WO2010123136A1 - Association of htra1 mutations and familial ischemic cerebral small-vessel disease - Google Patents

Abstract

The present invention provides a method of diagnosing a cerebrovascular disease in a human comprising the steps of: (a) measuring a mutation of HTRA1 gene in a test sample from said human; and (b) determining if the mutation of HTRA1 gene in said test sample correlates with a cerebrovascular disease in said human.

Description

DESCRIPTION

ASSOCIATION OF HTRAl MUTATIONS AND FAMILIAL ISCHEMIC CEREBRAL SMALL- VESSEL DISEASE

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the priority benefit of U.S. provisional Application Serial No. 61/170,762, filed April 20, 2009, which is hereby incorporated by reference herein in its entirety.

TECHNICAL FIELD

The present invention relates to a method of diagnosing cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL).

BACGROUND ART

Hypertension is a well-known risk factor for nonhereditary cerebral small-vessel disease.¹ Genetic causes have been identified for hereditary cerebral small- vessel diseases: cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy;² autosomal dominant retinal vasculopathy with cerebral leukodystrophy;³ brain small-vessel disease with hemorrhage;⁴ and familial cerebral amyloid angiopathies.⁵ Although arteriopathy in these cerebral small-vessel diseases is well documented, little is known about its genetic basis.

A cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL) is characterized by nonhypertensive cerebral small- vessel arteriopathy with subcortical infarcts, alopecia, and spondylosis, with onset in early adulthood.^6"8 On neuropathological examination, arteriosclerosis associated with intimal thickening and dense collagen fibers, loss of vascular smooth muscle cells, and hyaline degeneration of the media was observed in cerebral small arteries.^7"9 These pathological findings resemble those observed in patients with nonhereditary ischemic cerebral small- vessel disease.^7"11

DISCLOSURE OF INVENTION SUMMARY OF INVENTION

In the present invention, the present inventors show that mutations in HTRAl, a gene encoding HtrA serine peptidase 1, cause CARASIL.

The genetic cause of cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASJL), which is ischemic, nonhypertensive cerebral small-vessel disease with alopecia and spondylosis, has not been reported. We carried out a genomewide linkage analysis and fine mapped the implicated region of five families with CARASIL, followed by sequence analysis of a candidate gene. We carried out functional analysis of wildtype and mutant gene products, and assayed transforming growth factor β (TGF-β) family signaling, and gene and protein expression in the small arteries in the cerebrum of two patients with CARASIL.

We observed linkage of the disease to the 2.4-Mb region on chromosome 1Oq, which contains the HTRAl gene. HTRAl is a serine protease that represses signaling by TGF-β family members. Sequence analysis revealed two nonsense mutations and two missense mutations in HTRAl. The missense mutations and one of the nonsense mutations resulted in protein products that had comparatively low levels of protease activity and did not repress TGF-β family signaling. The other nonsense mutation resulted in the loss of HTRAl protein by nonsense-mediated mRNA decay. Immunohistochemistry of the cerebral small arteries in affected persons showed increased expression of ED-A fibronectin and versican in the thickened intima, and of TGF-β 1 in the media.

CARASIL is caused by mutations in the HTRAl gene. Our findings indicate a link between repressed inhibition of TGF-β family signaling and ischemic cerebral small-vessel disease, alopecia, and spondylosis.

The present invention describes the use in the diagnosis and detecting of cerebrovascular disease in a human. A mutation of HTRAl gene is indicative of some type of cerebrovascular disease.

In one embodiment, the present invention is drawn to a method of diagnosing a cerebrovascular disease in a human comprising the steps of: (a) measuring a mutation of HTRAl gene in a test sample from said human; and (b) determining if the mutation of HTRAl gene in said test sample correlates with a cerebrovascular disease in said human.

In one embodiment, the present invention is drawn to a method of detecting a cerebrovascular disease in a human comprising the steps of: (a) measuring a mutation of HTRAl gene in a test sample from said human; and (b) correlating the mutation of HTRAl gene in said test sample with a cerebrovascular disease in said human.

The test sample may be selected from the group including but not limited to blood, serum, plasma, saliva, cerebral spinal fluid, oral mucosa and nail. In a preferred embodiment, the test sample is blood.

In a preferred embodiment, the cerebrovascular disease is selected from the group including but not limited to cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL), acute cerebrovascular disease, ischemic cerebrovascular disease, Binswanger disease, leukoaraiosis, cerebral small vessel disease, and leukoencephalopatky.

In one embodiment, the present invention is drawn to a kit for diagnosing or detecting a cerebrovascular disease in a human, comprising a primer set for amplifying a mutant HTRAl gene and a wild type HTRAl gene.

In some embodiments, the determination or correlation step is a comparison between nucleotide sequence of test sample and a wild type nucleotide sequence of HTRAl gene.

In one embodiment, the present invention is drawn to a pharmaceutical composition comprising a substance that inhibits signaling by TGF-β family proteins. In a preferred embodiment, the substance that inhibits signaling by TGF-β family proteins is siRNA, shRNA or decoy nucleic acid that targets a gene coding for TGF-β.

BRIEF DESCRIPTION OF DRAWINGS

Figure 1. Pedigrees and HTRAl Mutations of Families with CARASIL.

Figure 2. Functional Consequences of HTRAl Mutations in CARASIL.

Figure 3. Nonsense-Mediated Decay of Nonsense-Mutated HTRA 1 mRNA.

Figure 4. Modulation of TGF-β Family-Mediated Transcriptional Responses by Mutated HTRAl Proteins.

Figure 5. Expression Level of Transfected HTRAl Proteins in TGF-β Family- Mediated Transcriptional Response Assay.

Figure 6. Expression Level of Transfected HTRAl Proteins in Assay of Phosphorylation of Smad proteins.

Figure 7. Effects of TGF-β on NOG mRΝA Expression in Cultured Skin Fibroblasts.

Figure 8. Immunohistochemical Analysis in Cerebral Small Arteries.

Figure 9. mRNA Expression of Extra Domain- A of Fibronectin in Cerebral Small Arteries.

