WO2003000890A1 - Genetic vasculopathy - Google Patents

Abstract

The invention relates to the field of vasculopathy and the diagnosis and treatment thereof. The invention provides a method for screening for a disease locus associated with vasculopathy in humans comprising testing genomic DNA via linkage analysis for the presence of a polymorphism or an allelic variation within a microsatellite region on chromosome 3p21.1-p21.3 and an isolated and/or recombinant nucleic acid, or fragment thereof, corresponding to or derived from a disease locus associated with vasculopathy which in humans is located on chromosome 3p21.1-p21.3.

Description

Title: Genetic vasculopathy

The invention relates to the field of vasculopathy, and in particular provides methods for screening for genetic components of autosomal dominant vasculopathy syndromes with prominent cerebroretinal manifestations and retinal vasculopathy as a first step towards the diagnosis and treatment thereof. Hereditary vascular retinopathy (HVR), Raynaud's phenomenon and migraine have been reported as a single syndrome uniquely present in a large Dutch family (Storimans et al, 1991; Terwindt et al., 1998). HVR shows autosomal dominant inheritance and is characterized by microangiopathy of the retina with microanaeurysms and telangiectatic capillaries, preferentially around the macula (Storimans et al., 1991). Abnormalities on fluorescein angiography can be detected in otherwise a-symptomatic family members in their late twenties, suggesting an age of onset in young adulthood. Later stages of the disease involve occlusion of branches of large retinal arteries, avascular areas in the retinal periphery, and sometimes, proliferative retinopathy with extensive avascular areas, even up to the optic disc. Eighty-percent of the HVR affected patients in this family also suffered from Raynaud's phenomenon, a pathological vasomotor reaction of the digital blood vessels to cold exposure, causing numb and white fingers. Migraine was present in 70% of the HVR patients and both migraine and Raynaud's phenomenon was observed in 55% of the HVR patients. A number of other vasculopathy disorders have been described with partly overlapping and partly distinctive clinical features (Grand et al., 1988; Gutmann et al, 1989; Jen et al., 1997; Weil et al, 1999; Niedermayer et al., 2000). Vascular retinopathy is a prominent feature in two previously described conditions: cerebroretinal vasculopathy (CRV; Grand et al., 1988) and hereditary endotheliopathy with retinopathy, nephropathy, and strokes (HERNS) (Jen et al., 1997). CRV is an autosomal dominant vasculopathy of adult onset affecting retinal and intracranial vessels, characterized by retinal microangiopathic changes with progressive visual loss and subsequent development of subcortical contrast-enhancing lesions with surrounding edema, mimicking tumors ('pseudotumor') (Grand et al, 1988; Gutmann et al, 1989). HERNS is also an autosomal dominantly inherited vasculopathy of adult onset, in which affected family members present with visual impairment due to vascular retinopathy, renal dysfunction with proteinuria, followed by progressive neurologic deterioration and findings of pseudotumor on neuroimaging studies. UltrastructuraL studies in HERNS systemically revealed abnormal multilamihated basement membrane (Jen et al., 1997).

Despite the overlapping features of retinal vasculopathy, there are also clear differences in the clinical manifestation and severity in HVR, CRV, and HERNS. Like the vascular retinopathy in HVR, CRV and HERNS are characterized by microangiopathy of the posterior pole. In contrast to HVR, however, the retinal periphery is not affected. Other features distinguish this syndrome from HVR such as the presence of a cerebral frontal pseudotumor, progressive cognitive deterioration, psychiatric disturbances, extensive cerebral white matter lesions on MRI, and stroke (Grand et al., 1988; Jen et al., 1997). A remarkable similarity between these three families is the high prevalence of migraine-like headaches. Also, Raynaud's phenomenon is only reported in HVR (Terwindt et al., 1998), while pseudotumor, seen in both CRV and HERNS, is not reported in HVR. Renal insufficiency has only been described in HERNS (Jen et al., 1997). Furthermore, while HVR has no apparent impact on life expectancy, at least one in two patients with HERNS has died from neurologic complications by their mid 40s (range 32-60). Of note, some patients with CRV or HERNS develop migraine-like headaches. Identification of a causative or related 'neurovascular' or 'retinovascular' locus would be likely to also help to further unravel the pathophysiology of retinal vasculopathies and more common neurovascular disorders such as migraine, stroke and Raynaud's phenomenon.

