilised PCR product is challenged with 18 labelled sequence-specific probes (described in Middleton et al., 1993 and listed in Table 1 herein) . Each specific probe comprises an oligonucleotide of complementary sequence to the particular defining polymorphic region of exon 2 of the HLA-DRB locus. These probes are directed against the most highly polymorphic areas of the gene and are therefore specific for each HLA-DR type. Under conditions of a certain stringency (as defined in Middleton et al., 1993), each oligonucleotide will only hybridize to the polymorphic DNA sequence against which it is raised and thus will provide HLA-DR typing with much more accuracy than is possible using serological methods. A further probe capable of specific binding to all HLA types may be used as a control.

The method of detection of bound probes may be by any one of the methods commonly used in the art. Preferably the probes themselves are labelled, either by radiolabelling, or by chemical modification eg. using digoxigenin (Kimura and Sasazuki, 1992; Boehringer Mannheim catalogue) . Detection may be by autoradiography, or by chemiluminescence respectively, depending on the system chosen. Most preferably, the invention uses digoxigenin-labelled oligonucleotides .

When using digoxigenin-labelled oligonucleotides, a labelled anti-digoxigenin antibody-enzyme conjugate is used for the detection of oligonucleotide. This specific reaction can be visualised by chemiluminescent detection using an AMPPD TABLE 1

π n

ι σ>

TABLE 1 CONT/ .

(A

C

DJ 0⁾

H

C H m en NJ x m m

H c m r-

N3 O)

substrate in accordance with the manufacturer's instructions (Boehringer Mannheim) . In the preferred embodiment of the invention, the conjugated enzyme comprises an alkaline phosphatase conjugate.

A preferred method of detection is by direct sequencing of the PCR products. This method is commonplace and will be well-known to those of skill in the art. Briefly, the initial PCR product is subjected to a second amplification employing an Applied Biosystems sequencing kit, as described in Morrison et al. 1993. The product is purified twice using phenol/chloroform and then precipitated using ethanol. For the sequencing reaction, the DNA is loaded onto a 6% polyacrylamide gel, before direct sequencing is performed in both forward and reverse directions (in triplicate) using fluorescence-labelled dideoxynucleotide termination on an Applied Biosystem 373A Automated DNA Sequencer. Alternative sequencing kits, PCR purification kits and automated sequencers are readily commercially available and may be employed in the present invention.

All three detection methods also lend themselves readily to the formulation of kits which can be used in diagnosis. The reagents suitable for applying the method of the invention to detect the appropriate polymorphisms may be packaged into convenient kits providing the necessary materials to carry out the molecular biological reactions that are described above. These are packaged into suitable containers or supports useful for performing the assay.

The essential components of the assay vary depending upon which embodiment of the invention is to be utilised. Regarding the detection of RFLPs, the essential components of the assay include the restriction enzyme associated with the polymorphism and the specific probe. Additionally, packages containing concentrated forms of reagents and buffers used for hybridization, prehybridization, DNA extraction and the like may be included. In particular however, labelled probe, or reagents suitable to form conveniently labelled probe are useful in facilitating the conduct of this method of the invention.

In connection with the amplification of DNA fragments using PCR and their subsequent analysis using specific probes, the essential components of the assay kit will include the thermostable . DNA polymerase enzyme associated with amplification of the DNA fragment and a suitable probe. For direct sequencing of PCR products, the essential components are the specific primers, a suitable thermostable DNA polymerase enzyme, ATP, the mixed nucleotide units for extension of the nucleotide chain, and fluorescent-labelled dideoxynucleotide termination products.

All documents mentioned in the text are incorporated by reference.

Various aspects and embodiments of the present invention will now be described by way of example and illustrated with reference to the figures. It will be appreciated that modification of detail may be made without departing from the scope of the invention.

Examples:

DNA was extracted from the frozen brain tissue of 45 (16 males; 29 females) late-onset AD cases and 28 (13 males; 15 females) aged-matched control patients. The age at death of the AD cases ranged from 70-93 with a mean age of 81, while that of the control cases ranged from 66-94 with a mean age of 77. The catchment area for both AD cases and controls was the North East of England. All AD brains contained numerous NP and NFT and brains with a pathological diagnosis of Lewy body dementia or with significant vascular pathology were not included in this study. Control cases showed little or insignificant degrees of NP and NFT. For details of neuropathological methods and diagnostic criteria see Perry et al. , 1990.

The APOE genotype of all cases used in this study was determined according to the method of Benjamin et al., 1995) .

