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WO2013061342A1 - Variants conférant un risque d'anévrisme intracrânial et d'anévrisme de l'aorte abdominale - Google Patents

Variants conférant un risque d'anévrisme intracrânial et d'anévrisme de l'aorte abdominale Download PDF

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Publication number
WO2013061342A1
WO2013061342A1 PCT/IS2012/000007 IS2012000007W WO2013061342A1 WO 2013061342 A1 WO2013061342 A1 WO 2013061342A1 IS 2012000007 W IS2012000007 W IS 2012000007W WO 2013061342 A1 WO2013061342 A1 WO 2013061342A1
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allele
asporin
condition
mutant
subject
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PCT/IS2012/000007
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English (en)
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Sóveig GRETARSDOTTIR
Guðmar THORLEIFSSON
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Decode Genetics Ehf
Illumina Inc.
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Publication of WO2013061342A1 publication Critical patent/WO2013061342A1/fr

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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

Definitions

  • Degenerative changes of the arterial wall may cause localized dilatation, or aneurysm, of the artery, including abdominal aorta aneurysm (AAA) and intracranial aneurysm (IA).
  • AAA abdominal aorta aneurysm
  • IA intracranial aneurysm
  • Atherosclerotic changes of the vessel wall are found in the majority of AAA that are characterized histopathologically by chronic inflammation, destructive remodeling of elastic media and depletion of medial smooth muscle cells resulting in marked weakening of the aortic wall.
  • berry aneurysms of intracranial arteries are not associated with atherosclerosis.
  • the histopathological features of IA are different.
  • Both AAA and IA represent a degenerative process of the arteries leading to their enlargement that is usually asymptomatic with natural history culminating in either a therapeutic intervention or rupture. Rupture of IA leads to subarachnoid haemorrhage, and rupture of both IA and AAA have high morbidity and mortality. In the case of AAA the rupture risk increases with the growth rate as well as the size of the aneurysm.
  • Intracranial aneurysm also called cerebral aneurysm or brain aneurysm is a cerebrovascular disorder in which weakness in the wall of a cerebral artery or vein causes a localized dilation or ballooning of the blood vessel.
  • a common location of cerebral aneurysms is on the arteries at the base of the brain, known as the Circle of Willis. Approximately 85% of cerebral aneurysms develop in the anterior part of the Circle of Willis, and involve the internal carotid arteries and their major branches that supply the anterior and middle sections of the brain. It is believed that aneurysms may result from congenital defects, preexisting conditions such as high blood pressure and atherosclerosis, or head trauma. Cerebral aneurysms occur more commonly in adults than in children but they may occur at any age.
  • Cerebral aneurysms are classified both by size and shape. Small aneurysms have a diameter of less than 15mm. Larger aneurysms include those classified as large (15 to 25mm), giant (25 to 50mm), and super giant (over 50mm). Saccular aneurysms are those with a saccular
  • Berry aneurysms are saccular aneurysms with necks or stems resembling a berry. Fusiform aneurysms are aneurysms without stems.
  • a small, unchanging aneurysm will produce no symptoms. Before a larger aneurysm ruptures, the individual may experience such symptoms as a sudden and unusually severe headache, nausea, vision impairment, vomiting, and loss of consciousness, or the individual may be asymptomatic, experiencing no symptoms at all. Onset is usually sudden and without warning. Rupture of a cerebral aneurysm is dangerous and usually results in bleeding into the meninges or the brain itself, leading to a subarachnoid hemorrhage (SAH) or intracranial hematoma (ICH), either of which constitutes a stroke.
  • SAH subarachnoid hemorrhage
  • ICH intracranial hematoma
  • hydrocephalus the excessive accumulation of cerebrospinal fluid
  • vasospasm vasospasm, or narrowing, of the blood vessels
  • multiple aneurysms may also occur.
  • the risk of rupture from an unruptured cerebral aneurysm varies according to the size of an aneurysm, with the risk rising as the aneurysm size increases.
  • the overall rate of aneurysm rupture is estimated at 1.3% per year.
  • the risk of short term re- rupture increases dramatically after an aneurysm has bled, though after approximately 6 weeks the risk returns to baseline.
  • Emergency treatment for individuals with a ruptured cerebral aneurysm generally includes restoring deteriorating respiration and reducing intracranial pressure.
  • the prognosis for a patient with a ruptured cerebral aneurysm depends on the extent and location of the aneurysm, the person's age, general health, and neurological condition. Some individuals with a ruptured cerebral aneurysm die from, the initial bleeding. Other individuals with cerebral aneurysm recover with little or no neurological deficit. The most significant factors in determining outcome are severity of the aneurysm and age.
  • Abdominal aortic aneurysm is a localized dilatation of the abdominal aorta, that exceeds the normal diameter by more than 50%.
  • the normal diameter of the infrarenal aorta is 2 cm. It is caused by a degenerative process of the aortic wall.
  • the aneurysm is most commonly located infrarenally (90%), other possible locations are suprarenal and pararenal.
  • the aneurysm can extend to include one or both of the iliac arteries.
  • An aortic aneurysm may also occur in the thorax.
  • AAA is uncommon in individuals of African, African American, Asian, and Hispanic heritage. The frequency varies strongly between males and females. The peak incidence is among males around 70 years of age, the prevalence among males over 60 years totals 2-6%. The frequency is much higher in smokers than in non-smokers (8: 1). Other risk factors include hypertension and male sex. In the US, the incidence of AAA is 2-4% in the adult population. Rupture of the AAA occurs in 1-3% of men aged 65 or more, the mortality being 70-95%.
  • AAAs are commonly divided according to their size and symptomatology.
  • An aneurysm is usually considered to be present if the measured outer aortic diameter is over 3 cm (normal diameter of aorta is around 2 cm). The natural history is of increasing diameter over time, followed eventually by the development of symptoms (usually rupture). If the outer diameter exceeds 5 cm, the aneurysm is considered to be large. For aneurysms under 5 cm, the risk of rupture is low, so that the risks of surgery usually outweigh the risk of rupture.
  • Aneurysms less than 5cm are therefore usually kept under surveillance until such time as they become large enough to warrant repair, or develop symptoms.
  • the vast majority of aneurysms are asymptomatic.
  • the risk of rupture is high in a symptomatic aneurysm, which is therefore considered an indication for surgery.
  • Possible symptoms include low back pain, flank pain, abdominal pain, groin pain or pulsating abdominal mass.
  • the complications include rupture, peripheral embolisation, acute aortic occlusion, aortocaval or aortoduodenal fistulae.
  • a palpable abdominal mass can be noted.
  • Bruits can be present in case of renal or visceral arterial stenosis.
  • the main treatment options for asymptomatic AAA are immediate repair and surveillance with a view to eventual repair. Surveillance is indicated in small aneurysms, where the risk of repair exceeds the risk of rupture. As an AAA grows in diameter the risk of rupture increases.
  • Endovascular repair first became practical in the 1990's and although it is now an established alternative to open repair, its role is yet to be clearly defined. It is generally indicated in older, high-risk patients or patients unfit for open repair. However, endovascular repair is feasible for only a proportion of AAA's, depending on the morphology of the aneurysm. The main advantage over open repair is that the perioperative period has less impact on the patient.
  • the present inventors have discovered that variants on chromosome 9q in the human Asporin gene are associated with increased risk of certain conditions, including intracranial aneurysm and abdominal aortic aneurysm.
  • the present invention relates to the utilization of such variants in the risk management of these conditions.
  • the invention provides a method of determining a susceptibility to a condition selected from the group consisting of intracranial aneurysm and abdominal aortic aneurysm, the method comprising analyzing data representative of at least one allele of an Asporin gene in a human subject, wherein different alleles of the human Asporin gene are associated with different susceptibilities to intracranial aneurysm in humans, and determining a susceptibility to intracranial aneurysm for the human subject from the data.
  • Another aspect relates to a method of determining whether an individual is at increased risk of developing a condition selected from the group consisting of intracranial aneurysm and abdominal aortic aneurysm, the method comprising steps of (i) obtaining a biological sample containing nucleic acid from the individual; (ii) determining, in the biological sample, nucleic acid sequence about the human Asporin gene; and (iii) comparing the sequence information to the wild-type sequence of human Asporin (SEQ ID NO:7); wherein an identification of a mutation in Asporin in the individual is indicative that the individual is at increased risk of developing the condition.
  • the method comprises further a step of (iv) determining whether the comparison results in the identification of the presence of a mutation in the Asporin gene in the biological sample.
  • the invention also provides a method of determining whether a human subject is at increased risk of developing a condition selected from the group consisting of intracranial aneurysm and abdominal aortic aneurysm, the method comprising analyzing amino acid sequence data about an Asporin polypeptide from the subject, wherein a determination of the presence of an altered Asporin polypeptide compared with a wild-type Asporin polypeptide with sequence as set forth in SEQ ID NO:8 is indicative that the subject is at increased risk of developing the condition.
  • a method of selecting a human subject with a condition selected from the group consisting of intracranial aneurysm and abdominal aortic aneurysm for treatment with a therapeutic regimen for treating the condition comprising (i) determining the presence or absence of at least one allele of at least one polymorphic marker in a nucleic acid sample obtained from the individual, wherein the at least one allele is an allele that encodes a defective Asporin polypeptide, and (ii) selecting for treatment with the therapeutic regimen a subject identified as having the at least one allele in the nucleic acid sample.
  • the invention also provides computer-implemented aspects for carrying out the methods described herein.
  • a system for identifying a susceptibility to a condition selected from the group consisting of intracranial aneurysm and abdominal aortic aneurysm in a human subject comprising (i) at least one processor; (ii) at least one computer- readable medium; (iii) a susceptibility database operatively coupled to a computer-readable medium of the system and containing population information correlating the presence or absence of one or more alleles of the human Asporin gene and susceptibility to the condition in a population of humans; (iv) a measurement tool that receives an input about the human subject and generates information from the input about the presence or absence of at least one mutant Asporin allele indicative of an Asporin defect in the human subject; and (v) an analysis tool that (a) is operatively coupled to the susceptibility database and the the measurement tool; (b) is stored on a computer-readable medium of the system, and (c) is adapted to be executed on a processor of the system, to
  • FIG 1 provides a diagram illustrating a system comprising computer implemented methods utilizing risk variants as described herein.
  • FIG 2 shows an exemplary system for determining risk of a condition selected from intracranial aneurysm and abdominal aortic aneurysm as described further herein.
  • FIG 3 shows a system for selecting a treatment protocol for a subject diagnosed with a condition selected from intracranial aneurysm and abdominal aortic aneurysm.
  • nucleic acid sequences are written left to right in a 5' to 3' orientation.
  • Numeric ranges recited within the specification are inclusive of the numbers defining the range and include each integer or any non-integer fraction within the defined range.
  • all technical and scientific terms used herein have the same meaning as commonly understood by the ordinary person skilled in the art to which the invention pertains.
  • the marker can comprise any allele of any variant type found in the genome, including SNPs, mini- or microsatellites, insertion-deletions, translocations and copy number variations (insertions, deletions, duplications).
  • Polymorphic markers can be of any measurable frequency in the population. For mapping of disease genes, polymorphic markers with popu lation frequency higher than 5-10% are in general most usefu l. However, polymorphic markers may also have lower population frequencies, such as 1-5% frequency, or even lower frequency, such as 0.1 to 1% frequency. The term shall, in the present context, be taken to include polymorphic markers with any population frequency.
  • an "allele” refers to the nucleotide sequence of a given locus (position) on a chromosome.
  • a polymorphic marker allele thus refers to the composition (i.e., sequence) of the marker on a chromosome.
  • Genomic DIMA from an individual contains two alleles (e.g. , allele-specific sequences) for any given polymorphic marker, representative of each copy of the marker on each chromosome.
  • a nucleotide position at which more than one sequence is possible in a population is referred to herein as a "polymorphic site”.
  • a "Single Nucleotide Polymorphism” or "SNP” is a DNA sequence variation occurring when a sing le nucleotide at a specific location in the genome differs between members of a species or between paired chromosomes in an individual. Most SNP polymorphisms have two alleles. Each individual is in this instance either homozygous for one allele of the polymorphism (i.e. both chromosomal copies of the individual have the same nucleotide at the SNP location), or the ind ividual is heterozygous (i.e. the two sister chromosomes of the individual contain different nucleotides).
  • the SNP nomenclature as reported herein refers to the official Reference SNP (rs) ID identification tag as assig ned to each unique SNP by the National Center for Biotechnological Information (NCBI) .
  • a “variant”, as described herein, refers to a segment of DNA that comprises a polymorphic site.
  • a “marker” or a “polymorphic marker”, as defined herein, is a variant.
  • a "microsatellite” is a polymorphic marker that has multiple small repeats of bases that are 2-8 nucleotides in length (such as CA repeats) at a particular site, in which the number of repeat lengths varies in the general population.
  • an “indel” or an “insertion-deletion” is a common form of polymorphism comprising a small insertion or deletion that is typically only a few nucleotides long.
  • haplotype refers to a segment of genomic DNA that is characterized by a specific combination of alleles arranged along the segment.
  • a haplotype comprises one member of the pair of alleles for two or more polymorphic markers or loci along the segment.
  • the haplotype can comprise two or more alleles, three or more alleles, four or more alleles, or five or more alleles.
  • Allelic identities are described herein in the context of the marker name and the particular allele of the marker, e.g. , "chr9 :94276933 allele A" refers to the A allele of marker chr9 :94276933.
  • Asporin refers to the asporin gene on chromosome 9q22.31.
  • the gene is sometimes abbreviated as ASPN, and is sometimes also referred to as periodontal ligament-associated protein 1 (PLAP1 ).
  • the name reflects the unique aspartate-rich N-terminus of the encoded asporin protein and the overall sequence similarity to decorin.
  • the sequence of the Asporin gene is set forth in SEQ ID NO:7 herein, and the sequence of the encoded asporin protein is set forth in SEQ ID NO:8 herein.
  • susceptibility refers to the proneness of an individual towards the development of a certain state ⁇ e.g. , a certain trait, phenotype or disease), or towards being less able to resist a particular state than the average ind ividual.
  • the term encompasses both increased susceptibility and decreased susceptibility.
  • particular alleles at polymorphic markers may be characteristic of increased susceptibility (i.e., increased risk) of a condition such as intracranial aneurysm and/or abdominal aortic aneurysm, as characterized by a relative risk (RR) or odds ratio (OR) of greater than one for the particular allele.
  • the markers and/or haplotypes of the invention are characteristic of decreased susceptibility (i.e., decreased risk) of the condition, as characterized by a relative risk of less than one.
  • look-up table is a table that correlates one form Of data to another form, or one or more forms of data to a predicted outcome to which the data is relevant, such as phenotype or trait.
  • a look-up table can comprise a correlation between allelic data for at least one polymorphic marker and a particular trait or phenotype, such as a particular d isease diagnosis, that an individual who comprises the particular allelic data is likely to display, or is more likely to display than individuals who do not comprise the particular allelic data.
  • Look-up tables can be multid imensional, i.e. they can contain information about multiple alleles for single markers simultaneously, or they can contain information about multiple markers, and they may also comprise other factors, such as particulars about diseases diagnoses, racial information, biomarkers, biochemical measurements, therapeutic methods or drugs, etc.
