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US20050191685A1 - Method for determining the risk of developing a neurological disease - Google Patents

Method for determining the risk of developing a neurological disease Download PDF

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US20050191685A1
US20050191685A1 US11/063,716 US6371605A US2005191685A1 US 20050191685 A1 US20050191685 A1 US 20050191685A1 US 6371605 A US6371605 A US 6371605A US 2005191685 A1 US2005191685 A1 US 2005191685A1
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mbl
risk
variant
disease
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Eugeen Vanmechelen
Lieve Nuytinck
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Fujirebio Europe NV SA
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Innogenetics NV SA
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Priority to US11/063,716 priority Critical patent/US20050191685A1/en
Assigned to INNOGENETICS N.V. reassignment INNOGENETICS N.V. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: NUYTINCK, LIEVE, VANMECHELEN, EUGEEN
Publication of US20050191685A1 publication Critical patent/US20050191685A1/en
Priority to US12/157,494 priority patent/US8062842B2/en
Priority to US13/301,189 priority patent/US20120208185A1/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/172—Haplotypes
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/14—Heterocyclic carbon compound [i.e., O, S, N, Se, Te, as only ring hetero atom]
    • Y10T436/142222—Hetero-O [e.g., ascorbic acid, etc.]
    • Y10T436/143333—Saccharide [e.g., DNA, etc.]

Definitions

  • the present invention relates to the field of diagnosis of neurological diseases. More particularly, the present invention provides a method for determining the risk of developing a neurological disease, such as Alzheimer's disease or multiple sclerosis. The method of the invention is based on the detection of the presence or absence of one or more nucleic acid variants in the MBL genes of the subject under diagnosis.
  • Mannan-binding lectin is a plasma collectin (protein with both collagen-like and C-type lectin domains) synthesised by hepatocytes and secreted into the blood stream.
  • MBL is thought to have an important role in the innate immune system via the MBL pathway of complement activation.
  • MBL is a multimeric molecule that can bind to a wide variety of bacteria and other microbes, neutralising them and/or opsonizing them by activating complement using the lectin pathway of complement activation.
  • MBL binds to repeating mannose and N-acetylglucosamine sugar motifs characteristically displayed in high density on bacteria, fungi, viruses and protozoa but not on mammalian cells (Minchinton et al., 2002). It has been suggested that MBL plays an important role in the first hours/days of any primary immune response to a sugar-decorated pathogen. This provides the host with a first-line of defence before the adaptive immune system becomes operative. In addition, in humans MBL may be particularly important between 6 and 18 months of age when the adaptive system is still immature.
  • the human collectin genes are all located in a cluster on chromosome 10 (q21-24) (Hansen and Holmskov, 1998).
  • MBL-1 a pseudogene
  • MBL-2 which encodes a protein product.
  • MBL-2 comprises four exons with exon 1 ( FIG. 1 ) encoding a signal peptide, a cysteine-rich region and part of the glycine-rich collagenous region.
  • Exon 2 encodes the remainder of the collagenous region and exon 3 encodes an a-helical coiled-coil structure that is known as the ‘neck’ region.
  • the fourth exon encodes the carbohydrate-recognition domain that adopts a globular configuration.
  • MBL deficiency has been reported in many different populations and is largely explained by three structural and three promoter mutations.
  • the structural mutations occur at high frequency (generally 15% or greater cumulative allele frequency in most population studies) and are single base changes in codons 52, 54 and 57 of exon 1 (nucleotides+154, +161 and +170 respectively).
  • the changes are: Arg-52 to Cys (R52C, MBL D variant), Gly-54 to Asp (G54D, MBL B variant) and Gly-57 to Glu (G57E, MBL C variant).
  • the A variant is wild-type MBL and O refers to all the variants combined (Madsen et al., 1994; 1995).
  • the B and C variants have disrupted Gly-Xaa 1 -Xaa 2 repeats of the co-lagenous region resulting in an altered capacity to form the collagen triple helix.
  • the D variant introduces an additional cysteine residue and so may disrupt oligomer formation by generation of additional disulphide bands.
  • the B variant mutation occurs in 22-28% of Eurasian populations, whereas the C variant mutation is characteristic of sub-Saharan African populations in whom it reaches frequencies of 50-60%. The D mutation reaches frequencies of 14% in European populations but can be much lower elsewhere.
  • HXPA in three African-American patients with systemic lupus erythematosus (Sullivan et al., 1996) and LYPD recently found in an Euro-Brazilian individual (Boldt and Petzl-Erler, 2002).
  • the concentration of MBL2 in serum is highly variable between healthy individuals. This variation is highly genetically determined by the presence of promoter, 5′ UTR and exon 1 polymorphisms. Previous studies have indicated that the three structural variants B, C, and D and some of the promoter haplotypes have a dominant effect on the MBL concentrations in serum. Genotypes made up of combinations of seven haplotypes are mainly responsible for a 1000-fold concentration variation found in healthy human beings. All three exon 1 variants are associated with significantly decreased MBL levels compared with homozygotes of the wild-type gene.
  • the relatively common A/B heterozygotes generally have around a tenth of the MBL concentration found in A/A individuals, while B/B homozygotes or compound variant heterozygotes (B/C, etc.) typically possess MBL levels around the limit of detection by enzyme linked immunosorbent assay (ELISA) methods.
  • ELISA enzyme linked immunosorbent assay
  • the dimorphic HAL and Y/X loci allow modulation at the transcriptional level, with associated production H>L and Y>X (Kilpatrick, 2002a). It has been well established that high MBL2 producing haplotypes are HYP, followed by LYQ and LYP, whereas the LXP haplotype is associated with the lowest level of serum MBL2.
  • the LX promoter is shown to have an influence on the MBL2 level similar to that found in individuals with the B structural gene variant (Steffensen et al., 2000).
  • MBL2 deficiency virus
  • MBL deficiency may be protective and this might explain the high frequency of MBL mutations in sub-Saharan Africa and South America.
  • MBL is also able to modulate disease severity in both infectious and autoimmune disease.
  • the mechanism whereby MBL exerts such effects is unclear but one possibility is through a dose-dependent modulation of pro-inflammatory cytokines.
  • AD Alzheimer's disease
  • the incidence of AD increases with age, affecting 1 out of 10 persons older than age 65 and nearly 1 out of 2 persons older than age 85.
  • the natural history of the disease can be characterised as an irreversibly progressive brain disorder that ultimately results in devastating memory loss, profound behavioural and personality changes, and severely damaged cognitive abilities.
  • These impairments are related to the underlying death of brain cells and the breakdown of communication between them.
  • the impact of AD on society and on national economies is enormous.
  • AD Alzheimer's disease 2019
  • Apolipoprotein E Apolipoprotein E
  • the ⁇ 4 allele is recognised as a susceptibility gene for early-and late-onset familial AD as well as for sporadic AD (Corder et al., 1993; Tsai et al., 1994; Roses, 1996; Higgins et al., 1997).
  • AD amyloid precursor protein
  • PSEN1 presenilin 1
  • PSEN2 presenilin 2
  • the presenilin-1 (PSEN1) genotype and a CYP46 polymorphism have also been associated with a higher risk of late-onset sporadic AD (Wragg et al., 1996; Papassotiropoulos et al., 2003).
  • the relative contribution of APP, PSEN and CYP46 mutations is, however, the subject of considerable controversy and the involvement of other genetic factors is suggested.