The invention provides a method for screening for a disease locus associated with vasculopathy in humans comprising testing genomic DNA via linkage analysis for the presence of a polymorphism or an allelic variation within a microsateUite region on chromosome 3p21.1-p21.3, preferably wherein said region is identifiable by the presence of microsatellites D3S3521, , D3S3685, D3S3564, D3S3582, D3S1581, D3S1588, D3S1578, D3S1289, or D3S3616. The present gene region as defined by recombinants spans a genetic distance of ca 3.5 cM, corresponding to a physical distance of ca 12 Mb. This region contains more than 200 genes, including the laminin beta2 (LAMB2) gene. Mutation analysis provides a causative gene involved. Subsequent functional analyses, including immunohistochemistry, transfection assays and transgenesis, provides insight in the involvement and pathopysiological role of a causative gene. In particular, the invention provides a method for screening for genetic components of autosomal dominant vasculopathy syndromes with prominent cerebroretinal manifestations and retinal vasculopathy and to the diagnosis and treatment thereof. Considering the autosomal dominant character, it is likely that a single gene is predominantly involved in the various manifestations of retinal vasculopathy, either with variable expression of the gene defect, or with allelic mutations. Less likely is the possibility that multiple closely linked genes are involved, for example, in a microdeletion causing a contiguous gene syndrome. As a result from said screening as provided herein, the invention provides an isolated and/or recombinant nucleic acid, or fragment thereof, corresponding to or derived from a disease locus associated with vasculopathy which in humans is located on chromosome 3p21.1-p21.3.

We performed a genome-wide search for linkage in an extended Dutch family with autosomal dominant vascular retinopathy (HVR), associated with migraine and Raynaud's phenomenon. Patients with vascular retinopathy are characterized by microangiopathy of the retina with microaneurysms and telangiectatic capillaries. The genome search, using the new capillary electrophoresis system, the ABI PRISM 3700 DNA Analyzer, revealed significant evidence for linkage to chromosome 3p21.1-p21.3 with a maximum pair-wise LOD score of Z = 5.25 at marker D3S1578. In a preferred embodiment, the invention provides a method (and nucleic acid) wherein said locus is located within a 13-cM interval between D3S3521 and D3S3616, corroborating that vasculopathy related disease has a genetic component located at above identified locus at chromosome 3. Within this region over 50 genes (Table 4) and a larger number of ESTs are located. Analysis of three of these candidate genes (DAGl, LAMR and LAMB2) from the > 50 genes located within the interval showed no deleterious mutation by direct sequencing analysis. We can not exclude the possibility that these genes are causing retinal vasculopathy related disease, but that we did not identify the mutation using only said direct sequencing techniques. The disease region is of considerable size and contains a relatively large number of candidate genes. The invention also provides a vector comprising a nucleic acid according to the invention, and a host cell and/or animal comprising such a nucleic acid, or nucleic acids (each containing part of the disease locus). The invention also provides a method for localising or identifying a gene using a nucleic acid molecule or a fragment of fragments thereof according to the invention, in particular wherein said gene is related to retinal vasculopathy related disease. In particular, the invention also provides recombinant expression vectors comprising isolated and/or recombinant nucleic acid comprising alleles of said genes or fragments thereof, and provides host cells or animals that comprise such vectors or that are otherwise transformed with an isolated and/or recombinant nucleic acid according to the invention.

The present invention also provides methods and tests (such as PCR, but also other tests to detect or amplify nucleic acids are known in the art) to detect, identify and localize or distinguish genes and alleles of such genes, or fragments thereof located in the disease locus disclosed herein. The invention among others provided the insight of the presence of a common locus for vasculopathy-related disease among which at least three autosomal dominant vasculopathy syndromes with prominent cerebroretinal manifestations. The invention thus also provides a rationale and methods for the testing and the development of specific prophylactic medication for such vasculophatic disorders.. The invention for example provides cells or animals that comprise recombinant vectors that comprise variants of said genes from said locus or cells or animals that are transformed with said variants. Also, the invention provides means to identify naturally occurring variants of experimental animals with changes in said locus related to vasculopathy. Here, we report the mapping of the gene for HVR, one of at least three autosomal dominant vasculopathy syndromes with prominent cerebroretinal manifestations, by demonstrating linkage to the chromosome 3p21.1-p21.3 region at the short arm of chromosome 3 in an extended Dutch kindred.