  • databases refers to a collection of data organized for one or more purposes.
  • databases may be organized in a digital format for access, analysis, or processing by a computer.
  • the data are typically organized to model features relevant to the invention.
  • one component of data in a database may be information about variations in a population, such as genetic variation with respect to Asporin, but also variation with respect to other medically informative parameters, including other genetic loci, race, ethnicity, sex, age, behaviors and lifestyle (tobacco consumption (smoking), alcohol consumption (drinking), exercise, body mass indices), glucose tolerance/diabetes, blood pressure, lipid profile, cholesterol levels and any other factors that medical personnel may measure in the context of standard medical care or specific diagnoses.
  • Other components of the database may include one or more sets of data relating to susceptibility to a disease in a population, and/or suitability or success of a disease treatment, and/or suitability or success of a protocol for screening for or presenting a disease, such as intracranial aneurysm and/or abdominal aortic aneurysm.
  • the data is organized to permit analysis of how the biological variation in the population correlates with the susceptibility to disease and/or the suitability or success of the treatment, protocol, etc.
  • a look-up datable (or the information in a look-up table) may be stored in a database to facilitate aspects of the invention.
  • a "computer-readable medium” is ah information storage medium that can be accessed by a computer using a commercially available or custom-made interface.
  • Exemplary computer- readable, media include memory ⁇ e.g., RAM, ROM, flash memory, etc.), optical storage media (e.g., CD-ROM), magnetic storage media (e.g., computer hard drives, floppy disks, etc.), punch cards, or other commercially available media.
  • Information may be transferred between a system of interest and a medium, between computers, or between computers and the computer- readable medium for storage or access of stored information. Such transmission can be electrical, or by other available methods, such as IR links, wireless connections, etc.
  • biological sample refers to a sample obtained from an individual that contains nucleic acid and/or protein and/or fluid containing organic and/or inorganic metabolites and substances.
  • the biological sample comprises nucleic acid suitable for genetic analysis.
  • nucleic acid sample refers to a sample obtained from an individual that contains nucleic acid (DNA or RNA).
  • the nucleic acid sample comprises genomic DNA.
  • a nucleic acid sample can be obtained from any source that contains genomic DNA, including a blood sample, sample of amniotic fluid, sample of cerebrospinal fluid, or tissue sample from skin, muscle, buccal or conjunctival mucosa, placenta, gastrointestinal tract or other organs.
  • antisense agent or “antisense oligonucleotide” refers, as described herein, to molecules, or compositions comprising molecules, which include a sequence of purine an pyrimidine heterocyclic bases, supported by a backbone, which are effective to hydrogen bond to corresponding contiguous bases in a target nucleic acid sequence.
  • the backbone is composed of subunit backbone moieties supporting the purine and pyrimidine heterocyclic bases at positions which allow such hydrogen bonding. These backbone moieties are cyclic moieties of 5 to 7 atoms in size, linked together by phosphorous-containing linkage units of one to three atoms in length.
  • the antisense agent comprises an oligonucleotide molecule. Variants on chromosome 9q in the Asporin gene associate with intracranial aneurysm and abdominal aortic aneurysm
  • variants on chromosome 9q in the human Asporin gene are predictive of risk of conditions such as intracranial aneurysm and abdominal aortic aneurysm.
  • the inventors have discovered that certain variants in the Asporin gene confer increased risk of these conditions.
  • the inventors have furthermore identified novel missense and nonsense variants at several additional positions in the human Asporin gene, which are believed to represent further variations conferring increased risk of intracranial aneurysm and/or abdominal aortic aneurysm .
  • Identified variants are summarized in the variant table.
  • Variant Table Shown is identity of variant, its position in the Asporin gene (SEQ ID NO: 7), the encoded amino acid change in SEQ ID NO : 8, identity of mutated and wild-type alleles and reference to flanking sequence of the variant.
  • Asporin is an extracellu lar matrix protein (ECM) which belongs to the family of small leucine-rich proteoglycans (SLRPs).
  • SLRPs are biologically active components of the extracellular matrix and they consist of two different structural components, (i) a conserved protein core (with leucine rich-repeat motifs, LRRs) involved in protein/protein interactions and (ii) varying numbers and types of glycosaminoglycan (GAG) side chains. These components allow the SLRPs to interact with a host of different cell surface receptors, cytokines, g rowth factors and other ECM components thereby modulating a wide range of cell-matrix interactions.
  • ECM extracellu lar matrix protein
  • the SLRPs are divided into different classes based on genomic and protein homology and asporin belongs to the class I family.
  • the class I includes also decorin, biglycan, ECM 2 and ECMX.
  • the best characterized SLRP family members are decorin and biglycan.
  • Both decorin and biglycan have multiple functions and have been shown to be involved in control of fibrillogenesis, cell proliferation and survival, fibrosis, inflammation, modulation of cytokine bio-activity, adhesion and migration and receptor- binding associated with signal transduction (Merline, R. et a/. J Cell Commun Signal 3 : p. 323-35 (2009)) .
  • the amino acid sequence of asporin is highly similar to that of decorin and big lycan.
  • asporin lacks a serine/glycine dipeptide consensus sequence for GAG substitutions and is therefore not considered to be a proteoglycan. Instead asporin contains a unique N-terminal aspartic acid repeat (D-repeat) which is polymorphic in humans. It has been demonstrated that the asporin D-repeat polymorphism significantly associates with knee and hip osteoarthritis in the Japanese population (allele D14 is risk allele and D13 is protective) (Kizawa, H. et al. Nat Genet-44 (2005)).
  • D-repeat N-terminal aspartic acid repeat
  • Asporin The functional role of Asporin is not well known. Due to the association observed between Asporin D-repeat polymorphism and OA, most studies have focused on the function and regulation of Asporin in articular cartilage. Asporin mRNA is expressed in a number of d ifferent tissues with the highest expression in osteoarthritic articular cartilage, aorta, uterus, heart and liver (Lorenzo, P., et al. J Biol Chem 276 : 12201-11 (2001)) .
  • asporin like the other SLRP class I proteins, plays a structural and/or signalling function in the extracellular matrix of skeletal and other specialised connective tissues (Henry, S.P., et al. J Biol Chem 276: 12212-21 (2001)).
  • asporin physically binds to TGF- ⁇ through its LRR domains and inhibits TGF- ⁇ signalling (Kou, I., M . Nakajima, and S. Ikegawa, J Bone Miner Metab 28:395-402 (2010) ; Nakajima, M ., et al. J Biol Chem 282:32185-92 (2007)).
  • asporin negatively regulates chondrogenesis by inhibiting the canonical TGF- ⁇ /Smad signal.
  • TGF- ⁇ is a multifunctional cytokine involved in numerous essential biological processes including development, ECM synthesis, cell proliferation, differentiation and tissue repair. Perturbations of TGF- ⁇ signalling has been strongly implicated in the pathogenesis of several diseases e.g. in cancer initiation and progression and tissue fibrosis.
  • TGF- ⁇ signalling is associated with aortic aneurysm in a mouse model of Marfan syndrome (a systemic disorder of connective tissue caused by mutations in the gene encoding fibrillin- 1) and this can be prevented by TGF- ⁇ agonists (Habashi, J. ., et al. Science 312 : 117-21 (2006)).
  • Excess TGF- ⁇ sig nalling has also been demonstrated in other aortic aneurysm syndromes, including Loeys-Dietz syndrome (caused by mutations in the TGFRBl or TGFRB2) and arterial tortuosity syndrome (caused by mutation in SLC2A10) (Coucke, P.J., et al. Nat Genet 38 :452-7 (2006) ; Loeys, B.L., et a/. Nat Genet 37 :275-81 (2005)) .
  • TGFRBl TGFRB2 and ACVR1
  • TGF- ⁇ ligand TGFB1
  • ENG co-receptors endog lin
  • TGFBR3 betaglycan
  • Asporin is expressed in osteoblast progenitor cells and as these cells regulate intramembranous bone formation (as well as being involved in healing of bone fractu res) it has been proposed that asporin may be one of the key proteins implied in calcification of blood vessels (Kalamajski, S., et al. Biochem J 423 : 53-9 (2009)).
  • the invention provides a method of determining a susceptibility to a cond ition selected from the group consisting of intracranial aneurysm and abdominal aortic aneurysm, the method comprising analyzing data representative of at least one allele of an Asporin gene (SEQ ID NO:7) in a human subject, wherein d ifferent alleles of the human Asporin gene are associated with different susceptibilities to the condition in humans, and determining a susceptibility to the condition for the human subject from the data.
  • SEQ ID NO:7 Asporin gene
  • the data can be any type of data that is representative of polymorphic alleles in the Asporin gene.
  • the data is nucleic acid sequence data.
  • the sequence data is data that is sufficient to provide information about particular alleles in a nucleic acid sequence.
  • the sequence data identifies the nucleotides at one specific position in a nucleic acid .
  • the sequence data identifies nucleotides at two or more sequential position in a nucleic acid .
  • the nucleic acid sequence data is obtained from a biological sample comprising or containing nucleic acid from the human individual.
  • the nucleic acids sequence may suitably be obtained using a method that comprises at least one procedure selected from (i) amplification of nucleic acid from the biological sample; (ii) hybrid ization assay using a nucleic acid probe and nucleic acid from the biolog ical sample;
  • the nucleic acid sequence data may also be obtained from a preexisting record .
  • the preexisting record may comprise a genotype dataset for at least one polymorphic marker.
  • the determining comprises comparing the sequence data to a database containing correlation data between the at least one polymorphic marker and susceptibility to the condition.
  • the sequence data is provided as genotype data, identifying the presence or absence of particular alleles at polymorphic locations.
  • the analyzing comprises analyzing the data for the presence or absence of at least one mutant allele indicative of an Asporin defect.
  • the defect may for example be a missense mutation in the Asporin gene relative to a wild-type Asporin gene, such as the wild- type Asporin gene with sequence as presented in SEQ ID NO:7 herein.
  • the defect may be a premature truncation or frameshift of an encoded Asporin protein, relative to a wild- type amino acid sequence, such as the wild-type amino acid sequence presented in SEQ ID NO :8 herein.
  • the Asporin defect may also be expression of an Asporin protein with reduced activity compared to a wild-type Asporin protein.
  • the activity can for example be calcium binding activity and collagen binding activity.
  • the activity may also be TGF- ⁇ binding activity.
  • the Asporin defect is selected from defects that impair any of these activities. Determination of activities such as calcium binding, collagen binding and TGF- ⁇ binging can be performed using standard assays well known to the skilled person, some of which are described herein. As noted above, such assays can be used to confirm that a particular Asporin mutation impairs or eliminates an Asporin activity and therefore would be expected to carry an increased susceptibility for intracranial aneurysm and/or abdominal aortic aneurysm as described herein.
  • the data to be analyzed by the method of the invention is suitably obtained by analysis of a biolog ical sample from a human subject to obtain information about particular alleles in the genome of the individual.
  • the information is nucleic acid information which comprises sufficient sequence to identify the presence or absence of at least one allele in the subject (e.g. a mutant allele) .
  • the information can also be nucleic acid information that identifies at least one allele of a polymorphic marker that is in linkage disequilibrium with a mutant allele.
  • Linkage d isequilibrium may suitably be determined by the correlation coefficient between polymorphic sites. In one embodiment, the sites are correlated by values of the correlation coefficient r 2 of greater than 0.5.
  • the information may also be information about measurement of quantity of length of Asporin mRNA, wherein the measurement is ind icative of the presence or absence of the mutant allele.
  • mutant alleles may result in premature truncation of transcribed mRNA which can be detected by measuring the length of mRNA.
  • the information may further be measurement of quantity of Asporin protein, wherein the measurement of protein is ind icative of the presence or absence of a mutant allele. Truncated transcripts will result in truncated forms of translated polypeptides, which can be measured using standard methods known in the art.
  • truncated proteins or proteins arising from a frameshift may have fewer or different epitopes from wildtype protein and can be distinguished with immunoassays. Truncated proteins or proteins altered in other ways may migrate differently and be distinguished with electrophoresis.
  • the information obtained may also be measurement of Asporin activity, wherein the measurement is indicative of the mutant allele.
  • the activity is suitably selected from collagen binding activity or calcium binding activity. In one embodiment, the information is selected from any one of the above mentioned types of information.
  • analyzing data comprises analyzing a biological sample from the human subject to obtain information selected from the group consisting of (a) nucleic acid sequence information, wherein the nucleic acid sequence information comprises sequence sufficient to identify the presence or absence of the mutant allele in the subject; (b) nucleic acid sequence information, wherein the nucleic acid sequence information identifies at least one allele of a polymorphic marker in linkage disequilibrium (LD) with the mutant allele, wherein the LD is characterized by a value for r 2 of at least 0.5; (c) measurement of the quantity or length of Asporin mRNA, wherein the measurement is ind icative of the presence or absence of the mutant allele; (d) measurement of the quantity of Asporin protein, wherein the measurement is indicative of the presence or absence of the mutant allele; and (e) measurement of Asporin activity, wherein the measurement is indicative of the presence or absence of the mutant allele.
  • LD polymorphic marker in linkage disequilibrium
  • a biolog ical sample is obtained from the human subject prior to the analyzing steps.
  • the analyzing may a lso suitably be performed by analyzing data from a preexisting record about the human subject.
  • the preexisting record may for example include sequence information or genotype information about the individual, which can identify the presence or absence of mutant alleles.
  • information about risk for the human subject can be determined using methods known in the art. Some of these methods are described herein. For example, information about odds ratio (OR), relative risk (RR) or lifetime risk (LR) can. be determined from information about the presence or absence of particular mutant alleles of Asporin.
  • OR odds ratio
  • RR relative risk
  • LR lifetime risk
  • the mutant allele of Asporin is a missense mutation, a frameshift mutation or a nonsense mutation. In one preferred embodiment, the mutant allele is a missense mutation. In another preferred embodiment, the mutant allele is a nonsense mutation. In another preferred embodiment, the mutant allele is a frameshift mutation. In certain embodiments, the mutant allele is a missense mutation, a frameshift mutation or a nonsense mutation. In one preferred embodiment, the mutant allele is a missense mutation. In another preferred embodiment, the mutant allele is a nonsense mutation. In another preferred embodiment, the mutant allele is a frameshift mutation. In certain
  • the mutation is selected from the group consisting of chr9 :94261761 allele A, chr9 :94261811 allele T, chr9 :94268484 allele T, chr9 :94268497 allele G, chr9 :94272846 allele A and chr9 : 94276933 allele A.
  • the mutant allele is a missense mutation in Asporin that results in expression of an Asporin protein with reduced or no activity compared to a wild-type Asporin protein.
  • the activity may suitably be calcium binding activity and collagen binding activity.
  • the mutant allele may also be a promoter polymorphism that leads to decreased expression of Asporin.
  • the mutant allele in Asporin encodes one of the following amino acid substitutions in Asporin : G307STOP, R290H, G193E, D189H, R105STOP and H23L. In one preferred embodiment, the mutant allele encodes a H23L amino acid substitution in Asporin.