  • MS Multiple sclerosis
  • CNS central nervous system
  • MS The most obvious risk factor for MS is gender (female sex). In all studies, MS affects women more than men. Other risk factors include ethnicity (MS is most common in Caucasian people of northern European origin and extremely rare among Asians, Africans and Native Americans) and family history. People who have relatives with MS are more likely to develop the disease than people with no family history of MS. It seems clear from various population studies that there is a genetic susceptibility involved in contracting the disease.
  • Dementia with Lewy bodies is an illness that presents with progressive dementia or psychosis. Parkinsonian signs, which may be absent or mild at the onset, eventually become common and rigidity is usually severe. Lewy bodies are found profusely in the brainstem, basal forebrain, hypothalamic nuclei and neocortex. Dementia with Lewy bodies is characterized by the relative absence of tangles and hyperphosphorylated tau in the brain. Parkinson's disease (PD) is a type of Lewy Body disease occurring in the middle or late life, with very gradual progression and a prolonged course.
  • PD Parkinson's disease
  • the present invention provides methods and kits for determining whether a subject is at risk (has an enhanced or elevated risk) of developing a neurological disease.
  • the methods and kits of the invention are based on the detection of the presence or absence of one or more nucleic acid variants in the MBL genes of said subject. Based on the presence or absence of certain nucleic acid variants in said genes, it can be determined whether the subject is at risk of developing a neurological disease.
  • the methods and kits of the present invention particularly relate to the detection of nucleic acid variant sequences in the promoter region, in the 5′ untranslated region (5′ UTR) and in exon 1 of the MBL2 genes. More particularly, the present invention relates to the detection of nucleic acid variant sequences in the MBL2 genes at the nucleic acid positions ⁇ 550, ⁇ 221, +4, +154, +161 and/or +170. In a preferred embodiment of the invention, following nucleic acid variant sequences are detected in the MBL2 gene at positions ⁇ 550 (G>C), ⁇ 221 (G>C), +4 (C>T), +154 (C>T), +161 (G>A) and/or +170 (G>A). In another preferred embodiment of the invention, following nucleic acid variant sequences are detected in the MBL2 genes at positions ⁇ 221 (G>C), +154 (C>T), +161 (G>A) and/or +170 (G>A).
  • nucleotide T at position +154 (variant D), nucleotide A at position +161 (variant B) and nucleotide A at position +170 (variant C) of the MBL2 genes appeared to be much lower in subjects suffering from AD compared to subjects in a control group. Accordingly, the absence of the nucleotide T at position +154 (variant D), nucleotide A at position +161 (variant B) and nucleotide A at position +170 (variant C), or the absence of the haplotypes HYPD, LYPB and LYQC, indicates that the subject is at risk of developing a neurological disease such as Alzheimer's disease.
  • nucleotide C at position ⁇ 221 (variant X) of the MBL2 genes appeared to be much higher in subjects suffering from AD compared to subjects in a control group. Accordingly, the presence of nucleotide C at position ⁇ 221 (variant X), or the presence of the haplotype LXPA indicates that the subject is at risk of developing a neurological disease such as Alzheimer's disease.
  • the prevalence of the LYPA haplotype appeared to be higher in subjects suffering from MS compared to subjects in a control group. Accordingly, the presence of the haplotype LYPA indicates that the subject is at risk of developing a neurological disease such as multiple sclerosis.
  • the nucleic acid variants in the MBL genes can also be detected by their phenotype.
  • Phenotypical detection includes the measurement of the concentration of one or more protein variants of the MBL product and/or measurement of the functional activity of the MBL product.
  • the risk for developing any neurological disease can be determined.
  • the risk is determined for developing Alzheimer's disease, Pick's disease, Parkinson's disease, dementia with Lewy bodies, Huntington disease, chromosome 13 dementias, Down's syndrome, cerebrovascular disease, multiple sclerosis, Rasmussen's encephalitis, viral meningitis, neuropsychiatric system lupus erythematosus (NPSLE, McCune and Golbus, 1988; Feinglass et al., 1989; Hanly and Liang, 1997; Croake et al., 1998), amyotrophic lateral sclerosis, Creutzfeldt-Jacob disease, Gerstmann-Straussler-Scheinker disease, transmissible spongiform encephalopathies, ischemic reperfusion damage (e.g. stroke), brain trauma, microbial infection or chronic fatigue syndrome.
  • the methods and kits of the present invention can be carried out in vivo or in vitro.
  • the methods and kits are carried out in vitro on a biological sample such as a tissue sample or a body fluid sample included but not limited to brain, blood, plasma, saliva, skin and cerebrospinal fluid.
  • the methods and kits of the present invention can also be carried out in combination with other methods for determining the risk of developing a neurological disease.
  • the methods and kits are carried out in combination with a method for Apo E genotyping and/or other markers.
  • FIG. 1 Structure and organization of part of the MBL2 gene. The localization of the main polymorphisms is shown.
  • the present invention relates to methods and kits for determining whether a subject is at risk of developing a neurological disease.
  • the methods and kits of the invention are based on the detection of the presence or absence of one or more nucleic acid variants in the MBL genes of the subject.
  • the present invention has identified that certain nucleic acid variants in the MBL genes are more frequently present in patients suffering from AD or MS compared to control subject while other nucleic acid variants in the MBL genes are more frequently absent in patients suffering from AD or MS compared to control subject. Accordingly, the present invention provides a method for determining whether a subject is at risk of developing a neurological disease, comprising:
  • Nucleic acid variant sequences are preferably detected in the promoter region, in the 5′ untranslated region (5′ UTR), and/or in exon 1 of the MBL2 gene. More particularly, nucleic acid variant sequences in the MBL2 gene are detected at the nucleic acid positions ⁇ 550, ⁇ 221, +4, +154, +161 and/or +170. In a preferred embodiment of the invention, following nucleic acid variant sequences are detected in the MBL2 gene at positions ⁇ 550 (G>C), ⁇ 221 (G>C), +4 (C>T), +154 (C>T), +161 (G>A) and/or +170 (G>A). In another preferred embodiment of the invention, following nucleic acid variant sequences are detected in the MBL2 gene at positions ⁇ 221 (G>C), +154 (C>T), +161 (G>A) and/or +170 (G>A).
  • the present invention provides a method for determining whether a subject is at risk of developing a neurological disease, comprising:
  • the present invention has identified that the prevalence of nucleotide T at position +154 (variant D), nucleotide A at position +161. (variant B) and nucleotide A at position +170 (variant C) of the MBL2 gene appeared to be much lower in subjects suffering from AD compared to subjects in a control group. Accordingly, the absence of nucleotide T at position +154 (variant D), nucleotide A at position +161 (variant B) and nucleotide A at position +170 (variant C), indicates that the subject is at risk of developing a neurological disease such as AD and the method of the invention comprises the following:
  • Nucleotide sequence T at position +154 (variant D), nucleotide A at position +161 (variant B) and nucleotide A at position +170 (variant C) correspond to the MBL2 haplotypes HYPD, LYPB and LYQC. Accordingly, the absence of the haplotypes HYPD, LYPB and LYQC indicates that the subject is at risk of developing a neurological disease such as AD.
  • the present invention has further identified that the prevalence of nucleotide C at position ⁇ 221 (variant X) of the MBL2 gene appeared to be much higher in subjects suffering from AD compared to subjects in a control group. Accordingly, the presence of nucleotide C at position ⁇ 221 (variant X) indicates that the subject is at risk of developing a neurological disease such as AD and the method of the invention comprises the following:
  • Nucleotide sequence C at position ⁇ 221 corresponds to the MBL2 hapolotype LXPA. Accordingly, the presence of the haplotype LXPA indicates that the subject is at risk of developing a neurological disease such as AD.
