METHODS AND COMPOSITIONS FOR PREDICTING AND TREATING INTRACRANIAL ANEURYSM

Abstract

The present invention relates to a method for predicting the risk of having or developing Intracranial aneurysms (IA) in a subject, by identifying at least one mutation in an angiogenic protein, such as Angiopoietin-Like 6 (ANGPTL6). In particular, inventors identified one rare nonsense variant (c.1378A>T) in the last exon of the ANGPTL6 gene which encodes a 10 circulating pro-angiogenic factor mainly secreted from the liver shared by the 4 tested affected members of a large pedigree with multiple IA carriers. They GC showed a 50% reduction of ANGPTL6 serum concentration in heterozygous c.1378A>T carriers compared to non-carrier relatives, due to the non-secretion of the truncated protein produced by the c.1378A>T transcripts. They observed a higher rate of individuals with a history of high blood pressure 15 among affected versus healthy carriers of ANGPTL6 variants, suggesting that ANGPTL6 could trigger cerebrovascular lesions when combined with other risk factors such as hypertension.

Claims

1. A method for predicting the risk of having or developing Intracranial aneurysms (IA) in a subject, comprising the steps of: i) identifying at least one mutation in an angiogenic protein; and ii) concluding that the subject is at risk of having or developing IA when at least one mutation is identified in said angiogenic protein.

2. The method according to claim 1, wherein, the angiogenic protein is angiopoietin-Like 6 (ANGPTL6).

3. A method for predicting the risk of having or developing Intracranial aneurysms (IA) in a subject comprises following steps: i) determining the expression level of ANGPTL6 in a biological sample obtained from said subject, ii) comparing the expression level of ANGPTL6 determined at step i) with a predetermined reference value and iii) concluding that the subject is at risk of having or developing IA when the expression level of ANGPTL6 determined at step i) is lower than the predetermined reference value, or concluding that the patient is not at risk of having or developing IA when the expression level of ANGPTL6 determined at step i) is higher than the predetermined reference value.

4. The method according to claim 1, wherein the method further comprises a step of detecting an angiogenic single nucleotide polymorphism (SNP) in a biological sample obtained from said subject.

5. A method of treating and/or preventing intracranial aneurysms in a subject in need thereof, comprising, i) determining the expression level of ANGPTL6 an angiogenic protein in a biological sample obtained from said subject, ii) comparing the expression level of ANGPTL6 the angiogenic protein determined at step i) with a predetermined reference value and iii) treating the subject by surgery and/or by one or more endovascular techniques when the expression level of the an angiogenic protein determined at step i) is lower than the predetermined reference value.

6. A kit for performing the methods according to claim 1, wherein said kit comprises means for measuring the expression level of an angiogenic protein and/or detecting angiogenic SNP that is indicative of subject at risk of having or developing Intracranial aneurysms (IA).

7. The method according to claim 5, wherein the angiogenic protein is angiopoietin-like 6 (ANGPTL6).

8. The method according to claim 5, further comprising a step of detecting a single nucleotide polymorphism (SNP) in a gene encoding the angiogenic protein.

Description

FIGURES

[0080] FIG. 1: Genetic investigations in a large family with multiple IA carriers Pedigree of family A showing the segregation pattern of the variant ANGPTL6 c.1378A>T (Filled, empty boxes and boxes with question marks indicate IA carriers, non-carriers and individuals with unknown status; signs + indicate the presence of the ANGPTL6 variant, signs its absence; the arrow indicates the index case, the asterisks indicate the individuals included in WES analysis).

[0081] FIG. 2: Expression of WT- and Lys460Ter-Lys460Ter-ANGPTL6 in cultured cells and individual sera

[0082] (A) Analysis by qPCR of ANGPTL6 transcripts in HEK293 cells expressing WT- and Lys460Ter-ANGPTL6. (B) Analysis of serum level of ANGPTL6 in controls (WT-ANGPTL6) and individuals expressing the Lys460Ter-ANGPTL6 (heterozygous) (**P<0.01).

[0083] FIG. 3: Familial cases of IA in the presence of rare coding variants in ANGPTL6

[0084] Filled, empty boxes and boxes with question marks indicate IA carriers, non-carriers and individuals with unknown status; signs + indicate the presence of the ANGPTL6 variant, signs its absence; black arrows indicates the index cases.

[0085] FIG. 4: Angptl6 mutant mice display dilated arteries under basal condition. The passive diameter of isolated basilar artery pressured at 50 mm Hg is significantly larger in Angptl6+/. and Angptl6/. mice than in controls.