  • another aspect of the invention may relate to a method of determining whether an individual is at increased risk of developing a condition selected from intracranial aneurysm and abdominal aortic aneurysm, the method comprising steps of (a) obtaining a biological sample containing nucleic acid from the individual; (b) determining, in the biological sample, nucleic acid sequence about the Asporin gene, and (c) comparing the sequence information to the wild-type sequence of Asporin, as set forth in SEQ ID NO:7 herein, wherein the identification of a mutation in Asporin in the individual is indicative that the individual is at increased risk of developing the condition.
  • the invention provides a method of determining whether an individual is at increased risk of developing a condition selected from intracranial aneurysm and abdominal aortic aneurysm, the method comprising steps of determining, in a biological sample from the individual, nucleic acid sequence about the Asporin gene, and comparing the sequence information to the wild-type sequence of Asporin, as set forth in SEQ ID NO:7 herein, wherein the identification of a mutation in Asporin in the individual is indicative that the individual is at increased risk of developing the condition.
  • the mutation may be a missense mutation, a promoter mutation, a nonsense mutation or a frameshift mutation in Asporin.
  • the mutation may further result in an Asporin defect as described in the above.
  • the human subject or human individual whose susceptibility is being assessed may be a male or a female.
  • the invention provides a method of determining a susceptibility to a condition selected from intracranial aneurysm and abdominal aortic aneurysm, the method comprising analyzing sequence data from a human subject for at least one variant in the human Asporin gene, or in an encoded human Asporin protein, wherein different alleles of the at least one variant are associated with different susceptibilities to the condition in humans, and determining a susceptibility to the condition for the human subject from the sequence data.
  • the variant is selected from the group consisting of chr9:94261761,
  • the variant is a variant that is correlated with at least one of these variants by a value for the correlation coefficient r2 > 0.5.
  • the data that is obtained is nucleic acid sequence data.
  • the nucleic acid sequence data is obtained from a biological sample comprising or containing nucleic acid from the human individual.
  • the nucleic acids sequence may suitably be obtained using a method that comprises at least one procedure selected from (i) amplification of nucleic acid from the biological sample; (ii) hybridization assay using a nucleic acid probe and nucleic acid from the biological sample; (iii) hybridization assay using a nucleic acid probe and nucleic acid obtained by amplification of the biological sample, and (iv) sequencing, in particular high-throughput sequencing.
  • the nucleic acid sequence data may also be obtained from a preexisting record.
  • the preexisting record may comprise a genotype dataset for at least one polymorphic marker.
  • the determining comprises comparing the sequence data to a database containing correlation data between the at least one
  • polymorphic marker and susceptibility to intracranial aneurysm and/or abdominal aortic aneurysm are polymorphic markers and susceptibility to intracranial aneurysm and/or abdominal aortic aneurysm.
  • Certain risk alleles have been found to be predictive of increased risk of intracranial aneurysm and abdominal aortic aneurysm. Thus, in certain embodiments, determination of the presence of at least one allele selected from the group consisting of chr9:94261761 allele A, chr9:94261811 allele T, chr9:94268484 allele T, chr9:94268497 allele G, chr9 :94272846 allele A and
  • chr9:94276933 allele A is indicative of an increased susceptibility of the condition for the human subject.
  • allele A of chr9 :94276933 is indicative of increased risk of intracranial aneurysm and abdominal aortic aneurysm.
  • determination of the presence of the allele is indicative of increased risk of these conditions the individual.
  • Determination of the absence of the risk variant (mutation) is indicative that the individual does not have the increased risk for the condition conferred by the allele.
  • the allele that is detected can be the allele of the complementary strand of DNA, such that the nucleic acid sequence data identifies an allele which is complement to any of the alleles of the polymorphic markers referenced above.
  • the allele that is detected may be the complementary T allele of the at-risk A allele of chr9 :94276933.
  • certain embodiments of the methods of the invention comprise a further-step of preparing a report containing results from the determination of risk, wherein said report is written in a computer readable medium, printed on paper, or displayed on a visual display.
  • Sequence data can be nucleic acid sequence data, which may be obtained by means known in the art. Sequence data is suitably obtained from a biological sample of genomic DNA, RNA, or cDNA (a "test sample") from an individual ("test subject). For example, nucleic acid sequence data may be obtained through direct analysis of the sequence of the polymorphic position (allele) of a polymorphic marker. Suitable methods, some of which are described herein, include, for instance, whole genome sequencing methods, whole genome analysis using SNP chips (e.g., Infinium HD BeadChip), cloning for polymorphisms, non-radioactive PCR-single strand
  • conformation polymorphism analysis denaturing high pressure liquid chromatography (DHPLC), DNA hybridization, computational analysis, single-stranded conformational polymorphism
  • SSCP restriction fragment length polymorphism
  • RFLP restriction fragment length polymorphism
  • CDGE clamped denaturing gel electrophoresis
  • DGGE denaturing gradient gel electrophoresis
  • CMC chemical mismatch cleavage
  • RNase protection assays use of polypeptides that recog nize nucleotide mismatches, such as E. coli mutS protein, allele-specific PCR, and direct manual and automated sequencing.
  • sequence data useful for performing the present invention may be obtained by any such sequencing method, or other sequencing methods that are developed or made available.
  • any sequence method that provides the allelic identity at particular polymorphic sites e.g., the absence or presence of particular alleles at particular polymorphic sites is useful in the methods described and claimed herein.
  • hybridization methods may be used (see Current Protocols in Molecular Biology, Ausubel et al., eds., John Wiley & Sons, including all su pplements) .
  • a biological sample of genomic DNA, RNA, or cDNA (a "test sample") may be obtained from a test subject. The subject can be an adult, child, or fetus. The DNA, RNA, or cDNA sample is then examined. The presence of a specific marker allele can be indicated by sequence-specific hybridization of a nucleic acid probe specific for the particular allele. The presence of more than one specific marker allele or a specific haplotype can be indicated by using several sequence-specific nucleic acid probes, each being specific for a particular allele.
  • a sequence-specific probe can be directed to hybridize to genomic DNA, RNA, or cDNA.
  • a "nucleic acid probe”, as used herein, can be a DNA probe or an RNA probe that hybrid izes to a complementary sequence.
  • One of skill in the art would know how to design such a probe so that sequence specific hybridization will occur only if a particular allele is present in a genomic sequence from a test sample.
  • determination of a susceptibility to intracranial aneurysm and abdominal aortic aneurysm comprises forming a hybridization sample by contacting a test sample, such as a genomic DNA sample, with at least one nucleic acid probe.
  • a test sample such as a genomic DNA sample
  • a probe for detecting mRNA or genomic DNA is a labeled nucleic acid probe that is capable of hybridizing to mRNA or genomic DNA sequences described herein.
  • the nucleic acid probe can be, for example, a full-length nucleic acid molecule, or a portion thereof, such as an oligonucleotide of at least 10, 15, 30, 50, 100, 250 or 500 nucleotides in length that is sufficient to specifically hybridize under stringent conditions to appropriate mRNA or genomic DNA.
  • the nucleic acid probe can comprise all or a portion of the nucleotide sequence of the Asporin gene, or the probe can be the complementary sequence of such a sequence.
  • Hybridization can be performed by methods well known to the person skilled in the art (see, e.g., Current Protocols in Molecular Biology, Ausubel et al., eds., John Wiley & Sons, including all supplements).
  • hybridization refers to specific hybridization, i.e., hybridization with no mismatches (exact hybridization).
  • the hybridization conditions for specific hybridization are high stringency.
  • Specific hybridization if present, is detected using standard methods. If specific hybridization occurs between the nucleic acid probe and the nucleic acid in the test sample, then the sample contains the allele that is complementary to the nucleotide that is present in the nucleic acid probe.
  • a peptide nucleic acid (PNA) probe can be used in addition to, or instead of, a nucleic acid probe in the hybridization methods described herein.
  • a PNA is a DNA mimic having a peptide-like, inorganic backbone, such as N-(2-aminoethyl)glycine units, with an organic base (A, G, C, T or U) attached to the glycine nitrogen via a methylene carbonyl linker (see, for example, Nielsen et al., Bioconjug. Chem. 5:3-7 (1994)).
  • the PNA probe can be designed to specifically hybridize to a molecule in a sample suspected of containing one or more of the at-risk alleles (mutations) shown herein to be associated with risk of intracranial aneurysm and abdominal aortic aneurysm.
  • Allele-specific oligonucleotides can also be used to detect the presence of a particular allele in a nucleic acid.
  • An "allele-specific oligonucleotide” (also referred to herein as an “allele-specific oligonucleotide probe”) is an oligonucleotide of any suitable size, for example an oligonucleotide of approximately 10-50 base pairs or approximately 15-30 base pairs, that specifically hybridizes to a nucleic acid which contains a specific allele at a polymorphic site (e.g., a polymorphic marker).
  • An allele-specific oligonucleotide probe that is specific for one or more particular alleles at polymorphic markers can be prepared using standard methods (see, e.g., Current Protocols in Molecular Biology, supra). PCR can be used to amplify the desired region. Specific hybridization of an allele-specific oligonucleotide probe to DNA from a subject is indicative of the presence of a specific allele at a polymorphic site (see, e.g., Gibbs et al., Nucleic Acids Res. 17 :2437-2448 (1989) and WO 93/22456). With the addition of analogs such as locked nucleic acids (LNAs), the size of primers and probes can be reduced to as few as 8 bases.
  • LNAs locked nucleic acids
  • LNAs are a novel class of bicyclic DNA analogs in which the 2' and 4' positions in the furanose ring are joined via an O-methylene (oxy-LNA), S-methylene (thio-LNA), or am ino methylene (amino-LNA) moiety.
  • oxy-LNA O-methylene
  • thio-LNA S-methylene
  • amino-LNA am ino methylene
  • Common to all of these LNA variants is an affinity toward complementary nucleic acids, which is by far the highest reported for a DNA analog .
  • particular all oxy-LNA nonamers have been shown to have melting temperatures (Tm) of 64°C and 74°C when in complex with complementary DNA or RNA, respectively, as opposed to 28°C for both DNA and RNA for the corresponding DNA nonamer.
  • LNA monomers are used in combination with standard DNA or RNA monomers.
  • the Tm could be increased considerably. It is therefore contemplated that in certain embod iments, LNAs are used to detect particular at-risk alleles, as described herein.
  • arrays of oligonucleotide probes that are complementary to target nucleic acid sequence segments from a subject can be used to identify particular alleles in a nucleic acid .
  • an oligonucleotide array can be used .
  • Oligonucleotide arrays typically comprise a plurality of different oligonucleotide probes that are coupled to a surface of a substrate in different known locations.
  • arrays can generally be produced using mechanical synthesis methods or lig ht d irected synthesis methods that incorporate a combination of photolithographic methods and solid phase oligonucleotide synthesis methods, or by other methods known to the person skilled in the art (see, e.g ., Bier et a ⁇ ., * Adv Biochem Eng
  • standard techniques for genotyping can be used to detect particular marker alleles, such as fluorescence-based techniques (e.g. , Chen et al., Genome Res. 9(5) : 492-98 (1999); Kutyavin et al., Nucleic Acid Res. 34 : el28 (2006)), utilizing PCR, LCR, Nested PCR and other techniques for nucleic acid amplification .
  • fluorescence-based techniques e.g. , Chen et al., Genome Res. 9(5) : 492-98 (1999); Kutyavin et al., Nucleic Acid Res. 34 : el28 (2006)
  • Specific commercial methodologies available for SNP genotyping include, but are not limited to, TaqMan genotyping assays and SNPlex platforms (Applied
  • Biosystems Biosystems
  • Gel electrophoresis Applied Biosystems
  • mass spectrometry e.g., MassARRAY system from Sequenom
  • minisequencing methods real-time PCR
  • Bio-Plex system BioRad
  • CEQ and SNPstream systems Beckman
  • array hybrid ization technology e.g., Affymetrix
  • Suitable biological sample in the methods described herein can be any sample containing nucleic acid (e.g., genomic DNA) and/or protein from the human individual.
  • the biological sample can be a blood sample, a serum sample, a leukapheresis sample, an amniotic fluid sample, a cerbrospinal fluid sample, a hair sample, a tissue sample from skin, muscle, buccal, or conjuctival mucosa, placenta, gastrointestinal tract, or other organs, a semen sample, a urine sample, a saliva sample, a nail sample, a tooth sample, and the like.
  • the sample is a blood sample, a saliva sample or a buccal swab.
  • nucleic acid sequence data may be obtained through indirect analysis of the nucleic acid sequence of the allele of the polymorphic marker, i.e. by detecting a protein variation.
  • one aspect of the invention relates to a method of determining whether a human subject is at increased risk of developing a condition selected from intracranial aneurysm and abdominal aortic aneurysm, the method comprising analyzing amino acid sequence data about a Asporin polypeptide from the subject, wherein a determination of the presence of an altered Asporin polypeptide compared with a wild-type Asporin polypeptide with sequence as set forth in SEQ ID NO:8 is indicative that the subject is at increased risk of developing the condition.
  • the altered Asporin polypeptide is an Asporin polypeptide that contains a missense mutation or a nonsense mutation compared with wild-type Asporin. In certain embodiments, the altered Asporin polypeptide has a reduced activity compared with wild-type Asporin, wherein the activity is selected from calcium binding activity and collagen binding activity.
  • Methods of detecting variant proteins are known in the art. For example, direct amino acid sequencing of the variant protein followed by comparison to a reference amino acid sequence can be used . Alternatively, SDS-PAGE followed by gel staining can be used to detect variant proteins of different molecular weights. Also, Immunoassays, e.g., antibody assays, e.g ., immunofluorescent immunoassays, immunoprecipitations, radioimmunoasays, ELISA, and Western blotting, in which an antibody specific for an epitope comprising the variant sequence among the variant protein and non-variant or wild-type protein can be used. In certain embodiments, the amino acid sequence data about Asporin protein is obtained or deduced from a preexisting record.
  • an amino acid substitution in the human Asporin protein is detected.
  • the amino acid substitution is selected from the group consisting of R290H, G193E, D189H and H23L
  • a truncated polypeptide encoded by an altered Asporin gene sequence is detected.
  • the truncated polypeptide is truncated at position 307.
  • the truncated polypeptide is truncated at position 105.
  • the detection may be suitably performed, for example using any of the methods described in the above, or any other suitable method known to the skilled artisan.
  • the methods can comprise obtaining sequence data about any number of polymorphic markers and/or about any number of genes.
  • the method can comprise obtaining sequence data for about at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 100, 500, 1000, 10,000 or more polymorphic markers.
  • the sequence data is obtained from a microarray comprising probes for detecting a plurality of markers.
  • the sequence data is obtained through nucleic acid sequencing, for example by high-throughput nucleic acid sequencing .
  • the sequence data may also be obtained by imputation of nucleic acid sequence using known methods, such as those described herein.
  • the polymorphic markers can be the ones of the group specified herein or they can be d ifferent polymorphic markers that are not specified herein.
  • the method comprises obtaining sequence data about at least two polymorphic markers.
  • each of the markers may be associated with a different gene.
  • the method comprises obtaining nucleic acid data about a human individual identifying at least one allele of a polymorphic marker, then the method comprises identifying at least one allele of at least one polymorphic marker.
  • the method can comprise obtaining sequence data about a human individual identifying alleles of multiple, independent markers, which are not in linkage disequilibrium.