  • the present invention has further identified that the prevalence of haplotype LYPA appeared higher in subjects suffering from MS compared to subjects in a control group. Accordingly, the presence of the haplotype LYPA indicates that the subject is at risk of developing a neurological disease such as MS and the method of the present invention comprises the following:
  • nucleic acid refers to a single stranded or double stranded nucleic acid sequence, which may contain from 8 nucleotides to the complete nucleotide sequence.
  • a nucleic acid may consist of deoxyribonucleotides or ribonucleotides, nucleotide analogues or modified nucleotides, or may have been adapted for therapeutic purposes.
  • nucleic acid variant means that the nucleic acid sequence at a certain position in the MBL gene differs relative to one or more reference nucleic acid sequences (Genebank NM — 000242.1 and NT — 024082).
  • nucleic acid polymorphism or “polymorphism” signifies the existence of two or more variants in the population present at a sequence of >1% of the population. The most simple nucleic acid polymorphism is a polymorphism affecting a single nucleotide, i.e. a single nucleotide polymorphism or SNP.
  • Nucleic acid polymorphisms further include any number of contiguous and/or non-contiguous differences in the primary nucleotide sequence of the nucleic acid under investigation relative to the primary nucleotide sequence of one or more reference nucleic acids.
  • the term “polymorphic position” or “position” refers to the nucleic acid position at which a nucleic acid polymorphism arises. Nucleic acid sequences comprising at least one such polymorphism are referred to as “polymorphic nucleic acid sequences”, “polymorphic polynucleotides”, “polymorphic sequences” or the like.
  • haplotype means a particular pattern-of sequential polymorphisms found on a single chromosome.
  • allele is one of several alternative forms of a gene or DNA sequence at a specific chromosomal location (locus). At each autosomal locus an individual possesses two alleles, one inherited from the father and one from the mother.
  • the MBL2 gene has four exons.
  • Exon 1 contains three single nucleotide polymorphisms:
  • the promotor and 5′ untranslated regions of the MBL2 gene are also polymorphic. Single nucleotide polymorphisms are present:
  • Promotor variants are in absolute linkage desequilibrium with coding variants, and only seven of the 64 possible haplotypes have been observed, i.e. HYPA, LXPA, LYQA, LYPA, HYPD, LYPB and LYQC (Minchinton et al, 2002). Two other, rare haplotype have also been described: HXPA (Sullivan et al., 1996) and LYPD (Boldt and Petzl-Erler (2002).
  • the subject on which the methods of the present invention is carried out can be any subject of which the risk for developing a neurological disease needs to be determined.
  • the subject may be a non-human subject such as (but not limited to) a cow, a pig, a sheep, a goat, a horse, a monkey, a rabbit, a hare, a chicken, a dog, a cat, a mouse, a rat, a hamster, an elk, a deer, a tiger, an elephant, a zebrafish, a pufferfish (Fugu), a fly, a worm or C. elegans. More preferably, the subject is a primate. Even more preferably, the subject is a human.
  • Immune and inflammatory responses in the central nervous system are observed in various chronic and acute neurological diseases such as Alzheimer's disease, myasthenia gravis, multiple sclerosis, microbial infections, head trauma and stroke, Pick's disease, Parkinson's disease, dementia with Lewy bodies, Huntington disease, chromosome 13 dementias, Down's syndrome, cerebrovascular disease, Rasmussen's encephalitis, viral meningitis, NPSLE, amyotrophic lateral sclerosis, Creutzfeldt-Jacob disease, Gerstmann-Straussler-Scheinker disease, transmissible spongiform encephalopathies, ischemic reperfusion damage.
  • CNS central nervous system
  • the risk is determined for developing Alzheimer's disease, Pick's disease, Parkinson's disease, dementia with Lewy bodies, Huntington disease, chromosome 13 dementias, Down's syndrome, cerebrovascular disease, multiple sclerosis, Rasmussen's encephalitis, viral meningitis, NPSLE, amyotrophic lateral sclerosis, Creutzfeldt-Jacob disease, Gerstmann-Straussler-Scheinker disease, transmissible spongiform encephalopathies, ischemic reperfusion damage (e.g. stroke), brain trauma, microbial infection or chronic fatigue syndrome.
  • Alzheimer's disease Pick's disease, Parkinson's disease, dementia with Lewy bodies, Huntington disease, chromosome 13 dementias, Down's syndrome, cerebrovascular disease, multiple sclerosis, Rasmussen's encephalitis, viral meningitis, NPSLE, amyotrophic lateral sclerosis, Creutzfeldt-Jacob disease, Gerstmann-Strauss
  • the term “developing a neurological disease” means that the subject, at the time that the method of the invention is carried out, does not show any clinical signs of a neurological disease, but that said subject will show clinical signs of a neurological disease later on during life time.
  • the term “developing a neurological disease” may further implicate that the subject, at the time that the method of the invention is carried out, shows already clinical signs of a neurological disease.
  • the method of the present invention is then carried out for the differential diagnosis of a neurological disease or for monitoring the course and severity of the disease progress.
  • the terms “risk”, “enhanced risk”, “elevated risk” or “likelihood” are interchangeable and are used with respect to the probability of developing a neurological disease.
  • the method of the present invention can be carried out in vivo or in vitro. Preferred, however, is in vitro detection of nucleic acid variants in the MBL gene in a biological sample obtained from the subject.
  • biological sample means a tissue sample or a body fluid sample.
  • a tissue sample includes (but is not limited to) a brain sample, bucal cells or a skin sample.
  • body fluid refers to all fluids that are present in the body including but not limited to blood, plasma, serum, lymph, urine, saliva or cerebrospinal fluid.
  • cerebrospinal fluid or “CSF” is intended to include whole cerebrospinal fluid or derivatives of fractions thereof well known to those skilled in the art.
  • a cerebrospinal fluid sample can include various fractionated forms of cerebrospinal fluid or can include various diluents added to facilitate storage or processing in a particular assay.
  • the biological sample may also be obtained by subjecting it to a pretreatment if necessary, for example, by homogenizing or extracting. Such a pretreatment may be selected appropriately by those skilled in the art depending on the biological sample to be subjected.
  • a nucleic acid comprising an intended sequence prepared from a biological sample may be prepared from DNA or RNA. Release, concentration and isolation of the nucleic acids from the sample can be done by any method known in the art. Currently, various commercial kits are available such as the QIAamp Blood Kit from Qiagen (Hilden, Germany) for the isolation of nucleic acids from blood samples, or the ‘High pure PCR Template Preparation Kit’ (Roche Diagnostics, Basel, Switzerland). Other, well-known procedures for the isolation of DNA or RNA from a biological sample are also available (Sambrook et al., 1989).
  • the nucleic acid may be amplified.
  • amplification procedures can be accomplished by those methods known in the art, including, for example, the polymerase chain reaction (PCR) and reverse transcription polymerase reaction (RT-PCR).
  • PCR polymerase chain reaction
  • RT-PCR reverse transcription polymerase reaction
  • the presence or absence of certain nucleic acid variants in the target sequence can be detected.
  • Numerous methods for detecting a single nucleotide anomaly in nucleic acid sequences are well-known in the art.
  • the present invention is not limited by any particular method used to detect the target sequences disclosed herein.
  • nucleic acid variants in the MBL genes of a subject may also be reflected phenotypically in the concentration, structure and functionality of the MBL product in, for example, the serum or plasma of said subject.
  • Promoter mutations in the MBL2 gene are associated with a decreased production of MBL2.