We thus provide a method for distinguishing between alleles of a gene related or associated with a disease locus for vasculopathy using a nucleic acid molecule or a fragment of fragments thereof according to the invention, in particular wherein the gene is of human origin, however, it is now also provided to select or detect a (non-human) cell or an animal other than a human, for example for research purposes, for displaying or carrying a locus according to the invention. For example, the invention provides a method for screening for genetic vasculopathy which comprises searching for the presence of genetic abnormalities via polymorphic analysis linked to genetic vasculopathy on the chromosome 3, in the region comprised between the microsatellites D3S3521 and D3S3616, of a family or of an possibly at risk individual, including for example a fetus. Such a method is preferred wherein the microsatellites used are selected from among the group consisting of: D3S3521, D3S3564, D3S3685, D3S3582, D3S1581, D3S1588, D3S1578, D3S1289, D3S3616 or other polymorphic markers between D3S3521 and D3S3616. These microsatellites are described in the microsatellites Genethon map (Weissenbach J. & al., Nature 359, 794-801, 1992 which is incorporated herein by reference) as well as in J. Weber et al, Am. J. Hum. Gen. 1993, 53, 1079-1095. The oHgonucleotide sequences serving as primers, which are specific of each microsateUite, are available in the Genome Data Bank (Accessing GDB.TM. and OMIM.TM. at Johns Hopkins University, Baltimore, Md., USA)). Also provided is a method for screening for genetic vasculopathy which comprises selecting polymorphisms via linkage analysis within microsateUite regions responsible for genetic vasculopathy on chromosome 3 with the aid of DNA probes to a linkage analysis with the microsateUites D3S3521, D3S3564, D3S3685, D3S3582, D3S1581, D3S1588, D3S1578, D3S1289, D3S3616 or other polymorphic markers between D3S3521 and D3S3616. Such a method for screening for a subgroup of genetic vasculopathy linked to chromosome 3 by a family or an at risk individual, including a fetus, the comprises the step of searching for a genetic linkage between DNA polymorphisms and a mutated region linked to the genetic vasculopathy, these DNA polymorphisms being located in the region of the chromosome 3 comprised between the microsatellites D3S3521 and D3S3616, including these microsatellites. It is useful to apply a method according to the invention on one or more members of a family whose status for genetic vasculopathy is known, including ill and healthy members, the method comprising the steps of determining if the family is linked to abnormalities on chromosome 3, determining the DNA polymorphisms which are most informative for the family, choosing primer pairs to the above-determined DNA polymorphisms, amplifying DNA fragments from an at risk individual to be tested and pertaining to this family with the selected primer pairs, and determining if this at risk individual is carrying the affected or healthy polymorphisms and calculating the risk of this individual having inherited the affected region. Then, such selected primers and testing protocol can advantageously be used to test subjects of unknown disease status, e.g, during screening efforts to find more individuals at risk for vasculopathies.

In particular, the invention provides a method of diagnosing susceptibility to genetic vasculopathy in a patient, the method comprising: determining the presence or absence of an aUele of a polymorphic marker in the DNA of the patient, wherein the polymorphic marker is within a segment of chromosome 3p21-p23 bordered by D3S3521 and D3S3616 having a variant form associated with a phenotype of genetic vasculopathy, whereby the presence of the allele in the patient indicates susceptibility to genetic vasculopathy.

The invention thus also provides a ceU or an animal selected for the presence of a polymorphism or aUelic variation related to vasculopathy by a method according to the invention, and provides a ceU or an animal comprising a genome in which nucleic acid sequences corresponding to a nucleic acid according to the invention have been modified. Use of such a ceU or an animal according to the invention is herein specificaUy provided to test or develop specific medication for the treatment of a vasculopathy Also, the invention provides a protein or peptide encoded by a nucleic acid, or a fragment or fragments thereof, according to the invention or a (be it a natural or synthetic) antibody directed against a protein or peptide according to the invention. Use of a nucleic acid, or fragment or fragments thereof, of protein or peptide or antibody according to the invention is herein also provided, for example to detect or diagnose a vasculopathy. Also, the invention provides use of a nucleic acid, or fragment or fragments thereof, or of a protein or peptide or antibody according to the invention for the preparation of a pharmaceutical composition, in particular for the preparation of a pharmaceutical composition for the treatment of a vasculopathy.