BEST MODE FOR CARRYING OUT THE INVENTION

1. Detection and diagnosis of genetic mutation of HTRAl gene (1) Genetic Mutations of HTRAl gene

Information on genetic mutations may be obtained by conventional methods for detecting genetic mutations. For example, the sequencing method, the PCR method, hybridization methods using a sequence-specific oligonucleotide as a template (e.g. TaqMan PCR method), and the like may be employed. The PCR method and the direct sequencing method may be used for detecting any type of genetic mutation.

In the present invention, preferred mutations for detecting or diagnosing cerebrovascular disease include but not limited to nonsense mutations and missense mutations of HTRAl gene (Accession number: NM 002775 (SEQ ID NO: I)).

In the present invention, "mutant" or "mutation" means a protein or DNA resulting from a modification such as deletion, addition or substitution of one or more (for example one to ten, preferably, one to five) amino acids or nucleotides and includes substances which undergo disinhibition of TGF-β family signaling. For example, analysis of CARASIL has lead to the discovery of HTRAl mutations such as, but not limited to A252T, V297M, R302X and R370X. A252T is a mutant resulting from substitution of the 252ⁿ alanine (Ala) by threonine (Thr) in the amino acid sequence of HTRAl (SEQ ID NO:2). V297M is a mutant resulting from substitution of the 297^th valine (VaI) by methionine (Met) in the amino acid sequence of HTRAl. R302X is a mutant resulting from substitution of the 302^nd arginine (Arg) by stop codone in the amino acid sequence of HTRAl. R370X is a mutant resulting from substitution of the 370^nd arginine (Arg) by stop codone in the amino acid sequence of HTRAl..

In the DNA sequence of HTRAl gene, the nucleotide at position 1108 was changed from C to T, compared with wild type HTRAl gene (SEQ ID NO:1). (Hereinafter, this mutation is expressed as "C1108— >T". Other mutations will also be expressed in the same manner.) Besides, when compared with wild type HTRAl gene (SEQ ID NO.l), the DNA sequence has mutations of G754→A, G889→A, C904→T or C1108→T

In the present invention, a detection or diagnosis is performed based on, for example, the above-mentioned mutations.

(2) Direct sequencing assay

Mutations of HTRAl gene can be detected by use of a direct sequencing method. In this assay, a DNA sample is first taken from a subject by an appropriate method. A target detection region is cloned into an appropriate vector and amplified through proliferation of a host cell (e.g., bacterial cell). Alternatively, DNA within the target detection region may be amplified by use of PCR. After the amplification, DNA within the target detection region is subjected to sequencing by an appropriate method. Examples of such a sequencing method include, but not limited to an automatic sequencing method. Examples of such an automatic sequencing method include a method using a Dye Terminator, and the like. The sequencing results are shown by an appropriate display method. Thereafter, the presence or absence of a predetermined mutation is determined.

(3) PCR assay

In the present invention, a mutation of HTRAl gene can be detected by use of an assay based on PCR. The PCR assay uses an oligonucleotide primer forming a hybrid only within a mutation type or wild type allele. A DNA sample is amplified by use of a primer set consisting of primers for a mutation type and a wild type. When only the mutation-type primer generates a PCR product, it is demonstrated that the subject has a mutation allele. When only the wild-type primer generates a PCR product, it is demonstrated that the subject has a wild-type allele.

RT-PCR can also be used to identify HTRAl niRNAs. In RT-PCR, mRNA from the diseased tissue is converted into cDNA by the enzyme reverse transcriptase, using methods well-known to those of ordinary skill in the art. The entire coding sequence of the cDNA is then amplified via PCR using a forward primer located in the 3' untranslated region, and a reverse primer located in the 5' untranslated region. The amplified products can be analyzed, for example by comparing the size of the amplified products with the size of the expected product from normal mRNA, e.g., by agarose gel electrophoresis. Thereafter, the presence or absence of a predetermined mutation is determined.

(4) Hybridization assay

In the present invention, a mutation of HTRAl gene can be detected by hybridization assay. The hybridization assay is a method of determining the presence or absence of a predetermined mutation based on the ability of the DNA derived from a sample to hybridize with a complementary DNA molecule (e.g., oligonucleotide probe). Hybridization assay is performed by various hybridization techniques and detection techniques. Whether or not a probe hybridizes with a target detection sequence (e.g., mutation) can be directly detected by visualizing a hybridized probe. This method is known as Northern or Southern assay (Current Protocols in Molecular Biology, John Wiley & Sons, NY (1991)). Thereafter, the presence or absence of a predetermined mutation is determined.

2. Inhibition of signaling by the TGF-β (l) siRNAand shRNA

In order to inhibit the signaling by the TGF-β, a method is employed that inhibits expression and/or function of TGF-β. For inhibition of signaling by the TGF-β, RNA interference (RNAi) may be utilized. siRNA (small interfering RNA) targeting TGF-β gene can be designed and synthesized for transduction of cells for RNAi. RNAi is a phenomenon in which dsRNA (double-strand RNA) specifically and selectively binds to a target gene, which is subsequently removed to efficiently inhibit the expression of the target. For example, when dsRNA is introduced into a cell, expression of a gene having a homologous sequence to the RNA is knocked down. siRNA is designed as follows.

(a) There is no limitation to the gene as long as the gene codes for TGF-β or pro- TGF-β and any domains can be used as candidates. For example, in the case of human, any domains in GenBank Accession number NM_000660 (SEQ ID NO:3 and 4) can be used as candidates.

(b) From the selected domains, sequences starting with AA with a length of 19 to 25 bases, preferably 19 to 21 bases are selected. The GC contents of the sequences are, for example, conveniently 40-60%. siRNA can be introduced into a cell by a method in which siRNA synthesized in vitro is linked to plasmid DNA and then introduced into the cell or a method in which two RNAs are annealed.

According to the present invention, shRNA may be used for providing RNAi effect. shRNA is an RNA molecule called short hairpin RNA that has a stem-loop structure for forming a complementary strand between one domain and the other domain of the single-stranded molecule. shRNA can be designed such that a part thereof forms a stem-loop. For example, when sequence A represents a sequence of one domain and sequence B represents a sequence complementary to sequence A, sequence A, a spacer and sequence B are provided in this order in one RNA strand with the whole length being 45 to 60 bases. The target domain is not particularly limited and any domain can be a candidate.