[0086] FIG. 5: Cerebral arteries of Angptl6 mutant mice abnormally dilate in high blood pressure condition. Hypertension did not modify the diameter of the basilar artery in control Angptl6+/+ mice (red dashed line) while it induces an increase of this diameter in Angptl6+/ and Angptl6/. mice (dotted line). The difference in the arterial diameter of Angptl6+/+ mice and that of Angptl6+/ and Angptl6/ mice is thus potentiated in high blood pressure condition (*P<0.05; **P<0.01).

[0087] FIG. 6: Change in mechanical properties of isolated basilar artery from Angptl6 mutant mice. Flow-mediated dilation, corresponding the increase in the diameter of the artery in response to a gradual increase in the intraluminal flow from 3 to 10 l/min is reduced in Angptl6/ mice compared with Angptl6+/+ mice. After L-NAME treatment to inhibit endothelial NO synthesis, flow-mediated dilation is similar in arteries from Angptl6+/+ and from Angptl6/ mice. This indicates that a reduced production of NO in response to flow in Angptl6/ mice. (*P<0.05; **P0.01).

EXAMPLES

Example 1

[0088] Material & Methods

[0089] Clinical Recruitment

[0090] Familial cases of IA are defined as at least two first-degree relatives both diagnosed with typical IA (defined as a saccular arterial dilatation of any size occurring at a bifurcation of the intracranial vasculature), without any age limitation. Index cases and their relatives were recruited following the French ethical guidelines for genetic research, and under approval from the French Ministry of Research (n DC-2011-1399) and the local ethical committee. Informed written consent was obtained from each individual agreeing to participate in the genetic study, to whom MRI screening and blood sampling were proposed.

[0091] The full recruiting process has been described previously (16). Briefly, neuroradiological phenotyping was performed in each recruiting center by interventional neuroradiologists, neurologists and neurosurgeons in order to recruit only cases with typical saccular bifurcation IA. Mycotic, fusiform-shaped or dissecting IA were systematically excluded, as well as IA in relation with an arteriovenous malformation and IA resulting from syndromic disorders such as Marfan disease or vascular forms of Elhers Danlos. Eye fundus, transthoracic echocardiography, non-invasive analysis of endothelial dysfunction, and Doppler echography analysis of peripheral arteries (sub clavians, radials, femorals, renals, and digestives) were carried out to check for any other vascular malformation or variation potentially linked to the presence of IA, thus constituting a syndrome yet unknown.

[0092] Whole Exome Sequencing (WES)

[0093] Genomic DNA was extracted from peripheral blood lymphocytes using the NucleoSpin Blood kit XL (Macherey Nagel, Germany). Briefly, coding exons from 3 g of genomic DNA were captured using the SureSelect Human All Exon V4 Kit (Agilent Technologies, Santa Clara, Calif.), following the manufacturer's protocol. DNA was sheared by acoustic fragmentation (Bioruptor Diagenode) and purified with the magnetic beads Agencourt AMPure XP (Beckmann Coulter genomics), and fragment quality was assessed (Tapestation 2200 Agilent). Exome-enriched genomes were paired-end sequenced (100-bp reads) on IIlumina HiSeq 1500 (Illumina Inc, San Diego, Calif.) to a mean depth above 30. Sequence reads were mapped to the human reference genome (Broad Institute human_glk_v37) using the Burrows-Wheeler Aligner (17). Duplicates were flagged using Picard software. Reads were realigned and recalibrated using the Genome Analysis Toolkit (GATK) (18). Variant detection was performed with GATK HaplotypeCaller. Functional annotation of high-quality variants was performed using Ensembl VEPv7.4. The sequencing quality was determined with the Depth Of Coverage Walker provided in GATK. Knime4Bio (19) was used for all merging and filtering steps. Variants with a sequencing depth of less than 10 or a genotype quality below 90 were excluded, as well as synonymous variants with no predicted effect on splicing sites. At last, from the resulting set of functional variants (as reported in FIG. 1), we filtered out any variant with a minor allele frequency (MAF) higher than 0.1% in the non-Finnish European (NFE) population from the ExAC database, as well as few remaining variants reported with a minor allele frequency (MAF) higher than 10% in our in-house database of 260 whole-exome sequences from individuals with various cardiac phenotypes.