Molecular DNA typing of the HLA-DR locus was carried out according to previously published methods (Kimura and Sasazuki, 1992; Nevinny-Stickel et al . , 1991; Middleton et al., 1993). Briefly, the highly polymorphic second exon of HLA-DRB was PCR-amplified from DNA samples extracted from each brain using the primers DRB AMP-A and DRB AMP-B. PCR products were then challenged with 19 digoxigenin-labelled sequence specific oligonucleotide probes. An alkaline phosphatase labelled anti-digoxigenin antibody was used to detect bound digoxigenin and this reaction was visualised by chemiluminescent detection, using an AMPPD substrate (Boehringer Mannheim) .

Specific HLA-DR types were deduced from the pattern of hybridization. While it was not possible to define and assign all the known DRB1 allele specificities to the samples tested due to the low resolution typing scheme employed, we could however assign each sample into broad DR antigen types (i.e. DR1, DR2, DR3, DR4 etc.) based on the allelic specificities obtained. The allele specificities DRB1^*13 and DRB1^*14 were grouped together as DR6.

Comparison of all AD cases (45) with controls (28) (see Table 2) showed that there were 18 (20%) DR1 antigens in the AD cases compared to only 3 (5.4%) in the controls (p=0.011). An inverse association was shown for DR6, there being only 5 antigens present in the 45 AD cases (5.6%) and 10 antigens present in the control cases (17.9%) (p=0.019). When cases with an APOE e4 allele were excluded from the analysis the -difference between AD and controls was even more marked. In the 12 AD cases there were 8 DR1 (33.3%) compared to 1 (2.3%) in the 22 control cases (p=0.001), while for DR6 there were 0 cases in the AD group but 9 (20.5%) in the control cases (p=0.014). In addition to DR1 and DR6 it was found that there were significant differences between AD and control cases lacking the APOE e4 allele in the frequency of DR2 , DR3 and DR4. There were 6 (25%) DR2, 5 (20.8%) DR3 and 1 (4.2%) DR4 antigens in the 12 AD cases compared with 7 (15.9%), 1 (2.3%) DR3 and 12 (27.3%) DR4 antigens in the 22 control cases (p=0.274 for DR2 and p=0.018 for DR3 and DR4 ) . There was no difference in DR antigen frequencies between AD and control groups possessing the APOE e4 allele. In the APOE e4 negative groups (Table 3) 10/12 AD cases had a DR1 and/or DR3 antigen compared to 2/22 in the control group (odds ratio 50, 95% Cl 6.1-407.9). DR4 and/or DR6 were present in 1/12 cases compared to 18/22 control cases (odds ratio 0.02, 95% Cl 0.002 - 0.203). TABLE 2

cn c

00 cn

H

m cn x m m -H c m r t

APOE e4(+) - cases possessing for the e4 allele. APOE e4(-) - cases not possessing the e4 allele.

Statistical analysis for HLA-DR8, 9, 10, 11 and 12 was not performed due to the low frequency of these antigen types.

Table 1: Comparison of HLA-DR antigen frequencies between late-onset AD cases and aged matched controls.

TABLE 3

APOE €4(-)AD APOE e4(-) Odds ratio n=12 CONTROLS (95% Cl) n=22 land/or 3 (present) 10 1 and/or 3 (absent) 50(6.1-407.9)

20

4 and/or 6 (present) 18 4 and/or 6 (absent) 0.02 (0.002- 0.203)

11

APOE e4(-) - cases not possessing the e allele.

^TaS^le^^2: _! ^RlS °^{f Alzhe}i*er's disease associated with DR in individuals with an APOE e4 nleeggaattiivvee σgeennoottvyrp,-e. ^ln

The odds ratio values obtained indicate that DR antigens are associated with substantial risk and protective effects for development of late-onset AD.

References

Barron K.D. (1995) The microglial cell. A historical review. J. Neurol. Sci. 134 (suppl) pp57-68.

Benjamin R. Leake A., Ince P.G., Perry R.H., McKeith I.G., Edwardson J.A. and Morris CM. (1995) Effects of apolipoprotein E type on cortical neuropathology in senile dementia of the Lewy body and Alzheimer's disease. Neurodegeneration 4 pp443-448.

Boyles J.K., Pitas R.E., Wilson E., Mahley R.W. and Taylor J.M. (1985) Apolipoprotein E associated with astrocytic glia of the central nervous system and with non-myelinating glia of the peripheral nervous system J. Clin. Invest. 76 ppl501- 1513.

Braak, H and Braak E. (1994), Pathology of Alzheimer's disease. In Calne D. B. (ed) Neurogenerative disease. Saunders, Philadelphia, pp585-613.