  • Linkage Disequilibrium refers to a non-random assortment of two genetic elements. For example, if a particu lar genetic element (e.g. , an allele of a polymorphic marker, or a haplotype) occurs in a population at a frequency of 0.50 (50%) and another element occurs at a frequency of 0.50 (50%), then the predicted occurrance of a person's having both elements is 0.25 (25%), assuming a random distribution of the elements.
  • a particu lar genetic element e.g. , an allele of a polymorphic marker, or a haplotype
  • Allele or haplotype frequencies can be determined in a population by genotyping individuals in a population and determining the frequency of the occurence of each allele or haplotype in the popu lation. For populations of diploids, e.g., human populations, individuals will typically have two alleles for each genetic element (e.g. , a marker, haplotype or gene).
  • the measure r 2 represents the statistical correlation between two sites, and takes the value of 1 if only two haplotypes are present. Markers which are correlated by an r 2 value of 1 are said to be perfectly correlated. In such an instance, the genotype of one marker perfectly predicts the genotype of the other.
  • the r 2 measure is arguably the most relevant measure for association mapping, because there is a simple inverse relationship between r 2 and the sample size required to detect association between susceptibility loci and SNPs. These measures are defined for pairs of sites, but for some applications a determination of how strong LD is across an entire region that contains many polymorphic sites might be desirable (e.g., testing whether the strength of LD differs significantly among loci or across populations, or whether there is more or less LD in a region than predicted under a particular model). Measuring LD across a region is not straightforward, but one approach is to use the measure r, which was developed in population genetics.
  • a significant r 2 indicative of markers being in linkage disequilibrium may be at least 0.1, such as at least 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99 or 1.0.
  • a significant r 2 indicates that the markers are highly correlated, and therefore in linkage disequilibrium. Highly correlated markers must, be definition, show highly comparable results in association mapping, since the genotypes for one marker predicts the genotype for another, correlated, marker.
  • the significant r 2 value can be at least 0.2.
  • the significant r 2 value can be at least 0.5. In one specific embodiment of invention, the significant r 2 value can be at least 0.8.
  • linkage disequilibrium as described herein refers to linkage disequilibrium characterized by values of r 2 of at least 0.2, such as 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.85, 0.9, 0.95, 0.96, 0.97, 0.98, 0.99.
  • linkage disequilibrium represents a correlation between alleles of distinct markers. It is measured by correlation coefficient or
  • Linkage disequilibrium can be determined in a single human population, as defined herein, or it can be determined in a collection of samples comprising individuals from more than one human population.
  • LD is determined in a sample from one or more of the HapMap populations. These include samples from the Yoruba people of Ibadan, Nigeria (YRI), samples from individuals from the Tokyo area in Japan (JPT), samples from individuals Beijing, China (CHB), and samples from U.S. residents with northern and western European ancestry (CEU), as described (The International HapMap Consortium, Nature 426:789-796 (2003)).
  • LD is determined in the Caucasian CEU population of the HapMap samples.
  • LD is determined in the African YRI population. In another embodiment, LD is determined in samples from the lOOOgenomes project (http://www.1000genomes.org). In yet another embodiment, LD is determined in samples from the Icelandic population.
  • Haplotype blocks can be used to map associations between phenotype and haplotype status, using single markers or haplotypes comprising a plurality of markers.
  • the main haplotypes can be identified in each haplotype block, and then a set of "tagging" SNPs or markers (the smallest set of SNPs or markers needed to distinguish among the haplotypes) can then be identified.
  • These tagging SNPs or markers can then be used in assessment of samples from groups of individuals, in order to identify association between phenotype and haplotype. If desired, neighboring haplotype blocks can be assessed concurrently, as there may also exist linkage disequilibrium among the haplotype blocks.
  • markers used to detect association thus in a sense represent "tags" for a genomic region (i.e., a haplotype block or LD block) that is associating with a given disease or trait, and as such are useful for use in the methods and kits of the invention.
  • the chr9:94276933 polymorphism may be detected directly to determine risk of intracranial aneurysm and/or abdominal aortic aneurysm.
  • any marker in linkage disequilibrium with the chr9:94276933 marker may be detected to determine risk.
  • chr9:94261761, chr9:94261811, chr9:94268484, chr9:94268497, chr9:94272846 and chr9:94276933 may be used to determine risk.
  • Suitable surrogate markers may be selected using public information, such as from the
  • the markers may also be suitably selected from results of whole-genome sequencing.
  • Markers with values of r 2 equal to 1 are perfect surrogates for the at-risk variants, i.e. genotypes for one marker perfectly predicts genotypes for the other. In other words, the surrogate will, by necessity, give exactly the same association data to any particular disease as the anchor marker. Markers with smaller values of r 2 than 1 can also be surrogates for the at- risk anchor variant.
  • the Fisher exact test can be used to calculate two- sided p-values for each individual allele. Correcting for relatedness among patients can be done by extending a variance adjustment procedure previously described (Risch, N. &Teng, J.
  • the method of genomic controls (Devlin, B. & Roeder, K. Biometrics 55:997 (1999)) can also be used to adjust for the relatedness of the individuals and possible
  • relative risk and the population attributable risk (PAR) can be calculated assuming a multiplicative model (haplotype relative risk model) (Terwilliger, J.D. & Ott, J., Hum. Hered.42:337-46 (1992) and Falk, C.T. & Rubinstein, P, Ann. Hum. Genet. 51 (Pt 3) :227 -33 (1987)), i.e., that the risks of the two alleles/haplotypes a person carries multiply.
  • a multiplicative model haplotype relative risk model
  • haplotypes are independent, i.e., in Hardy-Weinberg equilibrium, within the affected population as well as within the control population. As a consequence, haplotype counts of the affecteds and controls each have multinomial distributions, but with different haplotype frequencies under the alternative hypothesis.
  • risk(/?,)/risk(7 ; ) ⁇ fi/P i )/(f j / j ), where f and p denote, respectively, frequencies in the affected population and in the control population. While there is some power loss if the true model is not multiplicative, the loss tends to be mild except for extreme cases. Most importantly, p-values are always valid since they are computed with respect to null hypothesis. Risk assessment and Diagnostics
  • an absolute risk of developing a disease or trait defined as the chance of a person developing the specific disease or trait over a specified time-period.
  • a woman's lifetime absolute risk of breast cancer is one in nine. That is to say, one woman in every nine will develop breast cancer at some point in their lives.
  • Risk is typically measured by looking at very large numbers of people, rather than at a particular individual. Risk is often presented in terms of Absolute Risk (AR) and Relative Risk (RR).
  • AR Absolute Risk
  • RR Relative Risk
  • Relative Risk is used to compare risks associating with two variants or the risks of two different groups of people. For example, it can be used to compare a group of people with a certain genotype with another group having a different genotype.
  • a relative risk of 2 means that one group has twice the chance of developing a disease as the other group.
  • the creation of a model to calculate the overall genetic risk involves two steps: i) conversion of odds-ratios for a single genetic variant into relative risk and ii) combination of risk from multiple variants in different genetic loci into a single relative risk value.
  • allelic odds ratio equals the risk factor:
  • an individual who is at an increased susceptibility (i.e., increased risk) for a condition selected from intracranial aneurysm and abdominal aortic aneurysm is an individual who is carrying at least one at-risk variant as described herein.
  • the variant is within the human Asporin gene, or a variant encoded by a variation in the human Asporin gene.
  • significance associated with a marker is measured by a relative risk (RR).
  • significance associated with a marker or haplotye is measured by an odds ratio (OR).
  • the significance is measured by a percentage.
  • a significant increased risk is measured as a risk (relative risk and/or odds ratio) of at least 2.0, including but not limited to at least 3.0, at least 3.5, at least 4.0, at least 5.0, at least 6.0, at least 7.0, at least 8.0, at least 9.0, at least 10.0, at least 11.0, at least 12.0, at least 13.0, at least 14.0, at least 15.0, at least 16.0, at least 18.0, at least 20.0, at least 22.0, or at least 24.0.
  • a risk (relative risk and/or odds ratio) of at least 5.0 is significant.
  • a risk of at least 7.0 is significant.
  • An at-risk variant as described herein is one where at least one allele of at least one marker is more frequently present in an individual at risk for a condition such as intracranial aneurysm and abdominal aortic aneurysm (affected), or diagnosed with the condition, compared to the
  • control a comparison group
  • the control group may in one embodiment be a population sample, i.e. a random sample from the general population.
  • control group is represented by a group of individuals who are disease-free, i.e. individuals who have not been diagnosed with the condition.
  • markers with two alleles present in the population being studied such as SNPs
  • the other allele of the marker will be found in decreased frequency in the group of individuals with the trait or disease, compared with controls.
  • one allele of the marker (the one found in increased frequency in individuals with the trait or disease) will be the at-risk allele, while the other allele will be a protective allele.
  • Determining susceptibility can alternatively or additionally comprise comparing nucleic acid sequence data and/or protein sequence data (e.g., genotype data) to a database containing correlation data between polymorphic markers and susceptibility to a condition selected from intracranial aneurysm and abdominal aortic aneurysm.
  • the database can be part of a computer- readable medium described herein.
  • the database comprises at least one measure of
  • the database may comprise risk values associated with particular genotypes at such markers.
  • the database may also comprise risk values associated with particular genotype combinations for multiple such markers.
  • the database comprises a look-up table containing at least one measure of susceptibility to the condition for the polymorphic markers.
  • the method of determining a susceptibility to intracranial aneurysm and abdominal aortic aneurysm further comprises reporting the susceptibility to at least one entity selected from the group consisting of the individual, a guardian of the individual, a genetic service provider, a physician, a medical organization, and a medical insurer.
  • the reporting may be accomplished by any of several means.
  • the reporting can comprise sending a written report on physical media or electronically or providing an oral report to at least one entity of the group, which written or oral report comprises the susceptibility.
  • the reporting can comprise providing the at least one entity of the group with a login and password, which provides access to a report comprising the susceptibility posted on a password -protected computer system. Study population
  • the methods and kits described herein can be utilized from samples containing nucleic acid material (DNA or RNA) or protein material from any source and from any individual, or from genotype or sequence data derived from such samples.
  • the individual is a human individual.
  • the individual can be an adult, child, or fetus.
  • the individual is a female individual.
  • the nucleic acid or protein source may be any sample comprising nucleic acid or protein material, including biological samples, or a sample comprising nucleic acid or protein material derived therefrom.
  • the present invention also provides for assessing markers in individuals who are members of a target population.
  • Such a target population is in one embodiment a population or group of individuals at risk of developing a condition selected from intracranial aneurysm and abdominal aortic aneurysm, based on other genetic factors, biomarkers, biophysical parameters, or lifestyle factors.
  • the Icelandic population is a Caucasian population of Northern European ancestry.
  • a large number of studies reporting results of genetic linkage and association in the Icelandic population have been published in the last few years. Many of those studies show replication of variants, originally identified in the Icelandic population as being associating with a particular disease, in other populations (Sulem, P., et a ⁇ . Nat Genet May 17 2009 (Epub ahead of print); Rafnar, T., et al. Nat Genet 41 :221-7 (2009) ; Gretarsdottir, S., et al. Ann Neurol 64:402-9 (2008) ; Stacey, S.N., ef al.
  • the markers described herein to be associated with risk of intracranial aneurysm and abdominal aortic aneurysm will show similar association in other human populations. It is further contemplated that additional variants in the human Asporin gene may be conferring risk of intracranial aneurysm and/or abdominal aortic aneurysm in other populations.
  • Particular embodiments comprising individual human populations are thus also contemplated and within the scope of the invention. Such embodiments relate to human subjects that are from one or more human population including, but not limited to, Caucasian populations, European populations, American populations, Eurasian populations, Asian populations, Central/South Asian populations, East Asian populations, and African populations. In certain embodiments, the invention pertains to individuals from Caucasian populations.
  • the individuals are of ancestry that includes Caucasian ancestry.
  • the invention pertains to Icelandic individuals.
  • the invention relates to markers identified in specific populations, as described in the above.
  • measures of linkage disequilibrium (LD) may give different results when applied to different populations. This is due to different population history of different human populations as well as differential selective pressures that may have led to differences in LD in specific genomic regions.
  • certain markers e.g. SNP markers, have different population frequency in different populations, or are polymorphic in one population but not in another.
  • the person skilled in the art will however apply the methods available and as taught herein to practice the present invention in any given human population.
  • This may include assessment of polymorphic markers in the LD region of the present invention, so as to identify those markers that give strongest association within the specific population.
  • the at-risk variants of the present invention may reside on different haplotype background and in different frequencies in various human populations.
  • the invention can be practiced in any given human population.
  • Polymorphic markers associated with increased susceptibility of a condition selected from intracranial aneurysm and abdominal aortic aneurysm are useful in diagnostic methods. While methods of diagnosing the condition are known in the art, the detection risk markers for the condition advantageously may be useful for detection of the condition at its early stages and may also reduce the occurrence of misdiagnosis. In this regard, the invention further provides methods of diagnosing the condition comprising obtaining sequence data identifying at least one risk allele as described herein, in conjunction with carrying out one or more clinical diagnostic steps for the identification of the condition. Aneurysms have rapid onset. Small unchanging aneurysms may produce little if any symptoms.
  • aneurysms Before large aneurysms rupture, the individual with the aneurysm may experience symptoms such as sudden and severe headache, nausea, vision impairment, vomiting and loss of consciousness. Rupture of aneurysms is a life threatening condition. Therefore, it is imperative that the risk of a rupturing aneurysm be determined at an early stage.
  • the genetic variants described herein are thus useful to determine whether particular individuals are at high risk of developing an aneurysm, such that onset of an aneurysm may be monitored and appropriate ameliorating or preventive treatment be undertaken at an early stage.
  • a sample containing genomic DNA or protein from an individual is collected.
  • sample can for example be a buccal swab, a saliva sample, a blood sample, or other suitable samples containing genomic DNA or protein, as described further herein.
  • the sample is obtained by non-invasive means (e.g., for obtaining a buccal sample, saliva sample, hair sample or skin sample).
  • non-surgical means i.e. in the absence of a surgical intervention on the individual that puts the individual at substantial health risk.
  • Such embodiments may, in addition to noninvasive means also include obtaining sample by extracting a blood sample (e.g., a venous blood sample).
  • genomic DNA or protein obtained from the individual is then analyzed using any common technique available to the skilled person, such as high-throughput technologies for genotyping and/or sequencing. Results from such methods are stored in a convenient data storage unit, such as a data carrier, including computer databases, data storage disks, or by other convenient data storage means.
  • the computer database is an object database, a relational database or a post-relational database.
  • the genotype data is subsequently analyzed for the presence of certain variants known to be susceptibility variants for a particular human condition, such as the genetic variants described herein associated with risk of intracranial aneurysm and abdominal aortic aneurysm. Genotype and/or sequencing data can be retrieved from the data storage unit using any convenient data query method.
  • Calculating risk conferred by a particular genotype for the individual can be based on comparing the genotype of the individual to previously determined risk (expressed as a relative risk (RR) or and odds ratio (OR), for example) for the genotype, for example for an heterozygous carrier of an at- risk variant.
  • the calculated risk for the individual can be the relative risk for a person, or for a specific genotype of a person, compared to the average population with matched gender and ethnicity.