Also the invention provides a method for the identification of detection of an individual's proneness for acquiring vasculopathy-related disease comprising detecting a vasculopathy-related mutation in a nucleic-acid sample derived from said individual wherein in humans said mutation is located on chromosome 3p21.1-p21.3, preferably wherein said vasculopathy comprises vascular retinopathy. Such a method comprises the step of determining the presence or absence of an allele of a polymorphic marker in the patient, for example wherein the presence or absence of the aUele is determined by amphfying a segment of DNA within chromosome 3p21.1-3p21.3 that spans the polymorphic marker. In addition, the invention provides the step of determining the size of the amplified segment, a step of determining the sequence of the amplified segment, and, optionaUy, comprises the step of determining the presence or absence of a restriction enzyme site within the amplified segment. In another embodiment, a method according to the invention is provided wherein the presence or absence of the allele is determined by contacting the DNA from the patient with an oligonucleotide probe capable of hybridizing to the aUele under stringent conditions; determining whether hybridization has occurred thereby indicating the presence of the allele. To obtain a nucleic acid according to the invention, it suffices to isolate a sample of DNA from the patient, and subject it to one of many techniques known in the art, such as PCR, array, aUell-specific oligohybridizations, Southernblotting and so on. DNA is most practicaUy obtained from available family members whose status with respect to the disease is known; genomic DNA can for example be isolated from peripheral blood leucocytes, lymphoblastoid cell lines, cultured amniotic fluid cells, or chorionic viUi, by subjecting a sample to standard proteinase K treatment and phenolchlorophorm extraction techniques, and amplified and/or digested with the appropriate restriction enzymes if needed. The invention is further explained in the detailed description.

DetaUed description

Subjects and Methods

Genome scan and genotyping

Detailed clinical data on the extended Dutch family with over 300 individuals have been published previously (Storimans et al, 1991; Terwindt et al., 1998). In a prior genome-wide search for linkage in this family, using radioactively labeled primers and more than 300 markers, 75% of the genome was excluded and no locus was found (Terwindt et al., 1998). The current study focused genome-wide genotyping only on the 21 family members with the most distinctive phenotype, namely HVR, together with first-degree relatives for phase reconstruction. Genotyping was performed using 400 evenly distributed markers from the ABI PRISM Linkage Mapping Set version 2, analyzed via the new capiUary electrophoresis system, the ABI PRISM 3700 DNA Analyzer

(Applied Biosystems). PCR reactions were performed under standard conditions using an ABI PRISM 877 Integral Thermal Cycler (Applied Biosystems). AU genotypes were independently double scored by JDY and RAO using Genotyper 2.1 software and consensus tables were created for each marker. Discrepancies were corrected by assigning the genotype status "unknown" to ambiguous genotypes. Genotyping for foUow-up and high-resolution mapping was carried out by PCR under standard conditions using PE9700 instruments. PCR products were detected by use of an ABI377 sequencer and analyzed by Genescan and Genotyper software (Applied Biosystems).

Genetic Analysis

Analysis was carried out under the assumption of a dominant model for vascular retinopathy with 90% penetrance and 0.0001 frequency of the disease allele. The phenocopy rate was set at 0.1%. The affection status of family members without HVR symptoms was considered to be "unknown". The data were checked for Mendelian inheritance using the UNKNOWN program (Lathrop et al, 1984) and two-point linkage analyses were performed using MLINK program of the LINKAGE package, version 5.1 (Lathrop et al, 1984). Multipoint LOD scores were computed by the VITESSE algorithm (O'Connell and Weeks, 1995). Marker-aUele frequencies were estimated on the basis of data on the founders of the family used in this study. For foUow-up analysis, if desired a more stringent model may be used for linkage analysis with the same disease- aUele frequency, assuming complete penetrance and not aUowing for phenocopies. The Marshfield and Genethon genetic maps were used for selection of additional markers and to determine the genetic distances (Broman et al, 1998; Dib et al, 1996).