(2) Decoy nucleic acid

A decoy nucleic acid in the present invention implies a short decoy nucleic acid including the binding site for a transcription factor. If this nucleic acid is transfected into the cell, transcription factor binds to this nucleic acid competitively to inhibit binding to the original binding site on the genome the transcription factor. As a result, expression of the transcription factor is inhibited. Typically, a decoy nucleic acid is a nucleic acid and its analogs, which contains at least one nucleic acid sequence that can bind to the target binding sequence. Decoy nucleic acids can be designed based on the nucleotide sequences of TGF-β or pro-TGF-β, by forming a single strand or double strands comprising of its complementary strand. The length is not particularly limited, but a desirable length ranges from 15 to 60 bases and preferably from 20 to 30 bases.

The siRNA, shRNA or decoy nucleic acid used in the present invention can be produced by a chemical synthesis or a biochemical synthesis known in the art. For example, a nucleic acid synthesis method using a common DNA/RNA synthesis device can be employed as a gene recombinant technology.

3. Pharmaceutical composition containing siRNA, shRNA or decoy nucleic acid

The present invention relates to a pharmaceutical composition containing one or more said siRNA, shRNA or decoy nucleic acid for treating or preventing the cerebrovascular disease. Applicable diseases of the pharmaceutical composition of the present invention include CARASIL. When pharmaceutical composition of the present invention is applied to these diseases, said diseases can be present singly, or multiple diseases can be associated.

The pharmaceutical composition of the present invention can be used in such a form that siRNA, shRNA or decoy nucleic acids can be incorporated into the cellular lesions or into the tissue cells. The mode of administration of the pharmaceutical composition of the present invention can be either an oral or a parenteral route. In the case of oral administration, an appropriate drug form can be selected from tablets, pearls, sugarcoated tablets, capsules, liquid agents, gels, syrups, slurries and suspensions. In the case of parenteral administration, via pulmonary administration types (e.g., using a nebulizer, etc.), via nasal administration types, subcutaneous injection types (e.g., ointments, cream agents), and injection types are available. In the case of injection types, the pharmaceutical composition can be administered systemically or locally, directly or indirectly to the diseased areas via various drip fusions such as intravenous injection, intramuscular injection, intraperitoneal injection and subcutaneous injection.

When the pharmaceutical composition of the present invention is used as a gene therapy, in addition to direct administration by injection of the composition, a method of administering a vector incorporating the aforementioned siRNA, shRNA or decoy nucleic acid is available. As the aforementioned vectors, adenovirus vector, adeno- associated virus vector, herpes virus vector, vaccinia virus vector, retrovirus vector, lentivirus vector, and the like are available.

A pharmaceutical composition of the present invention can be introduced into a phospholipid vesicle, such as a liposome, and the vesicle can be administered. A vesicle retaining a pharmaceutical composition of the present invention is introduced into a specific cell by the lipofection method. The cells obtained are then administered systemically intravenously or intra-arterially. They can be administered locally, for example, to the brain, cerebral vessel, subarachnoid space or cerebral ventricle. In order to introduce the pharmaceutical composition of the present invention into the target tissues or organs, commercial gene transfection kits (e.g., Adeno Express: Clontech Corp.) can be used. As lipids to form a liposome structure, phospholipids, cholesterols and nitrolipids can be used.

The pharmaceutical composition of the present invention can be formulated by a conventional method and can contain pharmaceutically acceptable carriers. Such carriers can be additives or the following additives are available: water, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, sodium carboxymethylcellulose, sodium polyacrylate, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, methylcellulose, ethyl cellulose, xanthan gum, gum arabic, casein, agar, polyethylene glycol, diglycerin, glycerin, propylene glycol, vaseline, paraffin, stearyl alcohol, stearic acid, human serum albumin, mannitol, sorbitol, lactose, and surfactants that are acceptable as pharmaceutical additives.

The aforementioned additives can be selected singly or in combination according to the types of the pharmaceutical composition of the present invention. For example, when used as an injection formula, a purified nucleic acid is dissolved in a solvent (e.g., saline, buffer solution, glucose solution, etc.) and then mixed with Tween 80, Tween 20, gelatin, and human serum albumin, etc. Alternatively, it can be freeze- dried form to be dissolved before use. As an excipient for freeze dry, the following materials are available: sugars such as mannitol, glucose, lactose, sucrose, mannitol and sorbitol etc.; starch such as corn, wheat, rice, potato and other vegetable starch; celluloses such as methyl cellulose, hydroxypropylmethyl cellulose, or sodium carboxymethylcellulose; rubbers such as gum Arabic, traganto rubber; gelatin and collagen, etc. If desirable, disintegrants or solubilizers, such as cross-linked polyvinyl pyrrolidone, agar, alginic acid or its salts (e.g., sodium alginate) are available.

Doses of the pharmaceutical composition of the present invention vary with age, sex, symptoms, administration routes, frequency of administration, and types of formulas. A method of administration is appropriately selected based on patient's age and symptom. An effective dose is the amount of a nucleic acid that is required for reducing symptoms of the cerebrovascular diseases. A single dosage of the pharmaceutical composition of the present invention ranges from 0.1 μg to 100 mg per kg body weight and preferably from 1 to 10 μg. However, the aforementioned treatment agent is not limited by these dosages.

4. Kit

The term "kit" refers to a supply system for supplying primer set for amplifying wild type or mutated HTRAl gene. When it is used in a reaction assay, a system for storing, transporting or supplying a reaction reagent and/or auxiliary substance is included in such a supply system. Examples of such a reaction reagent include, but not limited to, an oligonucleotide and an enzyme contained in a container. Examples of such an auxiliary substance include, but not limited to, a buffer and an instruction leaflet. Examples of such a kit include at least one type of accommodation unit (e.g., box) containing a relevant reaction reagent and/or auxiliary substance, and the like.

EXAMPLES

Hereinbelow, the present invention will be described more specifically with reference to the following Example. However, the technical scope of the present invention is not limited to the Example.