  • the average population risk can be expressed as a weighted average of the risks of different genotypes, using results from a reference population, and the appropriate calculations to calculate the risk of a genotype group relative to the population can then be performed.
  • the risk for an individual is based on a comparison of particular genotypes, for example heterozygous carriers of an at-risk allele of a marker compared with non-carriers of the at-risk allele.
  • the calculated risk estimated can be made available to the customer via a website, preferably a secure website.
  • a method of assessing the responsiveness of a human individual to a therapeutic agent for a conditions selected from intracranial aneurysm and abdominal aortic aneurysm comprising determining the presence or absence of at least one allele of at least one polymorphic marker in a nucleic acid sample obtained from the individual, wherein the at least one allele is an allele that encodes a defective Asporin polypeptide, wherein a determination of the presence of the allele is indicative that the individual is responsive to the therapeutic agent.
  • the at least one allele is a missense mutation or a nonsense mutation in Asporin.
  • the therapeutic regimen is selected from the group consisting of medical hypotensive therapy, surgical clipping and endovascular coiling.
  • Another aspect provides a method of selecting a human subject with a condition selected from the group consisting of intracranial aneurysm and abdominal aortic aneurysm for treatment with a therapeutic regimen for treating the condition, the method comprising determining the presence or absence of at least one allele of at least one polymorphic marker in a nucleic acid sample obtained from the individual, wherein the at least one allele is an allele that encodes a defective Asporin polypeptide, and selecting for treatment with the therapeutic regimen a subject identified as having the at least one allele in the nucleic acid sample.
  • a further aspect provides a method of selecting a therapeutic regimen for a human subject with a condition selected from the group consisting of intracranial aneurysm and abdominal aortic aneurysm, the method comprising analyzing data representative of at least one allele of an Asporin gene in a human subject with the condition to identify the presence or absence of a mutant allele in Asporin that encodes alters the amino acid sequence of an encoded Asporin protein in the subject, and selecting a therapeutic regimen of a therapeutic agent for treating intracranial aneurysm and/or abdominal aortic aneurysm for a subject identified from the data as having the mutation.
  • the wild-type Asporin gene is preferably a gene with sequence as set forth in SEQ ID NO:7 herein, and the wild-type Asporin protein preferably a protein with sequence as set forth in SEQ ID NO:8 herein.
  • the analyzing suitably includes screening for presence or absence of mutant alleles in Asporin that are predictive of risk of the condition.
  • screening comprises obtaining information selected from the group consisting of (a) nucleic acid sequence
  • nucleic acid sequence information comprises sequence sufficient to identify the presence or absence of the mutant allele in the subject; (b) nucleic acid sequence information, wherein the nucleic acid sequence information identifies at least one allele of a polymorphic marker in linkage disequilibrium (LD) with the mutant allele, wherein the LD is characterized by a value for r 2 of at least 0.5; (c) measurement of the quantity or length of Asporin m NA, wherein the measurement is indicative of the presence or absence of the mutant allele; (d) measurement of the quantity of Asporin protein, wherein the measurement is indicative of the presence or absence of the mutant allele; and (e) measurement of Asporin activity, wherein the measurement is indicative of the presence or absence of the mutant allele.
  • the mutation results in an encoded Asporin protein with reduced or altered calcium binding or collagen binding activity.
  • polymorphic markers of the invention are useful in determining a prognosis of a human individual with intracranial aneurysm and abdominal aortic aneurysm.
  • the variants described herein are indicative of risk of these conditions.
  • mutant alleles that predispose to these conditions are at increased risk of the conditions.
  • Such mutant alleles are predicted to be indicative of prognosis of the conditions.
  • the prognosis predicted can be any type of prognosis relating to the progression of the condition and/or relating to the chance of recovering from the condition.
  • the prognosis can, for instance, relate to the severity of the cond ition, or how the condition will respond to therapeutic treatment.
  • the prognosis for an individual with a ruptured intracranial aneurysm depends on the extent and location of the aneurysm, the person's age, general health, and neurological condition. Some ind ividuals with a ru ptured aneurysm die from the initial bleed ing . Other individuals with intracranial aneurysm recover with little or no neurological deficit. The most significant factors in determining outcome are grade and age. Generally patients with low grade hemorrhage on admission to the emergency room and patients who are younger within the typical age range of vulnerability can anticipate a good outcome, without death or permanent disability. Older patients and those with poorer g rades on admission have a poor prognosis.
  • the invention provides a method of predicting prognosis of an individual diagnosed with, a condition selected from the group consisting of intracranial aneurysm and abdominal aortic aneurysm.
  • the method comprises analyzing data representative of at least one allele of a Asporin gene in a human subject, wherein different alleles of the human Asporin gene are associated with different susceptibilities to the condition in humans, and determining a prognosis of the human subject from the data.
  • the analyzing may comprise analysis for a mutation in Asporin that leads to reduced or altered activity of an encoded Asporin protein.
  • the analyzing comprises analyzing for the presence or absence of at least one mutant allele indicative of an Asporin defect selected from the group consisting of premature truncation or frameshift of an encoded Asporin protein, relative to the Asporin amino acid sequence set forth in SEQ ID NO:8, expression of an Asporin protein with reduced activity compared to a wild-type Asporin protein (SEQ ID NO :8), and reduced expression of Asporin protein, compared to wild-type Asporin.
  • the reduced activity is reduced collagen bind ing activity or reduced calcium binding activity.
  • the sequence data can be nucleic acid sequence data or amino acid sequence data.
  • determination of the presence of a missense mutation, a frameshift mutation or a nonsense mutation in Asporin is indicative of prognosis of the condition.
  • the determination of the presence of a mutation in Asporin that leads to reduced or altered activity of Asporin is in certain embodiments indicative of a worsened prognosis of the condition.
  • the presence of such mutations is in certain embodiments indicative that the individual has a worse prog nosis of the condition than do individuals with the condition who do not carry such mutations.
  • the prognostic method further includes one or more additional steps, such as a step relating to generating the data by analyzing a biological sample; and/or a step involving selecting or administering a medial protocol to the subject, as described elsewhere herein. Kits
  • Kits useful in the methods of the invention comprise components useful in any of the methods described herein, including for example, primers for nucleic acid amplification, hybridization probes (e.g. probes for detecting particular mutant alleles), restriction enzymes (e.g., for RFLP analysis), allele-specific oligonucleotides, antibodies, e.g., antibodies that bind to an altered
  • Asporin polypeptide e.g. a missense variant in Asporin or a truncated Asporin polypeptide
  • Asporin polypeptide means for amplification of nucleic acids
  • means for analyzing the nucleic acid sequence of nucleic acids means for analyzing the amino acid sequence of polynucleotides, etc.
  • the kits can for example include necessary buffers, nucleic acid primers for amplifying nucleic acids (e.g., a nucleic acid segment comprising one or more of the polymorphic markers as described herein), and reagents for allele-specific detection of the fragments amplified using such primers and necessary enzymes (e.g., DNA polymerase).
  • kits can provide reagents for assays to be used in combination with the methods of the present invention, e.g. , reagents for use with other diagnostic assays for intracranial aneurysm and abdominal aneurysm.
  • the invention pertains to a kit for assaying a sample from a subject to detect a susceptibility to a condition selected from intracranial aneurysm and abdominal aortic aneurysm in the subject, wherein the kit comprises reagents necessary for selectively detecting at least one at-risk variant for the condition in the individual, wherein the at least one at-risk variant is a polymorphic marker in the human Asporin gene or an amino acid substitution in an encoded Asporin protein.
  • the markers encodes an Asporin protein with a reduced or altered activity compared with wild-type Asporin.
  • the reagents comprise at least one contiguous oligonucleotide that hybridizes to a fragment of the genome of the individual comprising at least one polymorphism of the present invention.
  • the reagents comprise at least one pair of oligonucleotides that hybridize to opposite strands of a genomic segment obtained from a subject, wherein each oligonucleotide primer pair is designed to selectively amplify a fragment of the genome of the individual that includes at least one polymorphism associated with the condition risk.
  • the polymorphism is selected from chr9:94261761, chr9:94261811, chr9:94268484,
  • the fragment is at least 20 base pairs in size.
  • oligonucleotides or nucleic acids e.g., oligonucleotide primers
  • the kit comprises one or more labeled nucleic acids capable of allele- specific detection of one or more specific polymorphic markers or haplotypes, and reagents for detection of the label.
  • Suitable labels include, e.g., a radioisotope, a fluorescent label, an enzyme label, an enzyme co-factor label, a magnetic label, a spin label, an epitope label.
  • determination of the presence of a particular marker allele is indicative of a increased susceptibility of the condition. In another embodiment, determination of the presence of a particular marker allele is indicative of prognosis of the condition, or selection of appropriate therapy for the condition. In another embodiment, the presence of the marker allele or haplotype is indicative of response to therapy for the condition. In yet another embodiment, the presence of the marker allele is indicative of progress of treatment of the condition.
  • the kit comprises reagents for detecting no more than 100 alleles in the genome of the individual. In certain other embodiments, the kit comprises reagents for detecting no more than 20 alleles in the genome of the individual.
  • a pharmaceutical pack comprising a therapeutic agent and a set of instructions for administration of the therapeutic agent to humans diagnostically tested for an at-risk variant for a condition selected from intracranial aneurysm and abdominal aortic aneurysm.
  • the therapeutic agent can be a small molecule drug, an antibody, a peptide, an antisense or RNAi molecule, or other therapeutic molecules.
  • an individual identified as a carrier of at least one variant of the present invention is instructed to take a prescribed dose of the therapeutic agent.
  • an individual identified as a homozygous carrier of at least one variant of the present invention is instructed to take a prescribed dose of the therapeutic agent.
  • an individual identified as a non-carrier of at least one variant of the present invention ⁇ e.g., an at-risk variant
  • the kit may additionally or alternatively comprise reagents for detecting an amino acid variation in a human Asporin protein ⁇ e.g., an amino acid substitution, or a truncated or otherwise altered amino acid sequence of an encoded Asporin protein).
  • the kit comprises at least one antibody for selectively detecting a truncated or altered Asporin polypeptide compared with wild-type Asporin (SEQ ID NO:8).
  • SEQ ID NO:8 wild-type Asporin
  • Other reagents useful for detecting amino acid variations are known to the skilled person and are also contemplated.
  • the kit further comprises a set of instructions for using the reagents comprising the kit.
  • the kit further comprises a collection of data comprising correlation data between the at least one at-risk variant and susceptibility to intracranial aneurysm and/or abdominal aortic aneurysm.
  • antisense agents are comprised of single stranded oligonucleotides (RNA or DNA) that are capable of binding to a complimentary nucleotide segment. By binding the appropriate target sequence, an RNA-RNA, DNA-DNA or RNA-DNA duplex is formed.
  • the antisense oligonucleotides are complementary to the sense or coding strand of a gene. It is also possible to form a triple helix, where the antisense oligonucleotide binds to duplex DNA.
  • Several classes of antisense oligonucleotide are known to those skilled in the art, including cleavers and blockers. The former bind to target RNA sites, activate intracellular nucleases (e.g., RnaseH or Rnase L), that cleave the target RNA.
  • Blockers bind to target RNA, inhibit protein translation by steric hindrance of the ribosomes. Examples of blockers include nucleic acids, morpholino compounds, locked nucleic acids and methylphosphonates (Thompson, Drug
  • Antisense oligonucleotides are useful directly as therapeutic agents, and are also useful for determining and validating gene function, for example by gene knock-out or gene knock-down experiments. Antisense technology is further described in Lavery er a/. , Curr. Opin. Drug Discov. Devel. 6:561-569 (2003), Stephens et al., Curr. Opin. Mol. Ther. 5: 118-122 (2003), Kurreck, Eur. J. Biochem. 270 : 1628-44 (2003), Dias et al., Mol. Cancer Ter. 1 :347-55 (2002), Chen, Methods Mol. Med. 75:621-636 (2003), Wang er a/., Curr. Cancer Drug Targets 1 : 177-96 (2001), and Bennett, Antisense Nucleic Acid Drug. Dev. 12 :215- 24 (2002).
  • the antisense agent is an oligonucleotide that is capable of binding to a particular nucleotide segment.
  • the nucleotide segment comprises the human Asporin gene.
  • the antisense nucleotide is capable of binding to a nucleotide segment of a human Asporin transcript.
  • the antisense nucleotide is capable of binding the a nucleotide segment of a human Asporin transcript with an altered sequence, wherein the sequence is altered by the presence of at least one variant selected from the group consisting of chr9:94261761 allele A, chr9:94261811 allele T, chr9:94268484 allele T, chr9:94268497 allele G, chr9:94272846 allele A and chr9 :94276933 allele A.
  • Antisense nucleotides can be from 5-400 nucleotides in length, including 5-200 nucleotides, 5-100 nucleotides, 10-50 nucleotides, and 10-30 nucleotides. In certain preferred embodiments, the antisense nucleotides is from 14-50 nucleotides in length, including 14-40 nucleotides and 14-30 nucleotides.
  • the variants described herein can also be used for the selection and design of antisense reagents that are specific for particular variants.
  • antisense oligonucleotides or other antisense molecules that specifically target mRNA molecules that contain one or more variants of the invention can be designed. In this manner, expression of mRNA molecules that contain one or more variant of the present invention can be inhibited or blocked.
  • the antisense molecules are designed to specifically bind a particular allelic form of the target nucleic acid, thereby inhibiting translation of a product originating from this specific allele, but which do not bind other or alternate variants at the specific polymorphic sites of the target nucleic acid molecule.
  • the antisense molecule is designed to specifically bind to nucleic acids comprising a variant selected from chr9:94261761 allele A, chr9 :94261811 allele T, chr9:94268484 allele T, chr9:94268497 allele G, chr9 :94272846 allele A and chr9:94276933 allele A.
  • antisense molecules can be used to inactivate mRNA so as to inhibit gene expression, and thus protein expression, the molecules can be used for disease treatment.
  • the methodology can involve cleavage by means of ribozymes containing nucleotide sequences complementary to one or more regions in the mRNA that attenuate the ability of the mRNA to be translated.
  • mRNA regions include, for example, protein-coding regions, in particular protein-coding regions corresponding to catalytic activity, substrate and/or ligand binding sites, or other functional domains of a protein.
  • RNA interference also called gene silencing, is based on using double-stranded RNA molecules (dsRNA) to turn off specific genes.
  • dsRNA double-stranded RNA molecules
  • siRNA small interfering RNA
  • siRNA molecules are typically about 20, 21, 22 or 23 nucleotides in length.
  • one aspect of the invention relates to isolated nucleic acid molecules, and the use of those molecules for RNA interference, i.e. as small interfering RNA molecules (siRNA).
  • the isolated nucleic acid molecules are 18-26 nucleotides in length, preferably 19-25 nucleotides in length, more preferably 20-24 nucleotides in length, and more preferably 21, 22 or 23 nucleotides in length.
  • RNAi-mediated gene silencing originates in endogenously encoded primary microRNA (pri-miRNA) transcripts, which are processed in the cell to generate precursor miRNA (pre-miRNA). These miRNA molecules are exported from the nucleus to the cytoplasm, where they undergo processing to generate mature miRNA molecules (miRNA), which direct
  • RNAi Clinical applications of RNAi include the incorporation of synthetic siRNA duplexes, which preferably are approximately 20-23 nucleotides in size, and preferably have 3' overlaps of 2 nucleotides. Knockdown of gene expression is established by sequence-specific design for the target mRNA. Several commercial sites for optimal design and synthesis of such molecules are known to those skilled in the art.