  • All three exon 1 variants are associated with a significant decrease of the concentration and functionality of the MBL2 level resulting in different protein variants. Therefore, the present invention also encompasses a method for determining whether a subject has a risk of developing a neurological disease wherein the nucleic acid variants in the MBL genes are detected by their phenotype.
  • Phenotypic detection of nucleic acid variants in the MBL genes may encompass the measurement of one or more protein variants of the MBL product in said subject. Accordingly, the present invention relates to a method for determining whether a subject is at risk of developing a neurological disease, comprising:
  • one or more protein variants of the MBL2 product are detected.
  • the method of the present invention comprises the following:
  • concentration refers to the presence or absence and/or amount of a certain protein variant.
  • a change in the concentration of a protein variant refers to a measurable increase or decrease, including total absence or presence, in the protein variant concentration when compared to a control subject.
  • Phenotypic detection of nucleic acid variants in the MBL genes may also encompass the assessment of the MBL functional activity in said subject. Accordingly, the present invention relates to a method for determining whether a subject is at risk of developing a neurological disease, comprising:
  • the “functional activity of the MBL product” or the “MBL functional activity” refers to the ability of the MBL product to initiate the MBL-dependent lectin pathway of complement (MBL pathway).
  • MBL protein variants or MBL functional activity in vivo or in vitro.
  • a biological sample obtained from the subject.
  • the MBL protein variants or MBL functional activity are detected in the brain, blood, serum, plasma, tissue, bucal cells or CSF of said subject.
  • a “control subject”, as defined in the present invention is a subject of the same species as the subject under examination which is free from, or not at risk of developing, the neurological disease.
  • concentration of any given protein variant or the functional activity of the MBL product obtained upon analyzing the subject under examination relative to the concentration or functional activity obtained upon analyzing a control subject will depend on the particular analytical protocol and detection technique that is used. Accordingly, those skilled in the art will understand that, based on the present description, any laboratory can establish, for a given MBL protein variant, a suitable “reference range”, “reference level range”, “concentration range or “range of levels” (those terms are used interchangeable) or a “reference functional activity” characteristic for control subjects according to the analytical protocol and detection technique in use.
  • the concentration or functional activity obtained for the subject under examination can then be compared with this reference and based on this comparison, a conclusion can be drawn as to whether the subject has a risk of developing a neurological disease.
  • Those skilled in the art will also know how to establish a cut-off value suitable for determining whether a subject is at risk of developing a neurological disease. Methods for defining cut-off values include (but are not limited to) the methods described by IFCC (1987).
  • the MBL products that are detected in the method of the present invention may be detected by any method known to those skilled in the art. They can be identified by their structure, by partial amino acid sequence determination, by functional assay, by enzyme assay, by various immunological methods, or by biochemical methods.
  • the functional assay may encompass the measurement of the ability to opsonize heat-killed baker's yeast (Miller et al., 1968), the assessment of phagocytosis of various microorganisms (Kuhlman et al., 1989) and/or detection of complement activation (Super et al., 1989; 1990; Yokota et al., 1995).
  • a serum test for complement activation described by Seelen et al. (2003), for example, the lectin pathway function is assessed using plates coated with mannan, followed by incubation of the serum in buffer containing Ca 2+ , Mg 2+ and an inhibitory antibody directed against C1q. The formation of the membrane attack complex is subsequently detected by use of a specific monoclonal antibody directed against C5b-9. More assays for the measurement of the functional activity of the MBL product are described by Seelen et al. (2005).
  • Biochemical methods include (but are not limited to) capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, two-dimensional liquid phase electrophoresis (2-D-LPE; Davidsson et al. 1999) or detection of the migration pattern in gel electrophoreses.
  • Sodium dodecyl sulfate-polyacrylamide gel electrophoresis SDS-PAGE
  • SDS-PAGE is a widely used approach for separating proteins from complex mixtures (Patterson and Aebersold, 1995). It can be performed in one- or two-dimensional (2-D) configuration. For less complicated protein preparation, one-dimensional SDS-PAGE is preferred over 2-D gels, because it is simpler.
  • 2-D gel electrophoresis incorporates isoelectric focusing (IEF) in the first dimension and SDS-PAGE in the second dimension, leading to a separation by charge and size (O'Farrell, 1975).
  • IEF isoelectric focusing
  • 2-D PAGE is a powerful technique for separating very complex protein preparations, resolving up to 10 000 proteins from mammalian tissues and other complex proteins (Klose and Kobalz, 1995; Celis et al., 1996; Yan et al., 1997).
  • the protein variants of MBL of the present invention can be identified by their isoelectric focusing point (pI) and their molecular weight (MW) in kilodaltons (kD).
  • the level of MBL protein variant can also be detected by an immunoassay.
  • an “immunoassay” is an assay that utilizes an antibody to specifically bind to the antigen (i.e. the MBL protein variant). The immunoassay is thus characterized by detection of specific binding of a MBL protein variant to an antibody.
  • Immunoassays for detecting MBL protein variants may be either competitive or noncompetitive. Noncompetitive immunoassays are assays in which the amount of captured analyte (i.e. the MBL protein variant) is directly measured. In competitive assays, the amount of analyte (i.e.
  • the MBL protein variant) present in the sample is measured indirectly by measuring the amount of an added (exogenous) analyte displaced (or competed away) from a capture agent (i.e. the antibody) by the analyte (i.e. the MBL protein variant) present in the sample.
  • a competition assay a known amount of the (exogenous) MBL protein variant is added to the sample and the sample is then contacted with the antibody.
  • the amount of added (exogenous) MBL protein variant bound to the antibody is inversely proportional to the concentration of the MBL protein variant in the sample before the exogenous MBL protein variant is added.
  • the antibodies can be bound directly to a solid substrate where they are immobilized.
  • immunological methods include but are not limited to fluid or gel precipitation reactions, immunodiffusion (single or double), agglutination assays, immunoelectrophoresis, radioimmunoassays (RIA), enzyme-linked immunosorbent assays (ELISA), TRIFMA (Christiansen et al., 1999), Western blots, liposome immunoassays (Monroe et al., 1986), complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays or immunoPCR.
  • An overview of different immunoassays is given in Wild (2001), Ghindilis et al. (2002) and Kilpatrick (2002b).
  • the level of MBL protein variant is determined by an immunoassay comprising at least the following steps:
  • the MBL protein variant can be detected by a sandwich ELISA comprising the following steps:
  • the secondary antibody itself carries a marker or a group for direct or indirect coupling with a marker.
  • the term “specifically recognizing”, “specifically binding with”, “specifically reacting with” or “specifically forming an immunological reaction with” refers to a binding reaction by the antibody to the MBL protein variant which is determinative of the presence of said MBL protein variant in the sample in the presence of a heterogeneous population of other proteins and/or other biologics.
  • the specified antibody preferentially binds to a particular MBL protein variant while binding to other MBL protein variants and other proteins does not occur in significant amounts.
  • Any antibody that recognizes the MBL protein variant under examination can be used in the above method.
  • antibody as used herein also includes antibodies and antibody fragments either produced by the modification of whole antibodies or synthesized using recombinant DNA methodologies.
  • the humanized versions of the mouse monoclonal antibodies are also made by means of recombinant DNA technology, departing from the mouse and/or human genomic DNA sequences coding for H and L chains or from cDNA clones coding for H and L chains.
  • the monoclonal antibodies used in the method of the invention may be human monoclonal antibodies.
  • the term ‘humanized antibody’ means that at least a portion of the framework regions of an immunoglobulin is derived from human immunoglobulin sequences.