METHODS

SUBJECTS AND GENETIC ANALYSIS

We enrolled five probands of consanguineous families of Asian (Japanese) ancestral origin and some of their family members for linkage analysis (families 2285, 1872, 2321, 2402, and 2520 in Table 1 and Figure IA). After identification of the causative gene for CARASIL, we enrolled one additional subject in family 3119 with pathologically confirmed CARASIL (Table 1). On neuropathological examination in the patient in family 3119, arteriosclerosis associated with intimal thickening and dense collagen fibers was observed in cerebral small arteries (Figure II). Ancestry was determined by self-report form the participant or family members. Probands showed diffuse white matter lesions on magnetic resonance imaging, autosomal recessive inheritance, onset of symptoms between their second and fifth decade, and spondylosis or alopecia (Table land Figure IE, IF, IQ and IH).^6"8

Table 1

Clinical Features of the Six Patients with CARASIL

The numbers in parentheses refer to the age at onset for each symptom. None of the patients had any malignancy, abnormalities in the retinal artery, or macular degeneration.

Although the affected persons of family 2520 did not have alopecia and cognitive impairment, we enrolled this family because the patients had diffuse white matter lesions on magnetic resonance imaging and spondylosis, and an affected sibling had pathological findings identical to those of CARASIL.⁸ We isolated genomic DNA from eleven subjects, including the five probands from these five families with CARASIL. We performed a genomewide linkage analysis using 763 microsatellite markers (Applied Biosy stems). Pairwise logarithm of the odds (LOD) scores were calculated with the MLINK program of the LINKAGE 5.2 and FASTLINK 4.1P package.¹²' ¹³ We established five new microsatellite markers, namely, Ml 236, Ml 238, Ml 241, Ml 260, and Ml 264, on the basis of simple-repeat information from the UCSC Genome Browser on Human. Primer sequences of these markers are summarized in Table 2.

Primer Sequences for Originally Established Polymorphic Markers.

To narrow the candidate interval, we established five new microsatellite markers, namely, M1236, M1238, M1241, M1260, and M1264, based on simple repeat information obtained from the 2006 human reference sequence in the University of California Santa Cruz Genome Browser Database (http://genome.ucsc.edu/index.html).

The following table 2 is a summary of amplification primers for the markers.

Table 2

We designed primer pairs for amplification of the nine coding exons of HTRAl. Control subjects to provide DNA and fibroblasts were recruited from healthy individuals of Asian (Japanese) ancestral origin, as determined by self-report. Control subjects were between 74 and 90 years of age, with no signs of dementia, as defined by the mini-mental state examination. We obtained written informed consent from the affected persons and their family members and written and oral informed consent from the control persons. The institutional review board of Niigata University approved this study.

Assay of HTRAl Protease Activity

We subcloned wild-type or mutant HTRAl complementary DNA (cDNA) lacking 1-140 codons into the vector pGEX 6P-3 to express polypeptides in Escherichia coli as fusions with glutathione S-transferase (GST) (GE Healthcare). Amino acid substitution of the serine protease motif S328A, which abolishes the protease activity in HTRAl, was used as a negative control.¹⁴ GST fusion proteins were overexpressed and purified. Protease activity using FITC-labeled substrate β-casein was evaluated with a QuantiCleave Fluorescent Protease Assay Kit (Pierce) by using recombinant GST- HTRAl. To eliminate the possibility that the lack of an N-terminus for GST-HTRAl affects the result of protease activity, we also performed the identical protease assay using the conditioned media from cells stably expressing full-length wild-type or mutant HTRAl tagged with a green fluorescent protein (GFP). GFP-tagged HTRAl proteins were detected by using anti-GFP antibody (MBL).

To assay formation of a stable complex with αl -antitrypsin, we transiently expressed αl -antitrypsin and either wild-type or mutated HTRAl cDNAs with a V5 tag at the C-terminus in HEK293 cells. These cells were grown in serum-free medium, and conditioned media were then immunoblotted with anti-V5 antibody.¹⁴

Expression of HTRAl and NOG mItNA

Total RNA was isolated from whole blood or cultured skin fibroblasts. cDNA was synthesized with the High-Capacity cDNA Reverse Transcription kit (Applied Biosystems). We assayed the expression of HTRAl mRNA in whole blood using gene- specific primers for HTRAl. To assay HTRAl mRNA levels in cultured skin fibroblasts in relation to the expression of glyceraldehyde 3 -phosphate dehydrogenase, we performed real-time quantitative reverse transcription-polymerase chain reaction (RT- PCR) using specific TaqMan^® probes and primer sets (Applied Biosystems). We assayed TVOG niRNA levels in cultured skin fibroblasts in relation to the expression of β-actin by real-time quantitative RT-PCR using SYBR Green assay (Applied Biosystems).

Reverse Transcription-Polymerase Chain Reaction Assay to Amplify HTRAl and NOG mRNA.

To determine whether R370X nonsense mutation in HTRAl resulted in loss of mRNA from a mutant allele, we analyzed mRNA from whole blood.- Total RNA was isolated with the PAX Gene Blood RNA kit (Pre-Analytix). cDNA was synthesized with the High-Capacity cDNA Reverse Transcription kit (Applied Biosystems). PCR was performed with the following primer pair.

Forward primer: 5'-CGCC ATC ATC AACTATCG-3' (SEQ ID NO: 15 ) Reverse primer: S'-GTCAAAAGTCTTGAGTGTCC-S ' (SEQ ID NO: 16) RT-PCR products were analyzed on a 2% agarose gel and sequenced with the use of the same primers.

To quantify HTRAl mRNA levels in cultured skin fibroblasts, we performed real-time RT-PCR using TaqMan^® Gene expression assays (Applied Biosystems) (Hs01O16151 ml for HTRAl and Hs99999905 ml for glyceraldehyde 3-phosphate dehydrogenase as a control). Real-time RT-PCR amplification was carried out on an ABI Prism 7100 Sequence Detection System (Applied Biosystems). To quantify NOG mRNA levels in cultured skin fibroblasts in relation to the β-actin mRNA levels as control, the following primer pairs were designed. For TVOG mRNA

Forward primer: 5 '-CC AGC ACTATCTCC AC ATC-3¹ (SEQ ID NO: 17) Reverse primer: 5¹-GCAGCGTCTCGTTCAGATC-3^l (SEQ ID NO: 18) For β-actin mRNA

Forward primer: S'-CTTCTACAATGAGCTGCGTG-S' (SEQ ID NO: 19) Reverse primer: 5'-GTCTCAAACATGATCTGGGTC-S' (SEQ ID NO:20)