  • siRNA molecules typically 25-30 nucleotides in length, preferably about 27 nucleotides
  • shRNAs small hairpin RNAs
  • the latter are naturally expressed, as described in Amarzguioui er al. ⁇ FEBS Lett. 579:5974-81 (2005)).
  • Chemically synthetic siRNAs and shRNAs are substrates for in vivo processing, and in some cases provide more potent gene-silencing than shorter designs (Kim et al., Nature Biotechnol. 23:222-226 (2005); Siolas et al., Nature Biotechnol. 23:227-231 (2005)).
  • siRNAs provide for transient silencing of gene expression, because their intracellular concentration is diluted by subsequent cell divisions.
  • expressed shRNAs mediate long-term, stable knockdown of target transcripts, for as long as transcription of the shRNA takes place (Marques et al., Nature Biotechnol. 23 :559-565 (2006); Brummelkamp et al., Science 296: 550-553 (2002)).
  • RNAi molecules including siRNA, miRNA and shRNA
  • the variants presented herein can be used to design RNAi reagents that recognize specific nucleic acid molecules comprising specific alleles and/or haplotypes (e.g., the alleles and/or haplotypes of the present invention), while not recognizing nucleic acid molecules comprising other alleles or haplotypes.
  • RNAi reagents can thus recognize and destroy the target nucleic acid molecules.
  • RNAi reagents can be useful as therapeutic agents (i.e., for turning off disease-associated genes or disease-associated gene variants), but may also be useful for characterizing and validating gene function (e.g., by gene knock-out or gene knockdown experiments).
  • RNAi may be performed by a range of methodologies known to those skilled in the art. Methods utilizing non-viral delivery include cholesterol, stable nucleic acid-lipid particle (SNALP), heavy-chain antibody fragment (Fab), aptamers and nanoparticles. Viral delivery methods include use of lentivirus, adenovirus and adeno-associated virus.
  • the siRNA molecules are in some embodiments chemically modified to increase their stability. This can include modifications at the 2' position of the ribose, including 2'-0-methylpurines and 2'- fluoropyrimidines, which provide resistance to Rnase activity. Other chemical modifications are possible and known to those skilled in the art.
  • nucleic acids and polypeptides described herein can be used in methods and kits of the present invention.
  • An "isolated" nucleic acid molecule is one that is separated from nucleic acids that normally flank the gene or nucleotide sequence (as in genomic sequences) and/or has been completely or partially purified from other transcribed sequences (e.g., as in an RNA library).
  • an isolated nucleic acid of the invention can be substantially isolated with respect to the complex cellular milieu in which it naturally occurs, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized.
  • the isolated material will form part of a composition (for example, a crude extract containing other substances), buffer system or reagent mix.
  • the material can be purified to essential homogeneity, for example as determined by polyacrylamide gel electrophoresis (PAGE) or column chromatography (e.g., HPLC).
  • An isolated nucleic acid molecule of the invention can comprise at least about 50%, at least about 80% or at least about 90% (on a molar basis) of all macromolecular species present.
  • genomic DNA the term "isolated" also can refer to nucleic acid molecules that are separated from the chromosome with which the genomic DNA is naturally associated.
  • the isolated nucleic acid molecule can contain less than about 250 kb, 200 kb, 150 kb, 100 kb, 75 kb, 50 kb, 25 kb, 10 kb, 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of the nucleotides that flank the nucleic acid molecule in the genomic DNA of the cell from which the nucleic acid molecule is derived.
  • the invention also pertains to nucleic acid molecules that hybridize under high stringency hybridization conditions, such as for selective hybridization, to a nucleotide sequence described herein (e.g. , nucleic acid molecules that specifically hybridize to a nucleotide sequence containing a polymorphic site associated with a marker or haplotype described herein) .
  • nucleic acid molecules can be detected and/or isolated by allele- or sequence-specific
  • hybridization e.g. , under high stringency conditions.
  • Stringency conditions and methods for nucleic acid hybridizations are well known to the skilled person (see, e.g. , Current Protocols in Molecular Biology, Ausubel, F. et al, John Wiley & Sons, (1998), and Kraus, M. and Aaronson, S., Methods Enzymol. , 200 : 546-556 (1991), the entire teachings of which are incorporated by reference herein.
  • the percent identity of two nucleotide or amino acid sequences can be determined by aligning the sequences for optimal comparison purposes [e.g. , gaps can be introduced in the sequence of a first sequence) .
  • the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%, of the length of the reference sequence.
  • the present invention also provides isolated nucleic acid molecules that contain a fragment or portion that hybridizes under highly stringent conditions to a nucleic acid that comprises, or consists of, the nucleotide sequence of the human Asporin gene as set forth in SEQ ID NO: 7, or a nucleotide sequence comprising, or consisting of, the complement of the nucleotide sequence of SEQ ID NO:7.
  • the nucleotide sequence comprises at least one polymorphic allele as described herein (chr9 :94261761 allele A, chr9 :94261811 allele T, chr9 :94268484 allele T, chr9 :94268497 allele G, chr9 :94272846 allele A and chr9:94276933 allele A).
  • the nucleic acid fragments of the invention may suitably be at least about 15, at least about 18, 20, 23 or 25 nucleotides, and can be up to 30, 40, 50, 100, 200, 300 or 400 nucleotides in length.
  • probes or primers are oligonucleotides that hybridize in a base- specific manner to a complementary strand of a nucleic acid molecule.
  • probes and primers include polypeptide nucleic acids (PNA), as described in Nielsen, P. et al., Science 254: 1497-1500 (1991).
  • PNA polypeptide nucleic acids
  • a probe or primer comprises a region of nucleotide sequence that hybridizes to at least about 15, typically about 20-25, and in certain embodiments about 40, 50 or 75, consecutive nucleotides of a nucleic acid molecule.
  • the probe or primer comprises at least one allele of at least one polymorphic marker or at least one haplotype described herein, or the complement thereof.
  • a probe or primer can comprise 100 or fewer nucleotides; for example, in certain embodiments from 6 to 50 nucleotides, or, for example, from 12 to 30 nucleotides.
  • the probe or primer is at least 70% identical, at least 80% identical, at least 85% identical, at least 90% identical, or at least 95% identical, to the contiguous nucleotide sequence or to the complement of the contiguous nucleotide sequence.
  • the probe or primer is capable of selectively hybridizing to the contiguous nucleotide sequence or to the complement of the contiguous nucleotide sequence.
  • the probe or primer further comprises a label, e.g. , a radioisotope, a fluorescent label, an enzyme label, an enzyme co-factor label, a magnetic label, a spin label, ah epitope label.
  • the invention also provides antibodies which bind to an epitope comprising either an Asporin variant amino acid sequence (e.g., a polypeptide comprising an amino acid substitution or a truncated polypeptide) encoded by a variant allele or the reference amino acid sequence encoded by the corresponding non-variant or wild-type allele of Asporin.
  • Asporin variant amino acid sequence e.g., a polypeptide comprising an amino acid substitution or a truncated polypeptide
  • the term "antibody” as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain antigen-binding sites that specifically bind an antigen.
  • a molecule that specifically binds to a polypeptide of the invention is a molecule that binds to that polypeptide or a fragment thereof (e.g., an Asporin polypeptide with sequence as set forth in SEQ ID NO:8, or a fragment thereof), but does not substantially bind other molecules in a sample, e.g. , a biological sample, which naturally contains the polypeptide.
  • immunologically active portions of immunoglobulin molecules include F(ab) and F(ab')2 fragments which can be generated by treating the antibody with an enzyme such as pepsin.
  • the invention provides polyclonal and monoclonal antibodies that bind to a polypeptide of the invention.
  • monoclonal antibody or “monoclonal antibody composition”, as used herein, refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope of a polypeptide of the invention.
  • a monoclonal antibody composition thus typically displays a single binding affinity for a particular polypeptide of the invention with which it immunoreacts.
  • Polyclonal antibodies can be prepared as described above by immunizing a suitable subject with a desired immunogen, e.g. , polypeptide of the invention or a fragment thereof.
  • a desired immunogen e.g. , polypeptide of the invention or a fragment thereof.
  • the antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized polypeptide.
  • ELISA enzyme linked immunosorbent assay
  • the antibody molecules directed against the polypeptide can be isolated from the mammal (e.g., from the blood) and further purified by well-known techniques, such as protein A chromatography to obtain the IgG fraction.
  • protein A chromatography to obtain the IgG fraction.
  • antibody-producing cells when the antibody titers are highest, antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler and Milstein, Nature 256:495-497 (1975), the human B cell hybridoma technique (Kozbor et al., Immunol. Today 4: 72 (1983)), the EBV-hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,1985, Inc., pp. 77-96) or trioma techniques.
  • standard techniques such as the hybridoma technique originally described by Kohler and Milstein, Nature 256:495-497 (1975), the human B cell hybridoma technique (Kozbor et al., Immunol. Today 4: 72 (1983)), the EBV-hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,1985,
  • hybridomas The technology for producing hybridomas is well known (see generally Current Protocols in Immunology (1994) Coligan et al., (eds.) John Wiley & Sons, Inc., New York, NY). Briefly, an immortal cell line (typically a myeloma) is fused to lymphocytes
  • splenocytes typically splenocytes
  • the culture supernatants of the resulting hybridoma cells are screened to identify a hybridoma producing a monoclonal antibody that binds a polypeptide of the invention.
  • a monoclonal antibody to a polypeptide of the invention can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with the polypeptide to thereby isolate immunoglobulin library members that bind the polypeptide.
  • Kits for generating and screening phage display libraries are commercially available (e.g. , the
  • recombinant antibodies such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard
  • chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art.
  • antibodies of the invention e.g. , a monoclonal antibody
  • a polypeptide-specific antibody can facilitate the purification of natural polypeptide from cells and of recombinantly produced polypeptide expressed in host cells.
  • an antibody specific for a polypeptide of the invention can be used to detect the polypeptide (e.g. , in a cellular lysate, cell supernatant, or tissue sample) in order to evaluate the abundance and pattern of expression of the polypeptide.
  • Antibodies can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to, for example, determine the efficacy of a given treatment regimen.
  • the antibody can be coupled to a detectable substance to facilitate its detection. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials,
  • bioluminescent materials examples include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin ; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 125 I, 131 I, 35 S or 3 H .
  • Antibodies can furthermore be useful for assessing expression of proteins, e.g. Asporin expression. Antibodies specific Asporin, or variants or truncated forms of Asporin, may be used to determine the expression levels of Asporin in a sample from an individual.
  • Antibodies can be used in other methods. Thus, antibodies are useful as diagnostic tools for evaluating proteins, such as variant proteins of the invention, in conjunction with analysis by electrophoretic mobility, isoelectric point, tryptic or other protease digest, or for use in other physical assays known to those skilled in the art. Antibodies may also be used in tissue typing . In one such embodiment, a specific variant protein has been correlated with expression in a specific tissue type, and antibodies specific for the variant protein can then be used to identify the specific tissue type.
  • Subcellular localization of proteins can also be determined using antibodies, and can be applied to assess aberrant subcellular localization of the protein in cells in various tissues. Such use can be applied in genetic testing, but also in monitoring a particular treatment modality.
  • Antibodies are further useful for inhibiting variant protein function, for example by blocking the binding of a variant protein to a binding molecule or partner. Such uses can also be applied in a therapeutic context in which treatment involves inhibiting a variant protein's function.
  • An antibody can be for example be used to block or competitively inhibit binding, thereby modulating (i.e., agonizing or antagonizing) the activity of the protein.
  • Antibodies can be prepared against specific protein fragments containing sites required for specific function or against an intact protein that is associated with a cell or cell membrane.
  • an antibody may be linked with an additional therapeutic payload, such as radionuclide, an enzyme, an immunogenic epitope, or a cytotoxic agent, including bacterial toxins (diphtheria or plant toxins, such as ricin).
  • an additional therapeutic payload such as radionuclide, an enzyme, an immunogenic epitope, or a cytotoxic agent, including bacterial toxins (diphtheria or plant toxins, such as ricin).
  • bacterial toxins diphtheria or plant toxins, such as ricin.
  • kits for using antibodies in the methods described herein includes, but is not limited to, kits for detecting the presence or absence of a protein (e.g., Asporin, or variants or truncated forms thereof) in a test sample.
  • kits for detecting the presence or absence of a protein e.g., Asporin, or variants or truncated forms thereof
  • One preferred embodiment comprises antibodies such as a labelled or labelable antibody and a compound or agent for detecting proteins in a biological sample, means for determining the amount or the presence and/or absence of protein (e.g., Asporin, or variants or truncated forms thereof) in the sample, and means for comparing the amount of variant protein in the sample with a standard, as well as instructions for use of the kit.
  • a system of the invention includes one or more machines used for analysis of biological material (e.g., genetic material), as described herein. I some variations, this analysis of the biological material involves a chemical analysis and/or a nucleic acid amplification.
  • biological material e.g., genetic material
  • an exemplary system of the invention which may be used to implement one or more steps of methods of the invention, includes a computing device in the form of a computer 110.
  • a computing device in the form of a computer 110.
  • Components shown in dashed outline are not technically part of the computer 110, but are used to illustrate the exemplary embodiment of Fig. 1.
  • Components of computer 110 may include, but are not limited to, a processor 120, a system memory 130, a
  • memory/graphics interface 121 also known as a Northbridge chip
  • I/O interface 122 also known as a Southbridge chip
  • the system memory 130 and a graphics processor 190 may be coupled to the memory/graphics interface 121.
  • a monitor 191 or other graphic output device may be coupled to the graphics processor 190.
  • a series of system busses may couple various system components including a high speed system bus 123 between the processor 120, the memory/graphics interface 121 and the I/O interface 122, a front-side bus 124 between the memory/graphics interface 121 and the system memory 130, and an advanced graphics processing (AGP) bus 125 between the memory/graphics interface 121 and the graphics processor 190.
  • the system bus 123 may be any of several types of bus structures including, by way of example, and not limitation, such architectures include Industry Standard Architecture (ISA) bus, Micro Channel Architecture (MCA) bus and Enhanced ISA (EISA) bus.
  • ISA Industry Standard Architecture
  • MCA Micro Channel Architecture
  • EISA Enhanced ISA
  • the computer 110 typically includes a variety of computer-readable media.
  • Computer-readable media can be any available media that can be accessed by computer 110 and includes both volatile and nonvolatile media, removable and non-removable media.
  • Computer readable media may comprise computer storage media.
  • Computer storage media includes both volatile and nonvolatile, removable and non-removable media implemented in any method or technology for storage of information such as computer readable instructions, data structures, program modules or other data.
  • Computer storage media includes, but is not limited to, RAM, ROM, EEPROM, flash memory or other memory technology, CD-ROM, digital versatile disks (DVD) or other optical disk storage, magnetic cassettes, magnetic tape, magnetic disk storage or other magnetic storage devices, or any other physical medium which can be used to store the desired information and which can accessed by computer 110.