Mutation Screening All exons of DAGl, LAMR and LAMB2 genes (Genbank accession numbers NM_004393, U43901 and S77512) were amplified by PCR from genomic DNA of two affected subjects and an unrelated control sample. Genomic structure of the genes was established with help of completed and draft sequences available from the Human Genome Project obtained via GenBank. Double-strand sequencing was performed using the dideoxy termination method on an ABI377 sequencer (Prism Big Dye Terminators Cycle Sequencing kit, PE Applied Biosystems).

Results

Gene mapping

Initial results from the two-point genome-wide linkage analysis produced no conclusive proof of linkage (data not shown). However, three chromosomal regions were identified for further analysis. The first region, on chromosome 1, included marker D1S218 with a maximum LOD score of 2.01. The second region was on chromosome 3 with marker D3S3616 yielding the second highest LOD score of the screen, 1.83. The last candidate region was located on chromosome 20 in which there were 5 flanking markers (D20S115, D20S186, D20S112, D20S195, D20S107) with slightly elevated LOD scores up to 0.72. For the regions on chromosome 1 and 20, haplotype reconstruction did not support this region being linked to the disease locus. However, the region on chromosome 3p was compatible with linkage. Genotyping of additional markers and additional family members (Figure 1) further supported linkage with this region. Two- point LOD scores for the closely spaced markers in the more extended pedigree on chromosome 3p are shown in table 1. A maximum two-point LOD score Zma_x = 5.25 at zero recombination fraction was obtained for marker D3S1578. Six flanking markers also yielded LOD scores (Z = 3) with statistical significance for linkage (Table 1). Multipoint analysis with the most informative markers confirmed the assignment of the HVR locus to this region on chromosome 3p (data not shown).

Haplotype Analysis

The most likely haplotypes, co-segregating with the disease are displayed in figure 1. Haplotype analysis shows that the entire region delimited by markers D3S3521 and D3S3616 is consistent with linkage to the disease, exhibiting crossover events with the disease locus in patient III- 18 and the offspring of III-5, respectively.

Mutation Screening Online physical maps avaUable at GeneMap'99, Genome Database (GDB) and Online Mendelian Inheritance in Man (OMIM) identified a large number (n>50) of genes within the candidate region. The list of genes included the gene encoding the dystroglycan protein (DAGl) (Ibraghimov-Beskrovnaya et al, 1992), the laminin receptor (LAMR) (Gehlsen et al, 1988; Jackers et al, 1998), and the laminin beta 2 chain (LAMB2) (Durkin et al, 1999). Direct sequencing of all the coding sequences and flanking introns of these three genes was performed in affected individuals and a healthy unrelated control. A number of sequence variations (n=10) were detected in the DAGl, LAMR, and LAMB2 genes, with three synonymous substitutions in the coding sequence not affecting the amino acid residue, one in each gene. In DAGl, we identified a substitution (C_ T) at codon 752 (CAC_ CAT). The second substitution (G_ A) was found in LAMR at codon 173 (TTA_ TTG), the third variation in the coding sequence (C_ T) was identified at codon 588 of LAMB2 (CCC_ CCT). However, these variations did not co-segregate with the disease haplotype, or were also present in control chromosomes and should therefore be regarded as non-pathogenic sequence variations.

We have localized a gene on chromosome 3p21 for hereditary cerebroretinal vasculopathies associated with varying combinations of a vascular retinopathy and migraine, Raynaud's phenomenon, stroke, cerebral pseudotumor, and neuropsychiatric symptoms. Linkage analysis of the Dutch HVR family yielded highly significant LOD scores (Table 1) mapping the gene to a ~13-cM interval on the short arm of chromosome 3. DetaUed haplotyping indicates reduced penetrance of HVR in this famUy as several members of the famUy with the complete disease-containing haplotype (IV-9, -11, -28), or parts of the haplotype (IV-25, -25) did not show any abnormalities on fluorescein angiography. As the current ages of these individuals range from 35 to 55 years and the age of onset of the HVR symptoms have been reported to range from 26 to 62 (Terwindt et al, 1998), the asymptomatic subjects sharing the disease haplotype may still develop the disease.