  • the antibodies used in the method of the present invention may be labeled with an appropriate label.
  • the particular label or detectable group used in the assay is not a critical aspect of the invention, so long as it does not significantly interfere with the specific binding of the antibody used in the assay.
  • the detectable group can be any material having a detectable physical or chemical property.
  • detectable labels have been well developed in the field of immunoassays and, in general, almost any label used in such methods can be applied to the method of the present invention.
  • a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, radiological, optical, or chemical means.
  • Useful labels in the present invention include, but are not limited to, magnetic beads (e.g.
  • DynabeadsTM DynabeadsTM
  • fluorescent dyes e.g. fluorescein isothiocyanate, texas red, rhodamine
  • radiolables e.g. 3 H, 125 I, 35 S, 14 C, or 32 P
  • enzymes e.g. horseradish peroxidase, alkaline phosphatase, and others commonly used in an ELISA
  • colorimetric labels such as colloidal gold, colored glass or plastic (e.g. polystyrene, polypropylene, latex, etc.) beads.
  • the label may be coupled directly or indirectly to the desired component or the assay according to methods well known in the art.
  • a wide variety of labels may be used, with the choice of label depending on the sensitivity required, the ease of conjugation with the compound, stability requirements, the available instrumentation, and disposal provisions.
  • Non-radioactive labels are often attached by indirect means.
  • a ligand molecule e.g. biotin
  • the ligand then binds to an anti-ligand (e.g. streptavidin) molecule, which is either inherently detectable or covalently bound to a signal system, such as a detectable enzyme, a fluorescent compound, or a chemiluminescent compound.
  • a number of ligands and anti-ligands can be used.
  • a ligand has a natural anti-ligand, for example, biotin, thyroxine, and cortisol, it can be used in conjunction with the labeled, naturally occurring anti-ligands.
  • a haptenic or antigenic compound can be used in combination with an antibody.
  • the antibodies can also be conjugated directly to signal generating compounds, for example, by conjugation with an enzyme or fluorophore.
  • Enzymes of interest as labels will primarily be hydrolases, particularly phosphatases, esterases and glycosidases, or oxidoreductases, particularly peroxidases.
  • Fluorescent compounds include fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umberlliferone, etc.
  • Chemiluminescent compounds include luciferin, and 2,3-dihydrophthalazinediones, for example, luminol.
  • Means for detecting labels are well known in the art.
  • means for detection include a scintillation counter or photographic film, as in autoradiography.
  • the label is a fluorescent label, it may be detected by exciting the fluorophore with the appropriate wavelength of light and detecting the resulting fluorescence. The fluorescence may be detected visually, by means of a photographic film, by the use of electronic detectors such as charge coupled devices (CCDs) or photomultipliers and the like.
  • CCDs charge coupled devices
  • enzyme labels may be detected by providing the appropriate substrates for the enzyme and detecting the resulting reaction product.
  • simple colorimetric labels may be detected simply by observing the color associated with the label.
  • agglutination assays can be used to detect the presence of the target antibodies.
  • antigen-coated particles are agglutinated by samples comprising the target antibodies.
  • none of the components need be labeled and the presence of the target antibody is detected by simple visual inspection.
  • Apolipoprotein E Apolipoprotein E polymorphism for predicting AD.
  • APP amyloid precursor protein
  • PSEN1 presenilin 1
  • PSEN2 presenilin 2
  • the presenilin-1 (PSEN1) genotype and a CYP46 polymorphism have also been associated with a higher risk of late-onset sporadic AD (Wragg et al., 1996; Papassotiropoulos et al., 2003).
  • the present invention also relates to a method for determining whether a subject is at risk of developing a neurological disease, comprising the step of detecting the presence or absence of a nucleic acid variant in the MBL genes in combination with the detection of one or more other risk factors.
  • the presence or absence of a nucleic acid variant in the MBL genes is detected in combination with the ApoE genotype, with a nucleic acid variant in the APP gene, with a nucleic acid variant in the presenilin 1 gene, with a nucleic acid variant in the presenilin 2 gene and/or with a nucleic acid variant in CYP46.
  • nucleic acid variants in the MBL genes of a subject may influence the concentration of MBL in the serum of said subject.
  • Promoter mutations in the MBL2 gene are associated with a decreased production of MBL2.
  • All three exon 1 variants are associated with a significant decrease of the MBL2 level.
  • these exon 1 variants may have impact on the functionality of MBL2. Therefore based on the detection of the presence or absence of one or more MBL nucleic acid variants, protein variants or MBL functional activity in a subject, it can be determined whether a certain therapeutic agent or treatment might be suitable for preventing the neurological disease the subject is expected (has a risk) to develop or for ameliorating the course of the disease and/or reducing its severity.
  • the method of the present invention may also be used in determining whether and which therapeutic agent might be suitable for a patient in order to prevent or treat a neurological disease.
  • preventing a disease means inhibiting or reversing the onset of the disease, inhibiting or reversing the initial signs of the disease, inhibiting the appearance of clinical symptoms of the disease.
  • treating a disease includes substantially inhibiting the disease, substantially slowing or reversing the progression of the disease, substantially ameliorating clinical symptoms of the disease or substantially preventing the appearance of clinical symptoms of the disease.
  • the method of the present invention also relates to the treatment or prevention of a neurological disease in a subject, comprising the following:
  • the present invention further relates to the use of MBL for the preparation of a medicament for the treatment or prevention of a neurological disease.
  • MBL is used for the preparation of a medicament for the treatment or prevention.
  • Alzheimer's disease Pick's disease, Parkinson's disease, dementia with Lewy bodies, Huntington disease, chromosome 13 dementias, Down's syndrome, cerebrovascular disease, multiple sclerosis, Rasmussen's encephalitis, viral meningitis, NPSLE, amyotrophic lateral sclerosis, Creutzfeldt-Jacob disease, Gerstmann-Straussler-Scheinker disease, transmissible spongiform encephalopathies, ischemic reperfusion damage (e.g. stroke), brain trauma, microbial infection or chronic fatigue syndrome.
  • Alzheimer's disease Pick's disease, Parkinson's disease, dementia with Lewy bodies, Huntington disease, chromosome 13 dementias, Down's syndrome, cerebrovascular disease, multiple sclerosis, Rasmussen's
  • MBL can be isolated from donor plasma or prepared by recombinant DNA techniques or synthetically.
  • MBL2 can be purified from pooled donor plasma by the procedure developed at Statens Serum Institut (Copenhagen, Denmark) (Valdimarsson et al., 1998).
  • Recombinant DNA techniques have been described by Jensenius et al. (2003) and further by Maniatis (1989) and in WO 96/29605.
  • Classical chemical synthesis is described by Houbenweyl (1974), Atherton and Shepard (1989) and in WO 96/29605.
  • MBL can be safely administered intravenously in doses sufficient for achieving normal concentrations in the blood.
  • Formulation of the MBL product for intravenous infusion has been described in Valdimarsson et al. (1998).
  • MBL is diluted to 200 ⁇ g/ml in 0.15M NaCl containing 1% (w/v) human serum albumin (Valdimarsson et al., 1998). It should be clear, however, that also other formulations can be used for administering the MBL product to the subject.
  • the preferred formulation of therapeutic compositions depends on the intended mode of administration and application.
  • compositions can also include, depending on the formulation desired, pharmaceutically-acceptable, non-toxic carriers or diluents, which are defined as vehicles commonly used to formulate pharmaceutical compositions for animal or human administration.
  • diluents are selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, physiological phosphate-buffered saline, Ringer's solutions, dextrose solution, and Hank's solution.