Assay of Signaling by TGF-β family Proteins

We used GeneTailor site-directed mutagenesis system (Invitrogen) to synthesize cDNA encoding HTRAl mutants and constitutively active TGF-βl proprotein (pro- TGF-βl containing the activating mutations C223S/C225S)¹⁷ and then individually subcloned these cDNAs into the pcDNA DEST-40 vector (Invitrogen). Constitutively active TGF-βl was synthesized from pro-TGF-βl containing the activating mutations C223S/C225S. We isolated SMAD2 cDNA from a human whole-brain cDNA library (Clontech) and subcloned it into the pcDNA DEST-40 vector. Luciferase assays were performed as previously described.¹⁵' ¹⁶ Mouse C2C12 myoblasts were cotransfected with pRL-TK renilla luciferase expression plasmid, HTRAl expression vectors, and the following constructs: (SBE )⁴-fϊrefly luciferase vector (TGF-β responsive reporter vector) and vectors containing SMAD2, SMAD4, and TGF-βl (encoding pro-TGF-βl with two point mutations (C223S, C225S));¹⁷ pGL3-Id985WT-firefly luciferase vector (BMP responsive reporter vector)¹⁶ and vectors containing SKdADl, SMAD4, and BMP- 4 (encoding pro-bone morphogenetic protein 4); pGL3-Id985WT-firefly luciferase vector (BMP responsive reporter vector)¹⁶ and vectors containing SMADl, SMAD4, and BMP-2 (encoding pro-BMP -2).¹⁸ Cell extracts were assayed for luciferase activity with the use of the Dual-Luciferase Reporter Assay System (Promega). The activity was corrected for transfection efficiency by using pRL-TK renilla luciferase activity. Every sample was transfected in triplicate, and every experiment was repeated three times.

Phosphorylation of Smad Proteins

Human embryonic kidney (HEK) 293 cells were cotransfected with vectors containing HTRAl and the following constructs: vectors containing SMAD2, SMAD4, and TGF-βl (encoding pro-TGF-βl with two point mutations (C223S, C225S)); vectors containing SMADl, SMAD4, and BMP-4; vectors containing SMADl, SMAD4, and BMP-2.¹¹' ¹⁸ The cells were lysed in RTPA buffer containing phosphatase inhibitor. We detected Smadl, phosphorylated Smadl, Smad2, and phosphorylated Smad2 proteins using anti-Smadl, anti-phospho-Smad 1/5/8, anti-Smad2/3, and anti-phospho-Smad2 (Cell Signaling) antibodies, respectively, through analysis by Western blot.

Immunohistochemistry and In Situ Hybridization.

We carried out immunoperoxidase staining on formalin-fixed, paraffin- embedded brains, obtained from two autopsied patients with CARASIL or autopsied control subjects (84-year-old female person with stroke, a person with schizophrenia, and a person with amyotrophic lateral sclerosis).⁸'⁹ The primary antibodies were against TGF-βl (1:50, Santa Cruz), versican (1:100, Seikagaku), and fibronectin ED-A (1:100, Abeam). Nonimmune immunoglobulin G was used as a negative control. We used cDNA encoding the ED-A domain of fibronectin (5404-5704 nucleotide fragment of fibronectin isoform 1: NM_212482.1) as a template for digoxigenin-labeled antisense and sense-complementary RNA probes. The sense probe was used as a negative control. We carried out in situ hybridization on the paraffin-embedded sections with the probes. After washing and blocking procedures, the sections were incubated with alkaline phosphatase-conjugated anti-digoxigenin antibodies. The signal was developed in 4- nitroblue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate solution (Roche). The sections were counterstained with Fast Red.

RESULTS Genetic Analysis

On carrying out genomewide linkage analysis of the five families, we obtained maximal cumulative pairwise LOD scores of 3.97 and 3.59 at D10S587 and D10S1656 (θ = 0.0); these markers were homozygous in all patients (Figure IA). We then fine- mapped the region between D10S597 and D10S575 using D10S1483 and five established polymorphic microsatellite markers (M1236, M1238, M1241, M1260, and Ml 264 see Table 2) (Figure IA and IB). The region between M1236 and M1260 was homozygous in all probands, suggesting that the causative gene was located within this 2.4-Mb region.

We first selected HTRAl as a candidate (Figure IB), because it is expressed in the blood vessels, skin, and bone.¹⁹ We identified two homozygous nonsense mutations: C1108→T (resulting in a stop codon at 370: R370X) in family 2285 and C904→T (resulting in a stop codon at 302: R302X) in family 1872 (Figure 1C). We also identified two homozygous missense mutations: G889— >A (predicted to result in the amino acid substitution V297M) in families 2321 and 2404, and G754-»A (predicted to result in the amino acid substitution A252T) in family 2520. In addition, we observed a homozygous nonsense mutation C904— ^T in the proband of family 3119. The missense mutations V297M and A252T were located in the genie region encoding the serine protease domain, and the amino acids predicted to be affected are either absolutely or strongly conserved among the HTRA homologues and HTRAl orthologues (Figure 1C and ID). We did not observe these changes in the 125 control persons.

PROTEASE ACTIVITY OF MUTANT HTRAl

The protease activities of the HTRAl encoded by constructs containing either of the missense mutations or of R370X were 21-50% of the activity of wild-type HTRAl. In contrast, HTRAl encoded by a construct containing the R370X mutation had a protease activity similar to that of wild-type HTRAl (Figure 2A and B). HTRAl attacks the reactive center loop of αl -antitrypsin, instigating serine protease activity of αl- antitrypsin which thereby mediates the formation of a covalent complex between the two molecules.¹⁴ We did not observe the formation of a stable complex between αl- antitrypsin and the mutant HTRAIs encoded by cDNAs containing either V297M, A252T, or R302X. In contrast, wild-type HTRAl, and that encoded by a cDNA containing R370X formed stable complexes with αl -antitrypsin (Figure 2C).