Haplotype analysis also reveals that there is no simple association between the HVR haplotype and the occurrence of migraine and Raynaud's phenomenon. FamUy members without HVR but with migraine or Raynaud's phenomenon did not all share the disease-related haplotype. For example, of the subjects who have the combination of migraine and Raynaud's phenomenon, subjects IV-5, -10, -12, -14, -15, -18, 20 do not have the HVR-related haplotype, whereas subjects IV-9 and IV- 11, do carry the disease haplotype. The high prevalence of migraine (RusseU et al. 1995; Launer et al. 1999) and Raynaud's phenomenon (Maricq et al, 1997) in the general population may at least partly contribute to this poor correlation. Fluorescein angiography clearly is crucial in establishing the diagnosis of this syndrome. Identification of the HVR gene may help to clarify the relation with migraine and Raynaud's phenomenon.

A number of genes are known to be located within the candidate region, for example, collagen type VII alphal (COL7A1). A large number of different types of mutations in the COL7A1 gene have been described in different famUies, causing dominant and recessive forms of epidermolysis of the skin (MIM 120120). None of the mutation-linked clinical features, as described so far, however, suggest that they are involved in the etiology of a vascular retinopathy, the cardinal feature of HVR, CRV and HERNS. Three genes were selected for further study, DAGl, LAMR and LAMB2.

DAGl encodes the 43-kD transmembrane and 156-kD extracellular dystrophin- associated glycoprotein. The protein is also caUed dystroglycan and is the laminin-binding glycoprotein that provides linkage between the sarcolemma and extracellular matrix. Dystroglycan has been suggested to play an important role in basement membrane assembly by binding soluble laminin and organizing it on the cell surface (Henry and Campbell, 1998). Dysfunction of DAGl or the other two genes LAMR and LAMB2, encoding for a laminin receptor and laminin a2 subunit, respectively, could well be implicated in the ultrastructural changes and phenotype observed (Noakes et al, 1995; Gehlsen et al, 1988). We found, though, no deleterious point mutation in the coding and splicing consensus sequences of the DAGl, LAMR or LAMB2 genes in these three families by direct sequencing. This analysis does not exclude variants in the noncoding regions such as regulatory elements, nor can we exclude deletions that are undetectable by our approach of exon-specific PCR amplification. The chromosomal 3p region has been implicated in other hereditary disorders in which the retina is affected. Spinocerebellar ataxia 7 (SCA7 [MIM 164500]) is associated with retinal degeneration in addition to cerebeUar degeneration. DetaUed genetic mapping of the SCA7 gene, however, located the gene between D3S1312 and D3S1600 (David et al, 1996), just proximal to marker D3S1300, outside the retinal vasculopathy candidate region. Analysis of the SCA7 repeat in the HVR famUy confirmed the exclusion of this gene (data not shown). Recently, linkage was found for Usher syndrome type II (USH2B [MIM 276905]) to chromosome 3p, entirely overlapping the HVR locus (Hmani et al, 1999). USH2B is characterized by congenital hearing loss, normal vestibular function, and appearance of retinitis pigmentosa in the late second to third decade of life. More studies need to be carried out to investigate whether these disorders are allekc.

The candidate region defined by haplotype reconstruction has a high density of genes and is physicaUy large for positional cloning Consequently, we can identify additional individuals with vascular retinopathy, (remotely) related to one of these famUies. Alternatively, narrowing down of the candidate region can be done by identification of new famUies with hereditary vascular retinopathy or related symptoms. There are at least three other families reported with cerebroretinal vasculopathy mimicking a brain tumor similar to the characteristics described for CRV (Gutmann et al, 1989; Weil et al, 1999; Niedermayer et al, 2000). Another disease that clinically resembles vascular retinopathy is familial exudative vitroretinopathy (FEVR [MIM 133780]). A locus for FEVR has been mapped to chromosome l lq and chromosome X but further locus heterogeneity has recently been demonstrated to exist (Bahashmus et al, 2000). Thus, involvement of the chromosome 3p21 locus in FEVR famUies not linked to the two known FEVR loci should be tested.