  • the pharmaceutical composition or formulation may also include other carriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenic stabilizers and the like.
  • the pharmaceutical compositions can also include large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids and copolymers (such as latex functionalized sepharose, agarose, cellulose, and the like), polymeric amino acids, amino acid copolymers, and lipid aggregates (such as oil droplets or liposomes).
  • macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids and copolymers (such as latex functionalized sepharose, agarose, cellulose, and the like), polymeric amino acids, amino acid copolymers, and lipid aggregates (such as oil droplets or liposomes).
  • compositions of the invention can be administered as injectable dosages of a solution or suspension of the substance in a physiologically acceptable diluent with a pharmaceutical carrier that can be a sterile liquid such as water, oils, saline, glycerol, or ethanol.
  • a pharmaceutical carrier that can be a sterile liquid such as water, oils, saline, glycerol, or ethanol.
  • encapsulation into biodegradable microparticles can be used as a parenteral delivery system (Brayden et al., 2001).
  • auxiliary substances such as wetting or emulsifying agents, surfactants, pH buffering substances and the like can be present in the therapeutic compositions.
  • Other components of pharmaceutical compositions are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, and mineral oil.
  • glycols such as propylene glycol or polyethylene glycol are preferred liquid carriers, particularly for injectable solutions.
  • compositions are prepared as injectables, either as liquid solutions or suspensions.
  • Solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared.
  • the preparation also can be emulsified or encapsulated in liposomes or micro particles such as polylactide, polyglycolide, or copolymer for enhanced adjuvant effect, as discussed above (Langer, 1990; Langer et al, 1997).
  • the pharmaceutical compositions can be administered in the form of a depot injection or implant preparation which can be formulated in such a manner as to permit a sustained or pulsatile release of the active ingredient.
  • binders and carriers include, for example, polyalkylene glycols or triglycerides. Such suppositories can be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1% to 2%.
  • Oral formulations include excipients, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, and magnesium carbonate. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations, or powders and contain 10% to 95% of active ingredient, preferably 25% to 70%.
  • Topical application can result in transdermal or intradermal delivery.
  • Topical administration can be facilitated by co-administration of the agent with cholera toxin or detoxified derivatives or subunits thereof or other similar bacterial toxins (Glenn et al., 1998).
  • Co-administration can be achieved by using the components as a mixture or as linked molecules obtained by chemical crosslinking or expression as a fusion protein.
  • transdermal delivery can be achieved using a skin path or using transferosomes (Paul et al., 1995; Cevc et al., 1998).
  • the present invention relates to the use of a nucleic acid containing one or more variants in the MBL gene (variant nucleic acid) for the manufacture of a medicament for the treatment or prevention of a neurological disease.
  • the variant sequence is comprised within the promoter region, the 5′ UTR or the exon 1 of the MBL2 gene. In another preferred embodiment, the variant sequence is in at least one of the positions ⁇ 550, ⁇ 221, +4, +154, +161 and/or +170 of the MBL2 gene. In another preferred embodiment, the variant sequence encompasses nucleotide T, at position +154 (variant D), nucleotide A at position +161 (variant B) and/or nucleotide A at position +170 (variant C) in the MBL2 gene. In another preferred embodiment, the variant sequence encompasses nucleotide G at position ⁇ 221 (variant Y) in the MBL2 gene.
  • MBL gene therapy is used for the preparation of a medicament for the treatment or prevention of Alzheimer's disease, Pick's disease, Parkinson's disease, dementia with Lewy bodies, Huntington disease, chromosome 13 dementias, Down's syndrome, cerebrovascular disease, multiple sclerosis, Rasmussen's encephalitis, viral meningitis, NPSLE, amyotrophic lateral sclerosis, Creutzfeldt-Jacob disease, Gerstmann-Straussler-Scheinker disease, transmissible spongiform encephalopathies, ischemic reperfusion damage (e.g. stroke), brain trauma, microbial infection or chronic fatigue syndrome.
  • Alzheimer's disease Pick's disease, Parkinson's disease, dementia with Lewy bodies, Huntington disease, chromosome 13 dementias, Down's syndrome, cerebrovascular disease, multiple sclerosis, Rasmussen's encephalitis, viral meningitis, NPSLE, amyotrophic lateral sclerosis, Creutzfeldt-J
  • nucleic acid molecule In order to facilitate the introduction of a nucleic acid molecule into cells, a number of different means for gene delivery can be used in association with the nucleic acid molecule.
  • the term “means for gene delivery” is meant to include any technique suitable for delivery of nucleic acid molecules across the blood brain barrier and/or for transmembrane delivery across cell membranes.
  • means for gene delivery are viral vectors (e.g., adeno-associated virus-based vectors, lipids/liposomes, ligands for cell surface receptors, etc).
  • the nucleic acids or a vector containing the same can be packaged into liposomes. Suitable lipids and related analogs are described by U.S. Pat. Nos.
  • Vectors and nucleic acids can also be adsorbed to or associated with particulate carriers, examples of which include polymethyl methacrylate polymers and polylactides and poly(lactide-co-glycolides) (McGee et al., 1997).
  • Gene therapy vectors or naked nucleic acids can be delivered in vivo by administration to an individual patient, typically by systemic administration (e.g., intravenous, intraperitoneal, nasal, gastric, intradermal, intramuscular, subdermal, or intracranial infusion) or topical application (see e.g., U.S. Pat. No. 5,399,346).
  • the nucleic acid can also be administered using a gene gun (Xiao and Brandsma, 1996).
  • the nucleic acid is precipitated onto the surface of microscopic metal beads.
  • the microprojectiles are accelerated with a shock wave or expanding helium gas, and penetrate tissues to a depth of several cell layers.
  • the AccelTM Gene Delivery Device manufactured by Agacetus, Inc. (Middleton Wis., US) is suitable.
  • naked nucleic acids can pass through skin into the blood stream simply by spotting the nucleic acid onto skin with chemical or mechanical irritation (WO 95/05853).
  • vectors encoding the variant nucleic acid can be delivered to cells ex vivo, such as cells explanted from an individual patient (e.g., lymphocytes, bone marrow aspirates, tissue biopsy) or universal donor hematopoietic stem cells, followed by reimplantation of the cells into a patient, usually after selection for cells which have incorporated the vector.
  • cells ex vivo such as cells explanted from an individual patient (e.g., lymphocytes, bone marrow aspirates, tissue biopsy) or universal donor hematopoietic stem cells, followed by reimplantation of the cells into a patient, usually after selection for cells which have incorporated the vector.
  • Effective doses of the therapeutic compositions of the present invention vary depending upon many different factors, including means of administration, target site, physiological state of the subject, whether the subject is a human or an animal, other medications administered, and whether treatment is prophylactic or therapeutic. Treatment dosages need to be titrated to optimize safety and efficacy. Examples of effective doses are described in Valdimarsson et al. (1998), Garred et al. (2002) and Validmarsson (2003).
  • kits for determining whether a subject is at risk of developing a neurological disease can be based on the detection of nucleic acid variants in the MBL genes of said subject or it can be based on the measurement of MBL protein variants or MBL functional activity.
  • the kit may comprise:
  • the kit comprises:
  • the kit comprises:
  • the kit comprises:
  • the kit comprises:
  • the kit comprises:
  • the means in step (a) of said kit may comprise:
  • hybridization buffer means a buffer allowing a hybridization reaction between the probes and the polynucleic acids present in the sample, or the amplified products, under the appropriate stringency conditions.
  • wash solution means a solution enabling washing of the hybrids formed under the appropriate stringency conditions.