  • the system memory 130 includes computer storage media in the form of volatile and/or nonvolatile memory such as read only memory (ROM) 131 and random access memory (RAM) 132.
  • the system ROM 131 may contain permanent system data 143, such as identifying and manufacturing information.
  • a basic input/output system (BIOS) may also be stored in system ROM 131.
  • RAM 132 typically contains data and/or program modules that are immediately accessible to and/or presently being operated on by processor 120.
  • Fig. 1 illustrates operating system 134, application programs 135, other program modules 136, and program data 137.
  • the I/O interface 122 may couple the system bus 123 with a number of other busses 126, 127 and 128 that couple a variety of internal and external devices to the computer 110.
  • a serial peripheral interface (SPI) bus 126 may connect to a basic input/output system (BIOS) memory 133 containing the basic routines that help to transfer information between elements within computer 110, such as during start-up.
  • BIOS basic input/output system
  • a super input/output chip 160 may be used to connect to a number of 'legacy' peripherals, such as floppy disk 152, keyboard/mouse 162, and printer 196, as examples.
  • the super I/O chip 160 may be connected to the I/O interface 122 with a bus 127, such as a low pin count (LPC) bus, in some embodiments.
  • a bus 127 such as a low pin count (LPC) bus, in some embodiments.
  • LPC low pin count
  • Various embodiments of the super I/O chip 160 are widely available in the commercial marketplace.
  • bus 128 may be a Peripheral Component Interconnect (PCI) bus, or a variation thereof, may be used to connect higher speed peripherals to the I/O interface 122.
  • PCI Peripheral Component Interconnect
  • a PCI bus may also be known as a Mezzanine bus.
  • Variations of the PCI bus include the Peripheral Component Interconnect-Express (PCI-E) and the Peripheral Component Interconnect - Extended (PCI-X) busses, the former having a serial interface and the latter being a backward compatible parallel interface.
  • bus 128 may be an advanced technology attachment (ATA) bus, in the form of a serial ATA bus (SATA) or parallel ATA (PATA).
  • ATA advanced technology attachment
  • the computer 110 may also include other removable/non-removable, volatile/nonvolatile computer storage media.
  • Fig. 1 illustrates a hard disk drive 140 that reads from or writes to non-removable, nonvolatile magnetic media.
  • the hard disk drive 140 may be a conventional hard disk drive.
  • Removable media such as a universal serial bus (USB) memory 153, firewire (IEEE 1394), or CD/DVD drive 156 may be connected to the PCI bus 128 directly or through an interface 150.
  • a storage media 154 may coupled through interface 150.
  • Other removable/non-removable, volatile/nonvolatile computer storage media that can be used in the exemplary operating environment include, but are not limited to, magnetic tape cassettes, flash memory cards, digital versatile disks, digital video tape, solid state RAM, solid state ROM, and the like.
  • the drives and their associated computer storage media discussed above and illustrated in Fig . 1, provide storage of computer readable instructions, data structures, program modules and other data for the computer 110.
  • hard disk drive 140 is illustrated as storing operating system 144, application programs 145, other program modules 146, and program data 147. Note that these components can either be the same as or different from operating system 134, application programs 135, other program modules 136, and program data 137. Operating system 144, application programs 145, other program modules 146, and program data 147 are given different numbers here to illustrate that, at a minimum, they are different copies.
  • a user may enter commands and information into the computer 20 through input devices such as a mouse/keyboard 162 or other input device combination.
  • Other input devices may include a microphone, joystick, game pad, satellite dish, scanner, or the like. These and other input devices are often connected to the processor 120 through one of the I/O interface busses, such as the SPI 126, the LPC 127, or the PCI 128, but other busses may be used. In some embodiments, other devices may be coupled to parallel ports, infrared interfaces, game ports, and the like (not depicted), via the super I/O chip 160.
  • the computer 110 may operate in a networked environment using logical connections to one or more remote computers, such as a remote computer 180 via a network interface controller (NIC) 170.
  • the remote computer 180 may be a personal computer, a server, a router, a network PC, a peer device or other common network node, and typically includes many or all of the elements described above relative to the computer 110.
  • the logical connection between the NIC 170 and the remote computer 180 depicted in Fig. 1 may include a local area network (LAN), a wide area network (WAN), or both, but may also include other networks.
  • LAN local area network
  • WAN wide area network
  • Such networking environments are commonplace in offices, enterprise-wide computer networks, intranets, and the Internet.
  • the remote computer 180 may also represent a web server supporting interactive sessions with the computer 110, or in the specific case of location-based applications may be a location server or an application server.
  • the network interface may use a modem (not depicted) when a broadband connection is not available or is not used.
  • the network connection shown is exemplary and other means of establishing a communications link between the computers may be used.
  • the invention is a system for identifying susceptibility to a condition selected from intracranial aneurysm and abdominal aortic aneurysm in a human subject.
  • the system includes tools for performing at least one step, preferably two or more steps, and in some aspects all steps of a method of the invention, where the tools are operably linked to each other.
  • Operable linkage describes a linkage through which components can function with each other to perform their purpose.
  • a system of the invention is a system for identifying susceptibility to a condition selected from the group consisting of intracranial aneurysm and abdominal aortic aneurysm in a human subject, and comprises:
  • a susceptibility database operatively coupled to a computer-readable medium of the system and containing population information correlating the presence or absence of one or more alleles of the human Asporin gene and susceptibility to the condition in a population of humans;
  • (Hi) is adapted to be executed on a processor of the system, to compare the information about the human subject with the population information in the susceptibility database and generate a conclusion with respect to susceptibility to the condition for the human subject.
  • Exemplary processors include all variety of microprocessors and other processing units used in computing devices.
  • Exemplary computer-readable media are described above.
  • the system generally can be created where a single processor and/or computer readable medium is dedicated to a single component of the system; or where two or more functions share a single processor and/or share a single computer readable medium, such that the system contains as few as one processor and/or one computer readable medium.
  • some components of a system may be located at a testing laboratory dedicated to laboratory or data analysis, whereas other components, including components (optional) for supplying input information or obtaining an output communication, may be located at a medical treatment or counseling facility (e.g., doctor's office, health clinic, HMO, pharmacist, geneticist, hospital) and/or at the home or business of the human subject (patient) for whom the testing service is performed.
  • a medical treatment or counseling facility e.g., doctor's office, health clinic, HMO, pharmacist, geneticist, hospital
  • the allele of the Asporin gene is selected from the group consisting of chr9:94261761 allele A, chr9:94261811 allele T, chr9. -94268484 allele T, chr9:94268497 allele G, chr9:94272846 allele A and chr9:94276933 allele A.
  • an exemplary system includes a susceptibility database 208 that is operatively coupled to a computer-readable medium of the system and that contains population information correlating the presence or absence of one or more alleles of the human Asporin gene and susceptibility to the condition in a population of humans.
  • the one or more alleles of the Asporin gene include mutant alleles that cause, or are indicative of, an Asporin defect such as reduced or lost function, as described elsewhere herein.
  • the susceptibility database contains 208 data relating to the frequency that a particular allele of Asporin has been observed in a population of humans with the condition and a population of humans free of the condition. Such data provides an indication as to the relative risk or odds ratio of developing the condition for a human subject that is identified as having the allele in question.
  • the susceptibility database includes similar data with respect to two or more alleles of Asporin, thereby providing a useful reference if the human subject has any of the two or more alleles.
  • the susceptibility database includes additional quantitative personal, medical, or genetic information about the individuals in the database diagnosed with the condition or free of the condition.
  • Such information includes, but is not limited to, information about parameters such as age, sex, ethnicity, race, medical history, weight, diabetes status, blood pressure, family history of the condition, smoking history, and alcohol use in humans and impact of the at least one parameter on susceptibility to the condition.
  • the information also can include information about other genetic risk factors for the condition besides Asporin variants.
  • the system further includes a measurement tool 206 programmed to receive an input 204 from or about the human subject and generate an output that contains information about the presence or absence of the at least one Asporin allele of interest.
  • the input 204 is not part of the system per se but is illustrated in the schematic Figure 2.
  • the input 204 will contain a specimen or contain data from which the presence or absence of the at least one Asporin allele can be directly read, or analytically determined.
  • the input contains annotated information about genotypes or allele counts for Asporin in the genome of the human subject, in which case no further processing by the measurement tool 206 is required, except possibly transformation of the relevant information about the presence/absence of the Asporin allele into a format compatible for use by the analysis routine 210 of the system.
  • the input 204 from the human subject contains data that is unannotated or insufficiently annotated with respect to Asporin, requiring analysis by the measurement tool 206.
  • the input can be genetic sequence of the chromosome or chromosomal region on which Asporin resides, or whole genome sequence information, or unannotated information from a gene chip analysis of a variable loci in the human subject's genome.
  • the measurement tool 206 comprises a tool, preferably stored on a computer-readable medium of the system and adapted to be executed on a processor of the system, to receive a data input about a subject and determine information about the presence or absence of the at least one mutant Asporin allele in a human subject from the data.
  • the measurement tool 206 comprises a tool, preferably stored on a computer-readable medium of the system and adapted to be executed on a processor of the system, to receive a data input about a subject and determine information about the presence or absence of the at least one mutant Asporin allele in a human subject from the data
  • measurement tool 206 contains instructions, preferably executable on a processor of the system, for analyzing the unannotated input data and determining the presence or absence of the Asporin allele of interest in the human subject.
  • the measurement tool optionally comprises a sequence analysis tool stored on a computer readable medium of the system and executable by a processor of the system with instructions for determining the presence or absence of the at least one mutant Asporin allele from the genomic sequence information.
  • the input 204 from the human subject comprises a biological sample, such as a fluid (e.g., blood) or tissue sample, that contains genetic material that can be analyzed to determine the presence or absence of the Asporin allele of interest.
  • a biological sample such as a fluid (e.g., blood) or tissue sample, that contains genetic material that can be analyzed to determine the presence or absence of the Asporin allele of interest.
  • an exemplary measurement tool 206 includes laboratory equipment for processing and analyzing the sample to determine the presence or absence (or identity) of the Asporin allele(s) in the human subject.
  • the measurement tool includes: an
  • oligonucleotide microarray e.g., "gene chip” containing a plurality of oligonucleotide probes attached to a solid support; a detector for measuring interaction between nucleic acid obtained from or amplified from the biological sample and one or more oligonucleotides on the
  • oligonucleotide microarray to generate detection data; and an analysis tool stored on a computer-readable medium of the system and adapted to be executed on a processor of the system, to determine the presence or absence of the at least one Asporin allele of interest based on the detection data.
  • the measurement tool 206 includes: a nucleotide sequencer (e.g., an automated DNA sequencer) that is capable of determining nucleotide sequence information from nucleic acid obtained from or amplified from the biological sample; and an analysis tool stored on a computer-readable medium of the system and adapted to be executed on a processor of the system, to determine the presence or absence of the at least one mutant Asporin allele based on the nucleotide sequence information.
  • a nucleotide sequencer e.g., an automated DNA sequencer
  • an analysis tool stored on a computer-readable medium of the system and adapted to be executed on a processor of the system, to determine the presence or absence of the at least one mutant Asporin allele based on the nucleotide sequence information.
  • the measurement tool 206 further includes additional equipment and/or chemical reagents for processing the biological sample to purify and/or amplify nucleic acid of the human subject for further analysis using a sequencer, gene chip, or other analytical equipment.
  • the exemplary system further includes an analysis tool or routine 210 that: is operatively coupled to the susceptibility database 208 and operatively coupled to the measurement tool 206, is stored on a computer-readable medium of the system, is adapted to be executed on a processor of the system to compare the information about the human subject with the population information in the susceptibility database 208 and generate a conclusion with respect to susceptibility to the condition for the human subject.
  • the analysis tool 210 looks at the Asporin alleles identified by the measurement tool 206 for the human subject, and compares this information to the susceptibility database 208, to determine a susceptibility to the condition for the subject.
  • the susceptibility can be based on the single parameter (the identity of one or more Asporin alleles), or can involve a calculation based on other genetic and non- genetic data, as described above, that is collected and included as part of the input 204 from the human subject, and that also is stored in the susceptibility database 208 with respect to a population of other humans.
  • each parameter of interest is weighted to provide a conclusion with respect to susceptibility to the condition.
  • Such a conclusion is expressed in the conclusion in any statistically useful form, for example, as an odds ratio, a relative risk, or a lifetime risk for subject developing the condition.
  • system as just described further includes a
  • the communication tool is operatively connected to the analysis routine 210 and comprises a routine stored on a computer-readable medium of the system and adapted to be executed on a processor of the system, to: generate a communication containing the conclusion; and to transmit the communication to the human subject 200 or the medical practitioner 202, and/or enable the subject or medical practitioner to access the communication.
  • the communication tool 212 provides an interface for communicating to the subject, or to a medical practitioner for the subject (e.g., doctor, nurse, genetic counselor), the conclusion generated by the analysis tool 210 with respect to susceptibility to the condition for the subject.
  • the medical practitioner will share the communication with the human subject 200 and/or counsel the human subject about the medical significance of the communication.
  • the communication is provided in a tangible form, such as a printed report or report stored on a computer readable medium such as a flash drive or optical disk.
  • the communication is provided electronically with an output that is visible on a video display or audio output (e.g., speaker).
  • the communication is transmitted to the subject or the medical practitioner, e.g., electronically or through the mail.
  • the system is designed to permit the subject or medical practitioner to access the communication, e.g., by telephone or computer.
  • the system may include software residing on a memory and executed by a processor of a computer used by the human subject or the medical practitioner, with which the subject or practitioner can access the communication, preferably securely, over the internet or other network connection.
  • this computer will be located remotely from other components of the system, e.g., at a location of the human subject's or medical practitioner's choosing.
  • the system as described further includes components that add a treatment or prophylaxis utility to the system.
  • value is added to a determination of susceptibility to a condition selected from intracranial aneurysm and/or abdominal aortic aneurysm when a medical practitioner can prescribe or administer a standard of care that can reduce susceptibility to the condition; and/or delay onset of the condition; and/or increase the likelihood of detecting the condition at an early stage, to facilitate early treatment when the condition has not spread and is most curable.
  • Exemplary lifestyle change protocols include loss of weight, increase in exercise, cessation of unhealthy behaviors such as smoking, and change of diet.
  • Exemplary medicinal and surgical intervention protocols include administration of pharmaceutical agents for prophylaxis; and surgery.
  • the system further includes a medical protocol database 214 operatively connected to a computer-readable medium of the system and containing information correlating the presence or absence of the at least one Asporin allele of interest and medical protocols for human subjects at risk for the condition.
  • medical protocols include any variety of medicines, lifestyle changes, diagnostic tests, increased frequencies of diagnostic tests, and the like that are designed to achieve one of the aforementioned goals.
  • the information correlating an Asporin allele with protocols could include, for example, information about the success with which the condition is avoided or delayed, or success with which the condition is detected early and treated, if a subject has an Asporin susceptibility allele and follows a protocol.
  • the system of this embodiment further includes a medical protocol tool or routine 216, operatively connected to the medical protocol database 214 and to the analysis tool or routine 210.