Corder E.H., Saunders A.M., Strittmatter W.J., Schmechel D.E., Gaskell P.C., Small G. ., Roses A.D., Haines J.L. and Pericak-Vance M.A. (1993) Gene dose of Apolipoprotein E type 4 allele and the risk of Alzheimer's disease in late onset families Science 261 pp921-923.

Dickson D.W. and Rogers J. (1992) Neurobiology of Alzheimer's disease: a conference report Neurobiology of aging 13 pp793-798.

Khachaturian, Z.S. (1985) Diagnosis of Alzheimer's Disease. Arch Neurol. 42 ppl097-1105. Kimura A. and Sasazuki T. Eleventh International Histocompatibility Workshop reference protocol for the HLA DNA-typing technique. In: Tsuji K., Aizawa M. and Sasazuki T. (eds) 1991 1, pp397-419.

McKeith I.G. etal . , Neurology, (1996) 47 pplll3-1124.

Middleton D., Hughes D.J., Williams F., Graham C.A., Martin J., Savage D.A. A new DRB1 allele DRB1^*1107 - a combination of DRBl'll and DRB1^*03. (1993) 42 ppl60-163.

Morris, C. M., Massey, H. M., Benjamin, R. , Leake A., et al., Molecular Biology of APOE alleles in Alzheimer's and non-Alzheimer's Dementias. J. Neurol. Transm. (suppl.) 47 pp205-218.

Nevinny-Stickel C, Hinzpeter M., Andreas A. and Albert E.D., Non-radioactive oligotyping for HLA-DRl-DRxlO using PCR, digoxigenin-labelled oligonucleotides and chemiluminescence detection. 1991 18 pp323-332.

Perry R.H., Irving D., Blessed G., Fairburn A. and Perry E.K. (1990) Senile dementia of Lewy body type: a clinically and neuropathologically distinct form of Lewy body dementia in the elderly. J. Neurol. Sci . 95 ppll9-139.

Plum F. Dementia: an approaching epidemic (1979) 279 pp372- 373.

Strittmatter W.J., Saunders A.M., Schmecel D. et al . (1993) PNAS USA 90, ppl977-1981. Wragg M., Hutton, M., Talbot C, and the Alzheimer's disease collaborative group (1996) Genetic association between intronic polymorphism in presenilin-1 gene and late-onset Alzheimer's disease. Lancet 347 pp509-512.

Claims

1. A method of determining susceptibility to late-onset Alzheimer's disease and dementia with Lewy bodies in an APOE-e4 negative patient comprising determining the HLA-DR polymorphism type of the patient.

2. A method according to claim 1 comprising determining whether the patient expresses an HLA-DR polymorphism type selected from the group comprising HLA-DR 1, 2, 3, 4, and 6.

3. A method according to claim 1 or 2 comprising determining .whether the patient expresses HLA-DR polymorphism type DR1 and/or DR2 and/or DR3.

4. A method according to claim 1 or 2 comprising determining whether the patient expresses HLA-DR polymorphism type DR4 and/or DR6.

5. A method according to any preceding claim comprising analyzing the genotype of chromosome 6 of the genome of the patient .

6. The method of claim 5 .wherein the analysis is carried out by: a) digesting the genomic DNA from a patient to a diagnostic fragment length, b) probing the DNA fragment with a probe specific for an HLA-DR polymorhphism type and c) detecting the bound probe.

7. A method according to any preceding claim comprising analyzing the genotype of exon 2 of HLA-DRB on chromosome 6 of the genome of the patient.

8. The method of claim 6 wherein the analysis is carried out by: a) amplifying a diagnostic length DNA fragment of the second exon of HLA-DRB from DNA samples isolated from patients, b) performing a second^' (nested) amplification to produce greater quantities of specific DNA and c) sequencing the amplified DNA fragment in order to analyze the precise polymorphism type of the HLA- DRB gene.

9. The method of claim 6 wherein the analysis is carried out by: a) amplifying a diagnostic length DNA fragment of the second exon of HLA-DRB from DNA samples isolated from patients, b) probing the amplified DNA sample with a probe specific for an HLA-DR polymorphism type and c) detecting the bound probe.

10. The method of claim 7 or 8, wherein the probe is detected by chemiluminescent detection.

11. The method of claim 7 or 8, wherein the probe is detected by autoradiography.

12. A method of determining susceptibility of an individual to late-onset Alzheimer's disease and dementia with Lewy bodies comprising determining the APOE-e4 genotype of the individual and determining the HLA-DR polymorphism type of the individual according to any one of claims 1 to 11.

13. Use of HLA-DR typing in a method of determining susceptibility to late-onset AD and dementia with Lewy bodies in an APOE-e4 negative patient.