  • the means for detecting the presence or absence of nucleic acid variants in the MBL genes is a line probe assay (LiPA; Stuyver et al., 1996; Stuyver et al., 1997; Van Geyt et al., 1998).
  • the selected set of probes is immobilized to a membrane strip in a line fashion.
  • An alternative is immobilization in a dot fashion. Said probes may be immobilized individually or as mixtures to the delineated locations.
  • the amplified MBL polynucleic acids can be labelled with biotine, and the hybrid can then, via a biotine-streptavidine coupling, be detected with a non-radioactive colour developing system.
  • oligonucleotides may be coupled to microspheres or chips.
  • An example of an assay that provides for simultaneous detection includes (but is not limited to) the xMAPTM technology (Luminex 100 IS, Austin, Tex., USA), the PamGene technology (PamGene, ‘s-Hertogenbosch, The Netherlands) and the Invader® platform (Third Wave Technologies, Inc., Madison, Wis., US).
  • a kit based on the detection of MBL protein variants may comprise an antibody that specifically recognizes the MBL protein variant that is detected.
  • a preferred kit for carrying out the method of the invention comprises:
  • the kit comprises:
  • the means in step (b) of said kit for determining, from the nucleic acid variants in the MBL gene, the MBL protein variant concentration and/or the MBL functional activity detected with the means of step (a), whether the subject is at risk of developing a neurological disease include a table, a chart, or similar, generally referred to as “a predisposition risk algorithm”, taking into account the MBL nucleic acid variant(s) or haplotype(s), the MBL protein variant concentration and/or the MBL functional activity to determine the risk for developing a neurological disease.
  • a predisposition risk algorithm taking into account the MBL nucleic acid variant(s) or haplotype(s), the MBL protein variant concentration and/or the MBL functional activity to determine the risk for developing a neurological disease.
  • MBL nucleic acid variant or haplotype may indicate the MBL nucleic acid variant or haplotype, the MBL protein variant concentration and/or the MBL functional activity that confer a risk for developing a neurological disease and/or it may indicate the MBL nucleic acid variant or haplotype, the MBL protein variant concentration and/or the MBL functional activity that confer protection for not developing a neurological disease.
  • the present invention also provides a method for determining whether a subject is at risk of developing a neurological disease making use of a computer.
  • information for example, on the MBL nucleic acid variant or haplotype, the MBL protein variant concentration and/or the MBL functional activity that confer a risk for developing a neurological disease and the MBL nucleic acid variant or haplotype, the MBL protein variant concentration and/or the MBL functional activity that confer protection for not developing a neurological disease is introduced into a computer by an operator.
  • this information is stored on a computer readable carrier.
  • Computer readable carriers or “computer readable media” include all carriers and media accessible and readable with a computer. Said carriers and media include magnetic tapes, floppy disks, hard disks, ZIP disks, CD-ROMs, electrical or electronical memories such as RAM and ROM and hybrid magnetic/optical storage media. After the correlation, the results of the comparison or assessment can be displayed on the computer on a display device such as, for example, a computer monitor or outputted on for example, a printer.
  • a display device such as, for example, a computer monitor or outputted on for example, a printer.
  • the kit of the present invention may include, in additions to the means of steps (a), a means for detection other risk factors for developing a neurological disease.
  • the kit additionally includes a means for detecting the Apo E genotype, a means for detecting a nucleic acid variant in the APP gene, a means for detecting a nucleic acid variant in the presenilin 1 gene, a means for detecting a nucleic acid variant in the presenilin 2 gene and/or a means for detecting a nucleic acid variant in CYP46.
  • the present invention relates to a kit comprising:
  • a part of the exon 1 and promoter sequences of MBL2 was amplified using biotinylated oligonucleotides.
  • the polymorphisms were detected by use of a reverse hybridization method (Line Probe Assay) with 12 probes designed to recognize the polymorphisms at positions ⁇ 550 (G>C), ⁇ 221 (G>C), +4 (C>T), +154 (C>T), +161 (G>A) and +170 (G>A) of MBL2. After stringent washing at 56° C., hybridized probes were incubated with a streptavidine-alkaline phosphatase conjugate.
  • the frequencies of the MBL2 haplotypes in the AD patients and in the control subjects are indicated in Table 2.
  • the promoter variant X (haplotype LXPA) is more frequently present in the AD group compared to the control group.
  • the odds ratios in Tables 1 and 2 clearly show that promoter variant X (haplotypes LXPA) has a significantly higher risk for developing AD.
  • Tables 1 and 2 show that the haplotypes with these variants (LYPB and LYQC) indeed have a lower risk of developing AD.
  • SP index of senile plaques
  • NFT neurofibrillary tangles
  • the healthy controls were volunteers without history, symptoms, or signs of psychiatric or neurological disease, malignant disease, or systemic disorders. Cognitive status was examined using MMSE, and individuals with scores below 28 were not included as controls.
  • a part of the exon 1 and promoter sequences of MBL2 was amplified using biotinylated oligonucleotides.
  • the polymorphisms were detected by use of a reverse hybridization method (Line Probe Assay) with 12 probes designed to recognize the polymorphisms at positions ⁇ 550 (G>C), ⁇ 221 (G>C), +4 (C>T), +154 (C>T), +161 (G>A) and +170 (G>A) of MBL2. After stringent washing at 56° C., hybridized probes were incubated with a streptavidine-alkaline phosphatase conjugate.
  • the frequencies of the MBL2 haplotypes and combined MBL2 haplotypes in the AD patients and in the control subjects are shown in Tables 3 and 4 respectively.
  • Model 1 shows that the MBL2-haplotype LXPA increased AD-incidence as compared with other pooled MBL2-haplotypes (P ⁇ 0.013, OR>1).
  • Model 2 shows that pooled MBL2-haplotypes HYPD, LYPB and LYQC decreased AD-incidence as compared with other pooled MBL2-haplotypes (P ⁇ 0.006, OR ⁇ 1).
  • the control group (C) consisted of 172 healthy individuals. From each blood sample, informed consent to participate in the study is available
  • the relevant coding sequences of the MBL2 gene were amplified using biotinylated oligonucleotides.
  • the polymorphisms were detected by use of a reverse hybridization method (Line Probe Assay) with 12 probes designed to recognize the polymorphisms at positions ⁇ 550 (G>C), ⁇ 221 (G>C), +4 (C>T), +154 (C>T), +161 (G>A) and +170 (G>A) of MBL2.
  • Line Probe Assay Line Probe Assay
  • hybridized probes were incubated with a streptavidine-alkaline phosphatase conjugate. The presence of a hybridized probe was revealed using NBIT/BCIP color development. Details on the reverse hybridization are described in Stuyver et al. (1996), Stuyver et al. (1997) and Van Geyt et al. (1998).
  • Alzheimer's disease a review of the disease, its epidemiology and economic impact. Arch. Gerontol. Geriatr. 27: 189-221.
  • Zhao L. P. Li S. S., Khalid, N. (2003) A Method for the assessment of disease associations with single-nucleotide polymorphism haplotypes and environmental variables in case-control studies. American Journal of Human Genetics 72: 1231-1250.