NONSENSE-MEDIATED DECAY

When a premature stop codon is located at least 50 to 55 nucleotides upstream of the 3 '-most exon-exon junction, messenger RNAs (mRNAs) may be degraded by nonsense-mediated decay.²⁰ Because the location of R370X fulfills these criteria (Figure 1C), we determined whether R370X-containing HTRAl mRNA is degraded by nonsense-mediated decay. The level of HTRAl mRNA expression in fibroblasts from the patient with the R370X mutation is 6.0% of that of control subjects, and treatment with cycloheximide, an inhibitor of nonsense-mediated decay, increased expression of R370X HTRAl mRNA to four times to that of the basal level (Figure 3A). We did not detect HTRAl protein in the culture medium of fibroblasts from the patient carrying the R370X mutation (Figure 3B). Furthermore, analysis of HTRAl of leukocytes from a heterozygous carrier of this mutation showed the presence of wildtype HTRAl mRNA only (Figure 3C).

Mutant HTRAl and Signaling by TGF-β Homologues.

The serine protease activity of HTRAl is necessary for the inhibition of TGF-β family signaling.¹⁵ We therefore tested the ability of mutant variants of HTRAl with "missense" amino acids to repress signaling by the TGF-β family members TGF-β, BMP-4 and BMP-2 (Figure 4A and Figure 5). As expected, none of the missense- mutated HTRAl proteins repressed signaling by these molecules. To exclude the possibility that HTRAl protease activity directly affects the reporter system, we assayed the phosphorylation of Smad in these assays (phosphorylated Smad is a downstream effector of the TGF-β family signaling pathway), and observed that none of the mutant HTRAl proteins repressed the subsequent phosphorylation of Smad proteins (Figure 4B and Figure 6).

We next investigated TGF-β signaling in fibroblasts from the subject with CARASIL with the R370X mutation and observed that the TGF-β signaling in these fibroblasts was more than three times that of control subjects (Figure 4C). In addition, NOG mRNA, which is induced by TGF-β signaling in fibroblasts, was also elevated in the probands (Figure 4D and Figure 7).²¹

Increased TGF-β signaling results in vascular fibrosis, with synthesis of extracellular matrix proteins including ED-A fibronectin and versican.^22"24 The intima of the CARASIL patients carrying the R302X or A252T mutation showed increased expression of ED-A fibronectin (Figure 4E, 4F, 4Q 4H, and Figure 8) and versican (Figure 4K and Figure 8) when compared with those of control subjects (Figure 4M, 4N and 40 ). The result was confirmed by an in situ hybridization assay that used a probe for ED-A fibronectin (Figure 41, 4 J and Figure 9). Moreover, the media of CARASIL patients exhibited elevated expression of TGF-β 1 (Figure 4L, 4P and Figure 8). These results indicate increased TGF-β signaling in the cerebral small arteries in CARASBL.

DISCUSSION

TGF-β family signaling is tightly associated with vascular angiogenesis and remodeling and has multifaceted roles in vascular endothelial cells and vascular smooth muscle cells, depending on the cell types and extracellular matrix.²⁵' ²⁶ Moreover, dysregulation of TGF-β family signaling results in hereditary vascular disorders.²⁶ Defective TGF-β signaling by mutations in the TGF-β receptors leads to hereditary hemorrhagic telangiectasia, whereas activation of TGF-β signaling contributes to Marfan syndrome and associated disorders.²⁶ Our findings extend the spectrum of diseases shown to be caused by the dysregulation of TGF-β signaling to include hereditary ischemic cerebral small-vessel disease. In addition, the pathological findings in CARASIL resemble those observed in nonhereditary ischemic cerebral small-vessel disease with hypertension; hypertension may increase TGF-β signaling.^7"11' ²⁷ Thus, TGF-β signaling might underlie the molecular basis of nonhereditary ischemic cerebral small-vessel disease with hypertension.

Disregulation of inhibition of TGF-β family signaling also has been linked to alopecia and spondylosis, the other cardinal clinical features of CARASIL. Transgenic mice overexpressing BMP-4, BMP-2, and TGF-β exhibit hair loss or retardation of the development of hair follicles.²⁸' ²⁹ BMP family members are well-known regulators of bone formation, repair, and regeneration.³⁰ Furthermore, HTRAl overexpression decreases BMP-2-induced mineralization, whereas reduced expression of HTRAl accelerates mineralization.³¹ Although it is possible that the loss of protease activity by HTRAl on other substrates associates with the pathogenesis of CARASIL, these findings strengthen the hypothesis that increased TGF-β family signaling contributes to the pathogenesis of CARASIL.¹⁴' ^31'33 It remains unclear why disinhibition of signaling by TGF-β family members caused by mutant HTRAl results in narrowly-restricted clinical phenotypes. Tissue-specific regulation of TGF-β family signaling or tissue- specific expression of HTRAl is an explanation.¹⁴'³³'^{34 25}' ²⁶

The molecular basis for regulation of TGF-β 1 signaling by HTRAl remains to be elucidated.¹⁵' ^{35> 36} TGF-β 1 is synthesized as a proprotein (pro-TGF-βl) and is subsequently cleaved into latency associated protein (LAP) and mature TGF-β 1 by proprotein convertase.²⁶ The mature TGF-β 1 is non-covalently bound to LAP and is sequestrated as a LAP-TGF-βl complex in an extracellular matrix.²⁶ The mature TGF- βl is released from the LAP-TGF-βl complex and is presented. Thus the TGF-β 1 signaling is regulated by balancing between maturation, sequestration, and presentation. Interestingly, Emilinl inhibits TGF-β 1 signaling by preventing the processing of pro- TGF-βl into mature TGF-β I.³⁷ The CARASIL patients demonstrated increased expression of mature TGF-β 1, suggesting that the HTRAl might also prevent the processing of pro-TGF-βl into mature TGF-β 1 depending on its protease activity. A single nucleotide polymorphism in the promoter region of HTRAl, which is associated with elevated expression levels of HTRAl, is a genetic risk factor for the neovascular form of age-related macular degeneration (AMD).³⁸' ³⁹ We observed no macular degeneration in the persons with CARASIL, consistent with the hypothesis that increased expression of HTRAl contributes to age-related macular degeneration.^6"8' ³⁸ That said, all of our patients were younger than the typical age of onset of neovascular form of AMD. Our results indicate that dysinhibition of TGF-β family signaling underlies the molecular basis for CARASIL and provide a basis for further investigation of therapeutic strategies for ischemic cerebral small-vessel disease, alopecia, and spondylosis.