In conclusion, we have mapped a disease locus on chromosome 3p21.1- p21.3, which is associated with a wide variety of cerebral, retinal, and vascular symptoms.

References

Electronic-Database Information

Accession numbers and URLs for data in this application are as follows:

Center for Medical Genetics, Marshfield Medical Research Foundation, http://www.marshmed.org/genetics

Genethon, http://www.genethon.fr/

The Genome Database, http://www.gdb.org/

National Center for Biotechnology Information (NCBI) for Online Mendelian Inheritance in Man, GenBank, GeneMap'99, and PubMed, http ://www . ncbi.nlm.nih.gov/

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Table 1

Two-Point LOD Score Results between the HVR Locus and Chromosome 3p

Markers

LOD i score at e =

Marker 0.00 0.01 0.05 0.10 0.20 0.30 0.40 Z max E (Zmax)

D3S1277 -4.48 -0.25 0.89 1.17 1.09 0.71 0.24 1.19 0.13

D3S3521 -0.01 2.62 3.00 2.88 2.31 1.54 0.66 3.00 0.05

D3S3564 4.28 4.20 3.89 3.48 2.61 1.65 0.61 4.28 0.00

D3S3685 4.48 4.41 4.10 3.70 2.84 1.86 0.76 4.48 0.00

D3S3582 1.07 1.04 0.94 0.81 0.56 0.30 0.09 1.07 0.00

D3S1581 3.77 3.69 3.40 3.03 2.29 1.54 0.73 3.77 0.00

D3S1588 1.32 1.28 1.13 0.98 0.60 0.31 0.10 1.32 0.00

D3S1578 5.25 5.17 4.80 4.33 3.31 2.20 0.98 5.25 0.00

D3S1289 4.71 4.62 4.29 3.85 2.92 1.90 0.79 4.71 0.00

D3S3616 0.30 2.92 3.28 3.14 2.51 1.67 0.71 3.28 0.05

D3S1547 -0.11 1.93 2.35 2.30 1.85 1.23 0.53 2.36 0.06

D3S1300 -2.73 -0.80 -0.23 -0.09 -0.07 -0.08 -0.05 0.00 0.50

Figure legends

Figure 1.

Partial pedigree of the Dutch HVR family (family 1). For privacy reasons, the order of the sibships has been randomized. Haplotypes for twelve microsateUite markers spanning ~20-cM on chromosome 3p are shown. FUled circles and squares indicate affected for aU three symptoms (HVR, Raynaud's, migraine): upper half indicates HVR, left bottom migraine, and right bottom Raynaud's phenomenon. The black bar indicates the haplotype segregating with the retinopathy phenotype.

Figure 2. Marker positions

Figure 3.

Marker positions

Claims

Claims

1. An isolated and/or recombinant nucleic acid, or fragment thereof, corresponding to or derived from a disease locus associated with vasculopathy which in humans is located on chromosome 3p21.1-p21.3.

2. A nucleic acid according to claim 1 wherein said vasculopathy comprises retinal vasculopathy.

3. A nucleic acid according to claim 1 or 2 wherein said vasculopathy comprises a hereditary vascular retinopathy commonly known as HVR

4. A nucleic acid according to claim 1, 2 or 3 wherein said locus is located within a 9-cM interval between D3S3521and D3S3616.

5. A vector comprising a nucleic acid according to anyone of claims 1 to 4.

6. A host ceU comprising a nucleic acid according to anyone of claims 1 to 4 or a vector according to claim 5.

7. A method for localizing or identifying a gene using a nucleic acid molecule or a fragment of fragments thereof according to any of claims 1-4.

8. A method distinguishing between alleles of a gene using a nucleic acid molecule or a fragment of fragments thereof according to any of claims 1-4.

9. A method for screening for a disease locus associated with vasculopathy in humans comprising testing genomic DNA via linkage analysis for the presence of a polymorphism or an allelic variation within a microsateUite region on chromosome 3p21.1-p21.3.

10. A method according to claim 9 wherein said region is identifiable by the presence of microsateUites D3S3521, D3S3564, D3S3685, D3S3582, D3S1581, D3S1588, D3S1578, D3S1289, or D3S3616 .

11. A method according to claim 9 or 10 wherein said vasculopathy comprises vascular retinopathy.