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US20100173417A1 (en) * 2009-01-04 2010-07-08 Lennart Minthon Diagnostic method
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US8980271B2 (en) 2013-01-18 2015-03-17 Ipierian, Inc. Methods of treating a tauopathy
US20150353249A1 (en) * 2005-12-09 2015-12-10 Dna Genotek Inc. Container system for releasably storing a substance
US10400018B2 (en) 2014-02-14 2019-09-03 Ipierian, Inc. Tau peptides, anti-tau antibodies, and methods of use thereof
US11002646B2 (en) 2011-06-19 2021-05-11 DNA Genotek, Inc. Devices, solutions and methods for sample collection
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Publication number Priority date Publication date Assignee Title
WO2005080594A2 (fr) 2004-02-24 2005-09-01 Innogenetics N.V. Methode de determination des risques qu'a un sujet de contracter une maladie neurologique
WO2007057119A2 (fr) * 2005-11-15 2007-05-24 F. Hoffmann-La Roche Ag Polymorphismes nucleotidiques simples (snp) du gene azgp
WO2008010082A2 (fr) * 2006-07-12 2008-01-24 Progenika Biopharma S.A. Méthode diagnostique
US8956859B1 (en) 2010-08-13 2015-02-17 Aviex Technologies Llc Compositions and methods for determining successful immunization by one or more vaccines
CN103076444B (zh) * 2013-01-04 2014-11-19 中国科学院东北地理与农业生态研究所 利用秀丽隐杆线虫鉴定白酒中塑化剂的方法
WO2016164474A1 (fr) * 2015-04-06 2016-10-13 The Trustees Of Columbia University In The City Of New York Valeur prédictive de données lipidomiques et enzymatiques génétiques combinées dans l'évaluation d'un risque de maladie pour la maladie à corps de lewy
MA44484A (fr) * 2016-03-25 2019-01-30 Genzyme Corp Biomarqueurs de protéinopathies et utilisations associées
JP6940107B2 (ja) 2018-10-30 2021-09-22 国立大学法人九州大学 発症リスク評価装置、発症リスク評価のための情報取得方法及びプログラム

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6562784B1 (en) * 1999-05-14 2003-05-13 Steffen Thiel Indications of mannan-binding lectin (MBL) in the treatment of immunocompromised individuals
US20030092019A1 (en) * 2001-01-09 2003-05-15 Millennium Pharmaceuticals, Inc. Methods and compositions for diagnosing and treating neuropsychiatric disorders such as schizophrenia

Family Cites Families (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4391904A (en) * 1979-12-26 1983-07-05 Syva Company Test strip kits in immunoassays and compositions therein
US5208036A (en) * 1985-01-07 1993-05-04 Syntex (U.S.A.) Inc. N-(ω, (ω-1)-dialkyloxy)- and N-(ω, (ω-1)-dialkenyloxy)-alk-1-yl-N,N,N-tetrasubstituted ammonium lipids and uses therefor
US5399346A (en) * 1989-06-14 1995-03-21 The United States Of America As Represented By The Department Of Health And Human Services Gene therapy
US5264618A (en) * 1990-04-19 1993-11-23 Vical, Inc. Cationic lipids for intracellular delivery of biologically active molecules
US6582908B2 (en) * 1990-12-06 2003-06-24 Affymetrix, Inc. Oligonucleotides
US5283185A (en) * 1991-08-28 1994-02-01 University Of Tennessee Research Corporation Method for delivering nucleic acids into cells
EP0714308A4 (fr) 1993-08-26 1998-07-29 Univ California Procede, compositions et dispositifs pour l'administration de polynucleotides nus qui codent des peptides a activite biologique
US5756662A (en) 1995-03-14 1998-05-26 Corixa Corporation Compounds and methods for the detection of T. cruzi infection
JP4176214B2 (ja) * 1998-11-11 2008-11-05 塩野義製薬株式会社 補体依存性細胞障害を抑制するモノクローナル抗体
EP1074634B1 (fr) 1999-07-09 2002-10-02 Institut Pasteur De Lille Procédé pour le diagnostic ou le prognostic de la maladie d'Alzheimer: Composition therapeutique pour la prévention ou le traitement de la maladie d'Alzheimer
AU2001278416A1 (en) * 2000-07-13 2002-01-30 Natimmune A/S Mannan-binding lectin (mbl) treatment of infections in individuals treated with tnf-alphainhibitors
US7022476B2 (en) * 2002-02-26 2006-04-04 New York Society For Ruptured And Crippled Maintaining The Hospital For Special Surgery Human FcγRIIB gene polymorphisms for assessing development of systemic lupus erythematosus and compositions for use thereof
CA2483441A1 (fr) * 2002-04-24 2003-11-06 The Council Of The Queensland Institute Of Medical Research Lectine de fixation du mannose et utilisations correspondantes
GB0212648D0 (en) * 2002-05-31 2002-07-10 Immunoclin Lab Ltd Treatment with cytokines
WO2005080594A2 (fr) 2004-02-24 2005-09-01 Innogenetics N.V. Methode de determination des risques qu'a un sujet de contracter une maladie neurologique

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6562784B1 (en) * 1999-05-14 2003-05-13 Steffen Thiel Indications of mannan-binding lectin (MBL) in the treatment of immunocompromised individuals
US20030092019A1 (en) * 2001-01-09 2003-05-15 Millennium Pharmaceuticals, Inc. Methods and compositions for diagnosing and treating neuropsychiatric disorders such as schizophrenia

Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150353249A1 (en) * 2005-12-09 2015-12-10 Dna Genotek Inc. Container system for releasably storing a substance
US20100173417A1 (en) * 2009-01-04 2010-07-08 Lennart Minthon Diagnostic method
US11592368B2 (en) 2011-06-19 2023-02-28 DNA Genotek, Inc. Method for collecting and preserving a biological sample
US11549870B2 (en) 2011-06-19 2023-01-10 DNA Genotek, Inc. Cell preserving solution
US11536632B2 (en) 2011-06-19 2022-12-27 DNA Genotek, Inc. Biological collection system
US11002646B2 (en) 2011-06-19 2021-05-11 DNA Genotek, Inc. Devices, solutions and methods for sample collection
US10040847B2 (en) 2012-08-16 2018-08-07 Ipierian, Inc. Methods of treating a tauopathy
US8926974B2 (en) 2012-08-16 2015-01-06 Ipierian, Inc. Methods of treating a tauopathy
US9567395B2 (en) 2012-08-16 2017-02-14 Ipierian, Inc. Methods of treating a tauopathy
US9051367B2 (en) 2013-01-18 2015-06-09 Ipierian, Inc. Methods of treating a tauopathy
US9777058B2 (en) 2013-01-18 2017-10-03 Ipierian, Inc. Methods of treating a tauopathy
US9447180B2 (en) 2013-01-18 2016-09-20 Ipierian, Inc. Methods of treating a tauopathy
US8980270B2 (en) 2013-01-18 2015-03-17 Ipierian, Inc. Methods of treating a tauopathy
US8980271B2 (en) 2013-01-18 2015-03-17 Ipierian, Inc. Methods of treating a tauopathy
US10400018B2 (en) 2014-02-14 2019-09-03 Ipierian, Inc. Tau peptides, anti-tau antibodies, and methods of use thereof
US12275685B2 (en) 2021-06-03 2025-04-15 Board Of Regents, The University Of Texas System Oligo-benzamide analogs and their use in cancer treatment

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US20120208185A1 (en) 2012-08-16
CA2555989A1 (fr) 2005-09-01
JP4884367B2 (ja) 2012-02-29
WO2005080594A3 (fr) 2006-02-09
WO2005080594A2 (fr) 2005-09-01
EP1721004A2 (fr) 2006-11-15
AU2005214091B2 (en) 2010-08-12
US20080261889A1 (en) 2008-10-23
JP2007528219A (ja) 2007-10-11
US8062842B2 (en) 2011-11-22

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