FIGURE LEGENDS

Figure 1. Pedigrees and HTRAl Mutations of Families with CARASBL.

Panel A shows pedigrees of families with CARASIL. Squares denote men; circles, women; solid symbols, affected family members; open symbols, unaffected members; double horizontal lines, consanguineous marriage. Microsatellite markers are shown in order from the centromere to the q-arm terminus. Originally developed microsatellite markers are indicated in blue type. Alleles where the phases are unequivocally determined are shown in parentheses. The region of homozygosity for each affected subject is boxed. Panel B shows the physical map of the candidate region for CARASIL on chromosome 1Oq. Panel C shows the distribution of mutations in HTRAl, which consists of nine exons (squares). Colored boxes represent exons corresponding to the insulin-like growth factor binding protein domain (green), Kazal- type serine protease inhibitor domain (red), trypsin-like serine protease domain (blue), PDZ domain (yellow), and untranslated regions (gray). The missense mutations are in black type, and the nonsense mutations are in red type. Panel D shows the conservation of HTRAl residues mutated in CARASIL. Conserved amino-acid residues are shaded (black, 100%; dark gray, 80%; gray, 60%). Sequences were obtained from GeneBank. T2-weighted magnetic resonance imaging of the brain (repetition time, 5000 msec; echo time, 150 msec) with a thickness of 5 mm showed an ischemic region in the basal ganglia and white matter (panels E and F; subject II-7, family 2321), and Tl -weighted lumbar magnetic resonance imaging (repetition time, 519 msec; echo time, 19 msec) with a thickness of 5 mm showed spondylotic changes of the lumbar spine (panel G; subject II-3, family 1872). Diffuse hair loss in the temporal and/or parietal area of the head was observed (subject II-2, family 2285; panel H). Cerebral small arteries in the arachnoid from subject II- 1, family 3119, showed marked intimal thickening, narrowing of the lumen, hyalinosis, and splitting of the internal elastic membrane (panel I, elastica van Gieson stain). Figure 2. Functional Consequences of HTRAl Mutations in CARASIL.

Panels A and B show FITC-labeled β-casein assay of mutated HTRAIs. The fluorescence units represent the protease activity. Conditioned media from HEK293 cells that stably expressed HTRAIs tagged with a green fluorescent protein (GFP) at the C-terminus (panel A) or recombinant N-terminal deleted HTRAl proteins expressed in E. coli (panel B) were incubated with FITC-labeled β-casein. The amount of HTRAl proteins was shown by immunoblotting with an anti-GFP antibody (panel A) or Coomassie brilliant blue (CBB) staining (panel B). The bars represent standard errors. Panel C shows a covalent complex formation (high molecular weight products; upper panel) between αl -antitrypsin and either wild-type (WT) or R370X HTRAl. Formation of the stable HtrA/αl -antitrypsin complex does not occur in the other mutant HTRAIs. The amount of HTRAl proteins was shown by immunoblotting with an anti-V5 antibody (lower panel).

Figure 3. Nonsense-Mediated Decay of Nonsense-Mutated HTRAl mRNA.

Panel A shows HTRAl mRNA levels in cultured skin fibroblasts from subject II- 2, family 2285, with R370X HTRAl as a percentage of levels in cells from control subjects (n = 4) with or without the nonsense-mediated decay inhibitor cycloheximide (CHX; 100 μg/ml) for 4 h. The bars represent the standard errors. Panel B shows Western blot analysis of HTRAl using the cultured skin fibroblasts of subject II-2, family 2285, with R370X and control subject with HTRAl antibody (MAB2916; R&D Systems). Panel C shows the results of RT-PCR assay. HTRAl PCR amplicons, the expected transcripts length of 600 bp, were obtained from cDNA prepared from peripheral blood of the heterozygote II- 1, family 2285, whereas they were not obtained from cDNA prepared from peripheral blood of subject II-2, family 2285. Electrophoregrams show wild-type and mutant (C1108-VT) alleles in the PCR products derived from the genomic DNA of the leukocytes of the unaffected heterozygous subject II- 1, family 2285, whereas only wild-type allele was detected in the reverse transcription PCR products derived from the RNA of the leukocytes of the same individual.

Figure 4. Modulation of TGF-β Family-Mediated Transcriptional Responses by Mutated HTRAl Proteins. In Panel A, C2C12 cells were cotransfected with pRL-TK renilla luciferase expression plasmid, wildtype (WT) or mutated HTRAl expression plasmid, and the following constructs: (left) (SBE )⁴-firefly luciferase vector (TGF-β responsive reporter vector) and vectors containing SMAD2, SMAD4, and TGF-β 1 (encoding pro-TGF-βl with two point mutations C223S/C225S);¹⁷ (middle) pGL3-Id985WT-firefly luciferase vector (BMP responsive reporter vector)¹⁶ and vectors containing SMADl, SMAD4, and BMP-4 (encoding pro-bone morphogenetic protein 4); (right) pGL3-Id985WT-firefly luciferase vector (BMP responsive reporter vector)¹⁶ and vectors containing SMADl, SMAD4, and BMP-2 (encoding pro-BMP-2).¹⁸ Data represent the mean with standard error of normalized firefly luciferase/renilla luciferase activities from three independent experiments. In Panel B, HEK293 cells were cotransfected with WT or mutated HTRAl -V5 expression vectors the following constructs: (left) vectors containing SMAD2, SMAD4, and TGF-β 1 (encoding pro-TGF-βl with two point mutations (C223S, C225S));¹⁷ (middle) vectors containing SMADl, SMAD4, and BMP-4 (encoding pro- bone morphogenetic protein 4); vectors containing SMADl, SMAD4, and BMP-2 (encoding pro-BMP-2).¹⁸ The ratio of phosphorylated Smad proteins was examined by immunoblotting of whole-cell lysates. Data represent the mean with standard error of four independent experiments. In panels A and B, the mean values for WT-HTRAl are significantly lower than others by the Tukey's multiple-comparison test (P < 0.05). In panel C, fibroblasts from two control subjects and subject II-2, family 2285, with R370X HTRAlwere cotransfected with pRL-TK renilla luciferase expression plasmid and vectors containing SMAD2, SMAD4, and TGF-β 1 (encoding pro-TGF-βl with two point mutations (C223S, C225S)).¹⁷ The mean values for R370X are significantly higher than others by the Tukey's multiple-comparison test (P < 0.05). Panel D shows NOG mRNA levels in fibroblasts from subject II-2, family 2285, with R370X HTRAl as a ratio of levels in fibroblasts from control subjects (n = 4). In panels E, F, Q H, I, J, K, and L, small cerebral arteries of autopsied subject II- 1, family 3119 (homozygous for the R302X mutation) shows marked intimal proliferation (panels E and Q elastica van Gieson stain), increased expression of an ED-A fibronectin in intima (panels F and H, IST-9 antibody), increased mRNA expression of an ED-A fibronectin in endothelial cells and subendothelial smooth muscle cells (panels I and J), and increased expression of a versican in intima (panel K) and TGF-β 1 in media (panel L). In panels M, N, O, and P, immunohistochemical analysis in the cerebral small arteries from autopsied control subject (40-year-old female with amyotrophic lateral sclerosis). Elastica van Gieson stain (panel M) and staining with anti-EDA-fibronectin antibody (IST-9: panel N), anti-versican antibody (panel O) and anti-TGF-βl antibody (panel P). The same results were obtained from two additional control subjects (84-year-old female, 62-year- old male with stroke, and 36-year-old female with schizophrenia).