  • the medical protocol tool or routine 216 preferably is stored on a computer-readable medium of the system, and adapted to be executed on a processor of the system, to: (i) compare (or correlate) the conclusion that is obtained from the analysis routine 210 (with respect to susceptibility to the condition for the subject) and the medical protocol database 214, and (ii) generate a protocol report with respect to the probability that one or more medical protocols in the medical protocol database will achieve one or more of the goals of reducing susceptibility to the condition; delaying onset of the condition; and increasing the likelihood of detecting the condition at an early stage to facilitate early treatment.
  • the probability can be based on empirical evidence collected from a population of humans and expressed either in absolute terms (e.g., compared to making no intervention), or expressed in relative terms, to highlight the comparative or additive benefits of two or more protocols.
  • the communication tool 212 Some variations of the system just described include the communication tool 212.
  • the communication tool generates a communication that includes the protocol report in addition to, or instead of, the conclusion with respect to susceptibility.
  • Information about Asporin allele status not only can provide useful information about identifying or quantifying susceptibility to conditions such as intracranial aneurysm and abdominal aortic aneurysm; it can also provide useful information about possible causative factors for a human subject identified with these conditions, and useful information about therapies for the patient with these conditions. In some variations, systems of the invention are useful for these purposes.
  • the invention is a system for assessing or selecting a treatment protocol for a subject diagnosed with a condition selected from intracranial aneurysm and abdominal aortic aneurysm.
  • An exemplary system schematically depicted in Figure 3, comprises:
  • a medical treatment database 308 operatively connected to a computer-readable medium of the system and containing information correlating the presence or absence of at least one Asporin allele and efficacy of treatment regimens for the condition;
  • a measurement tool 306 to receive an input (304, depicted in Fig. 3 but not part of the system per se) about the human subject and generate information from the input 304 about the presence or absence of the at least one Asporin allele indicative of an Asporin defect in a human subject diagnosed with the condition;
  • a medical protocol routine or tool 310 operatively coupled to the medical treatment database 308 and the measurement tool 306, stored on a computer-readable medium of the system, and adapted to be executed on a processor of the system, to compare the information with respect to presence or absence of the at least one Asporin allele for the subject and the medical treatment database, and generate a conclusion with respect to at least one of:
  • such a system further includes a communication tool 312 operatively connected to the medical protocol tool or routine 310 for communicating the conclusion to the subject 300, or to a medical practitioner for the subject 302 (both depicted in the schematic of Fig. 3, but not part of the system per se).
  • An exemplary communication tool comprises a routine stored on a computer-readable medium of the system and adapted to be executed on a processor of the system, to generate a communication containing the conclusion; and transmit the
  • the Asporin allele is selected from the group consisting of
  • Table 1 shows association results of chr9:94276933 with intracranial aneurysm (IA) and abdominal aortic aneurysm ( AAA) respectively (see text above).
  • 160.0-7 aneurysmal subarachnoid hemorrhage
  • 167.1 ruptured cerebral aneurysm
  • 169.0 sequele of subarachnoid haemorrhage
  • ICD9 diagnosis 430 subarachnoid hemorrhage from ruptured cerebral
  • Controls were drawn from other population based projects conducted by deCODE genetics.
  • Illumina SNP Chip Genotyping The Icelandic chip-typed samples were assayed with the Illumina Human Hap300, Hap CNV370, Hap 610, 1M or Omni-1 Quad bead chips at deCODE genetics. Only the 317,503 SNPs from the Human Hap300 chip were used in the long range phasing and the subsequent SNP imputations.
  • SNPs were excluded if they had (i) yield lower than 95%, (ii) minor allele frequency less than 1% in the population or (iii) significant deviation from Hardy- Weinberg equilibrium in the controls (P ⁇ 0.001), (iv) if they produced an excessive inheritance error rate (over 0.001), (v) if there was substantial difference in allele frequency between chip types (from just a single chip if the problem that resolved all differences, but from all chips otherwise). All samples with a call rate below 97% were excluded from the analysis. The final set of SNPs used for long range phasing was composed of 297,835 autosomal SNPs.
  • SNPs were imputed based on whole genome sequence data from 1176 Icelanders, selected for various neoplastic, cardiovascular and psychiatric conditions. All of the individuals were sequenced at a depth of at least 10X.
  • Sequencing adaptors containing overhangs were ligated to the DNA products followed by agarose (2%) gel electrophoresis. Fragments of about 400 bp were isolated from the gels (QIAGEN Gel Extraction Kit), and the adaptor-modified DNA fragments were PCR enriched for ten cycles using Phusion DNA polymerase (Finnzymes Oy) and PCR primers PE 1.0 and PE 2.0 (Illumina).
  • Enriched libraries were further purified using agarose (2%) gel electrophoresis as described above. The quality and concentration of the libraries were assessed with the Agilent 2100 Bioanalyzer using the DNA 1000 LabChip (Agilent) . Barcoded libraries were stored at -20 °C. All steps in the workflow were monitored using an in-house laboratory information management system with barcode tracking of all samples and reagents.
  • Imaging and analysis of the data was performed using either the SCS2.6 /RTA1.6 or SCS2.8/RTA1.8 software packages from Illumina, respectively.
  • Real-time analysis involved conversion of image data to base-calling in real-time.
  • c. Alignment For each lane in the DNA sequencing output, the resulting qseq files were converted into fastq files using an in-house script. All output from sequencing was converted, and the Illumina quality filtering flag was retained in the output. The fastq files were then aligned against Build 36 of the human reference sequence using bwa version 0.5.7. All genomic locations quoted refer to HG18 Build 36.
  • BAM file generation For each lane in the DNA sequencing output, the resulting qseq files were converted into fastq files using an in-house script. All output from sequencing was converted, and the Illumina quality filtering flag was retained in the output. The fastq files were then aligned against Build 36 of the human reference sequence using bwa version 0.5.7. All genomic locations quoted refer to HG18 Build 36.
  • SAM file output from the alignment was converted into BAM format using SAMtools version 0.1.8, and an in-house script was used to carry the Illumina quality filter flag over to the BAM file.
  • the BAM files for each sample were then merged into a single BAM file using SAMtools. Fina lly, Picard version 1.17 (see http ://picard.sourceforge. net/) was used to mark duplicates in the resulting sample BAM files.
  • SNP identification and genotype calling A two-step approach was applied. The first step was to detect SNPs by identifying sequence positions where at least one individual could be determined to be different from the reference sequence with confidence (quality threshold of 20) based on the SNP calling feature of the pileup tool in SAMtools.
  • SNPs that always differed heterozygous or homozygous from the reference were removed.
  • the second step was to use the pileup tool to genotype the SNPs at the positions that were flagged as polymorphic. Because sequencing depth varies and hence the certainty of genotype calls also varies, genotype likelihoods rather than deterministic calls were calculated.
  • genotype likelihoods rather than deterministic calls were calculated.
  • the HapMap2 CEU samples 96.3% were observed in the whole-genome sequencing data.
  • 89.4% were observed in the whole- genome sequencing data.
  • Long range phasing Long range phasing of all chip-genotyped individuals was performed with methods described previously (Kong, A. et al. Nat Genet 40, 1068-75 (2008) ; Holm, H . et al. Nat Genet 43, 316-20 (2011)). In brief, phasing is achieved using an iterative algorithm which phases a single proband at a time given the available phasing information about everyone else that shares a long haplotype identically by state with the proband. Given the large fraction of the Icelandic population that has been chip-typed, accurate long range phasing is available genome- wide for all chip-typed Icelanders.
  • Genotype imputation We imputed the SNPs identified and genotyped through sequencing into all Icelanders who had been phased with long range phasing using the same model as used by IMPUTE (Kong, A. et al. Nat Genet 40, 1068-75 (2008)). The genotype data from sequencing can be ambiguous due to low sequencing coverage. In order to phase the sequencing genotypes, an iterative algorithm was applied for each SNP with alleles 0 and 1. We let H be the long range phased haplotypes of the sequenced individuals and applied the following algorithm:
  • the genotype likelihood L g is the probability of the observed sequencing data at the SNP for a given individual assuming g is the true genotype at the SNP. If L 0 , Li and L 2 are the likelihoods of the genotypes 0, 1 and 2 in the individual that carries h,
  • step 3 when the maximum d ifference between iterations is greater than a
  • the algorithm also extends trivially to the X-chromosome. If source genotype data are only ambiguous in phase, such as chip genotype data, then the algorithm is still applied, but a ll but one of the Ls will be 0.
  • the reference set was intentionally enriched for carriers of the m inor a llele of a rare SN P in order to im prove im putation accuracy. In this case, expected a llele counts will be biased toward the minor alle le of the SN P. Call the enrichment of the minor allele E and let 9' be the expected minor allele count calcu lated from the
  • Genotype imputation information The informativeness of genotype im putation was estimated by the ratio of the variance of imputed expected a llele counts a nd the varia nce of the actual a llele counts :
  • Var ⁇ 9) ' where ⁇ e ⁇ 0, 1) is the allele count.
  • Var(E(9 ⁇ chip data)) was estimated by the observed varia nce of the imputed expected cou nts and Var ⁇ 6) was estimated by p(l - p), where p is the allele frequency.
  • the information value for chr9 :94276933 was 0.99.
  • Genealogy-based in silico genotyping In add ition to imputing sequence varia nts from the whole genome sequencing effort into chip genotyped individuals, we also performed a second imputation step where genotypes were imputed into relatives of chip genotyped ind ividuals, creating in silico genotypes.
  • the inputs into the second imputation step are the fully phased (in particular every allele has been assig ned its parent of origin (Kong, A. et al. Nature 462, 868-74 (2009)) imputed and ch ip type genotypes of the available chip typed ind ividual.
  • the algorithm used to perform the second imputation step consists of: For each acheotyped individual (the proband), find all chip genotyped individuals within two meioses of the individual. The six possible types of two meiotic distance relatives of the proband are (ignoring more complicated relationships due to pedigree loops) :
  • Haplotypes that are the same, except at most at a single SNP, are treated as identical.
  • haplotypes in the pedigree are incompatible over a bin, then a uniform probability distribution was used for that bin.
  • the most common causes for such incompatibilities are recombinations within the pedigree, phasing errors and genotyping errors.
  • the single point distributions are then convolved using the multipoint algorithm to obtain multipoint sharing probabilities at the center of each bin. Genetic distances were obtained from the most recent version of the deCODE genetic map (Kong, A. et al. Nature 467, 1099-103 (2010)).
  • ⁇ is the expected allele
  • Oc + (l - 0) ⁇ is an estimate of the allele count for the proband's paternal haplotype. Similarly, an expected allele count can be obtained for the proband's maternal haplotype.
  • Case control association testing: Logistic regression was used to test for association between SNPs and disease, treating disease status as the response and expected genotype counts from imputation or allele counts from direct genotyping as covariates. Testing was performed using the likelihood ratio statistic.
  • control association testing Logistic regression was used to test for association between SNPs and disease, treating disease status as the response and expected genotype counts from imputation or allele counts from direct genotyping as covariates. Testing was performed using the likelihood ratio statistic.
  • controls were matched to cases based on the informativeness of the imputed genotypes, such that for each case C controls of matching informativeness where chosen. Failing to match cases and controls will lead to a highly inflated genomic control factor, and in some cases may lead to spurious false positive findings.
  • the informativeness of each of the imputation of each one of an individual's haplotypes was estimate of
  • the mean informativeness values cluster into groups corresponding to the most common pedigree configurations used in the imputation, such as imputing from parent into child or from child into parent.
  • Inflation Factor Adjustment In order to account for the relatedness and stratification within the case and control sample sets we applied the method of genomic control based on chip typed markers (Devlin, B. & Roeder, K. Genomic control for association studies. Biometrics 55, 997-1004 (1999)). Quoted P values were adjusted accordingly.
  • Effective sample size estimation In order to estimate the effective sample size of the case control association analyses, we compared the variances of the logistic and generalized linear regression parameter estimates based on the in silico genotypes to their one step imputation counterparts.
  • Binding of collagen to wild-type or variant Asporin can be performed as described in Kalamajski et a/. (Biochem J 423 : 53-59 (2009)). Briefly, Maxisorb plates (Nunc) can be coated with lOug/mL acid-solubilized collagen, washed with PBS containing 0.5mg/mL BSA and 0.05% Tween20 and blocked with non-fat skimmed milk. Following washing, Asporin protein is added. The Asporin can include a tag, such as a His-tag. Following incubation, wells are washed and protein detected with a suitable antibody, for example a rabbit anti-H is antibody for detecting His-tagged Asporin. A secondary antibody, for example anti-rabbit alkaline phosphatase- conjugated antibody is used for detection, where enzyme activity is measured using PNPP as substrate, detecting colour formation at 405nm. EXAMPLE 3
  • Asporin protein is first spotted onto a nitrocellulose membrane and dried .
  • the membrane is then incubated in a buffer (lOm Hepes, pH 7.4, 60mM KCI, 5mM MgCI 2 ) with lmCi/ml of 45 Ca 2+ for lOmin.
  • the membrane is then washed with 50% ethanol, dried and isotope quantified by a bioimaging analyzer (for example FLA-3000 (Fuji)) .

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Abstract

Selon l'invention, il a été découvert que des variants particuliers dans le gène humain d'Asporine sont associés à un risque de présenter des états, tels qu'un anévrisme intracrânial et un anévrisme de l'aorte abdominale. La présente invention concerne des procédés de détermination de la probabilité de présenter ces états à l'aide de tels variants. L'invention concerne également des procédés mis en œuvre par informatique pour la détermination de la probabilité de présenter ces états.
PCT/IS2012/000007 2011-10-27 2012-10-29 Variants conférant un risque d'anévrisme intracrânial et d'anévrisme de l'aorte abdominale WO2013061342A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019018545A1 (fr) * 2017-07-18 2019-01-24 The Research Foundation For The State University Of New York Biomarqueurs d'anévrisme intracrânien
KR102158720B1 (ko) * 2019-04-03 2020-09-22 한림대학교 산학협력단 Lrrc3 유전자의 단일염기다형성을 포함하는 뇌동맥류 진단용 snp 마커

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011161700A1 (fr) * 2010-06-22 2011-12-29 Decode Genetics Ehf Marqueurs génétiques pour la gestion du risque d'une maladie vasculaire

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011161700A1 (fr) * 2010-06-22 2011-12-29 Decode Genetics Ehf Marqueurs génétiques pour la gestion du risque d'une maladie vasculaire

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
BIROS E. ET AL: "Association of an allele on chromosome 9 and abdominal aortic aneurysm", ATHEROSCLEROSIS, vol. 212, no. 2, October 2010 (2010-10-01), pages 539 - 542, XP027406889 *
DIDANGELOS, A ET AL.: "Proteomics characterization of extracellular space components in the human aorta", MOL CELL PROTEOMICS, vol. 9, no. 9, September 2010 (2010-09-01), pages 2048 - 2062, XP055066261 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019018545A1 (fr) * 2017-07-18 2019-01-24 The Research Foundation For The State University Of New York Biomarqueurs d'anévrisme intracrânien
US11685951B2 (en) 2017-07-18 2023-06-27 The Research Foundation For The State University Of New York Biomarkers for intracranial aneurysm
KR102158720B1 (ko) * 2019-04-03 2020-09-22 한림대학교 산학협력단 Lrrc3 유전자의 단일염기다형성을 포함하는 뇌동맥류 진단용 snp 마커

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