Figure 5

Expression Level of Transfected HTRAl Proteins in TGF-β Family-Mediated

Transcriptional Response Assay.

C2C12 cells were cotransfected with pRL-TK renilla luciferase expression plasmid, wild-type (WT) or mutated HTRAl expression plasmid, and the following constructs:

(Left) (SBE)₄-firefly luciferase vector (TGF-β responsive reporter vector) and vectors containing SMAD2, SMAD4, and TGF-β 1 (encoding pro-TGF-βl with two point mutations C223S/C225S);

(Middle) pGL3-Id985WT-firefly luciferase vector (BMP responsive reporter vector) and vectors containing SMADl, SMAD4, and BMP-4 (encoding pro-bone morphogenetic protein 4);

(Right) pGL3-Id985WT-firefly luciferase vector (BMP responsive reporter vector) and vectors containing SMADl, SMAD4, and BMP-2 (encoding pro-BMP-2).

Data represent the mean with standard error of normalized firefly luciferase/renilla luciferase activities from three independent experiments (top panel). The HTRAl proteins were stained with anti-V5 antibody (bottom panel).

Figure 6

Expression Level of Transfected HTRAl Proteins in Assay of Phosphorylation of

Smad proteins.

HEK293 cells were cotransfected with WT or mutated HTRAl -V5 expression vectors, and the following constructs:

(Left) vectors containing SMAD2, SMAD4, and TGF-β 1 (encoding pro-TGF-βl with two point mutations C223S/C225S);

(Middle) vectors containing SMADl, SMAD4, and BMP-4 (encoding pro-bone morphogenetic protein 4); (Right) vectors containing SMADl, SMAD4, and BMP-2 (encoding pro-BMP-2). The ratio of phosphorylated Smad proteins was examined by immunoblotting of whole cell lysates. Data represent the mean with standard error of four independent experiments (top panel), and the HTRAl proteins were stained with anti-V5 antibody (bottom panel).

Figure 7

Effects of TGF-β on NOG mRNA Expression in Cultured Skin Fibroblasts.

Cultured skin fibroblasts from healthy Japanese subjects (n=3) were treated with recombinant TGF-βl at 0.04-5.0ng/ml for 2 hours. NOG mRΝA levels as a fold change of levels in cells without TGF-βl. The bars represent the standard errors.

Figure 8

Immunohistochemical Analysis in Cerebral Small Arteries.

In panels A, B, and C, small cerebral arteries of autopsied subject II-3, family 5 (homozygous for the A252T mutation) show increased expression of an extra domain- A of fibronectin (panel A) and a versican in intima (panel B) and increased expression of TGF-βl in media (panel C).

Figure 9 mRΝA Expression of Extra Domain-A of Fibronectin in Cerebral Small Arteries.

In situ hybridization was carried out on the small cerebral arteries of autopsied subject π-1, family 6 (homozygous for the R302X mutation) with the use of antisense (panels A and C) and sense probes (panels B and D) derived from an extra domain-A of fibronectin. Panels E and F, in situ hybridization analysis of extra domain-A of fibronectin antisense probe in the cerebral small arteries of autopsied control subject (40-year-old female with amyotrophic lateral aclerosis).

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INDUSTRIAL APPLICABILITY

The present invention is useful for diagnosing or detecting cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL). SEQUENCE FREE TEXT SEQ ID NO:5-20: Synthetic DNA

Claims

1. A method of diagnosing a cerebrovascular disease in a human comprising the steps of:

(a) measuring a mutation of HTRAl gene in a test sample from said human; and

(b) determining if the mutation of HTRAl gene in said test sample correlates with a cerebrovascular disease in said human.

2. A method of detecting a cerebrovascular disease in a human comprising the steps of:

(a) measuring a mutation of HTRAl gene in a test sample from said human; and

(b) correlating the mutation of HTRAl gene in said test sample with a cerebrovascular disease in said human.

3. The method according to claim 1 or 2, wherein the test sample is selected from the group consisting of blood, serum, plasma, saliva, cerebral spinal fluid, oral mucosa and nail.

4. The method according to claim 1 or 2, wherein the test sample is blood.

5. The method according to claim 1 or 2, wherein the cerebrovascular disease is selected from the group consisting of cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy, acute cerebrovascular disease, ischemic cerebrovascular disease, Binswanger disease, leukoaraiosis, cerebral small vessel disease, and leukoencephalopatky.

6. A kit for diagnosing or detecting a cerebrovascular disease in a human, comprising a primer set for amplifying a mutant HTRAl gene and a wild type HTRAl gene.

7. A pharmaceutical composition comprising a substance that inhibits signaling by TGF-β family proteins.

8. The pharmaceutical composition according to claim 7, wherein the substance that inhibits signaling by TGF-β family proteins is siRNA, shRNA or decoy nucleic acid that targets a gene coding for TGF-β.