[0094] Identity-by-Descent Analysis

[0095] SNP genotyping was performed on population-optimized Affymetrix Axiom Genome-Wide CEU 1 array plates following the standard manufacturer's protocol. Fluorescence intensities were quantified using the Affymetrix GeneTitan Multi-Channel Instrument, and primary analysis was conducted with Affymetrix Power Tools following the manufacturer's recommendations. After genotype calling, all individuals had a genotype call rate above 97%. SNPs with an MAF <10%, a call rate <95% or with P<110-5 when testing for Hardy-Weinberg equilibrium were excluded. IBD estimation was performed with IBDLD v3.34, NoLD method (20). Shared regions were obtained by analyzing a set of independent SNPs (R.sup.2<0.2) using genotypes from French individuals (21) as a reference panel. The IBD status at every SNP locus was obtained for each pair of individuals, based on a hidden Markov model implemented in the IBDLD program. Still using IBDLD, we estimated the kinship coefficients between pairs of IA cases from distinct pedigrees. We invariably found values around 0.025, thus excluding non-documented close relatedness between mutation carriers.

[0096] Capillary Sequencing and Burden Testing

[0097] Validation experiments for each selected variant, familial segregation analyses and further screening for ANGPTL6 coding mutations were performed by capillary sequencing on an Applied Biosystems 3730 DNA Analyzer, using standard procedures. Sequences analyses were performed with SeqScape v2.5. ANGPTL6 variants were numbered according to the canonical transcript (ENST00000253109/NM_031917, protein accession number: ENSP00000253109/NP_114123). Burden test was performed using SKAT (22) and CAST (23), by comparing the proportion of individuals carrying at least one rare coding variant within ANGPTL6 (defined as a variant with an MAF below 1% among the 7,509 whole-genome sequenced individuals with NFE ancestry from the gnomAD database) among IA cases versus healthy individuals with French ancestry. ANGPTL6 status in control individuals was determined by whole-genome sequencing with a mean depth of coverage above 30. Rare variants were defined as variants with an MAF below 1% among the 7,509 whole-genome sequenced individuals with NFE ancestry from the gnomAD database. The count of alleles with rare coding variants in ANGPTL6 among cases was also compared with the same allele count among the 7,509 whole-genome sequenced individuals with NFE ancestry from gnomAD (24), through the use of Fisher's exact test.

[0098] Expression Analyses of ANGPTL6

[0099] HEK293 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. Stable HEK293 cell lines were obtained by transfection of pcDNA3.1 vector encoding WT-ANGPTL6 and Lys460Ter-ANGPTL6 (G418 selection). Recombinant proteins are expressed as Nter-FLAG fusion proteins. In HEK293, recombinant human proteins were detected by both anti-flag and anti-ANGPTL6 antibodies (Adipogen, AB_2490340) and ELISA (kit supplied by Adipogen).

[0100] In human subjects, serum ANGPTL6 levels were measured by ELISA. For transcript analysis, total RNA from stably transfected HEK293 cells was purified using Trizol (Life technology) according to the manufacturer's instructions then reverse-transcribed. Real-time quantitative PCR was performed using the TaqMan 7900 Sequence Detection System (Applied Biosystems). Primers used to assess ANGPTL6 mRNA expression were designed using the Primer Express 3.1 software (sequences available on request).

[0101] Results

[0102] A Nonsense Variant in ANGPTL6 Shared by Family Members with IA

[0103] The index case of family A (individual III-1; FIG. 1) was diagnosed after a subarachnoid hemorrhage (SAH) at the age of 51. This event revealed a ruptured anterior cerebral artery aneurysm and a second middle cerebral artery aneurysm (data not shown). She completely recovered from the subarachnoid hemorrhage and because of known familial history of ruptured IA (11-2, 11-5), a systematic screening was performed among relatives. Her cousin (III-5) and her niece (IV-1) were both diagnosed with respectively two and one IA. Her uncle (II-4) had an episode suggestive of aneurysmal SAH at the age of 36, and died before a CT scan or angiography could be performed. Her mother (II-1), who carries an ectasia measuring less than 2mm and diagnosed as uncertain (16), was classified as phenotype unknown.

[0104] Clinical information was collected for 28 individuals from family A (data not shown). IA was diagnosed on CT angiography or conventional angiography. DNA was available for 27 of them (DNA was unavailable for II-5 who died in 1974 after a rupture of IA). Individuals with IA (II-2, II-5, III-1, III-5 and IV-1) were all female. Noteworthy, all IA carriers except IV-1 suffered from high blood pressure.

[0105] We combined WES and IBD analysis to identify any rare genetic variant likely explaining this familial form of IA. Whole-exome sequencing applied to the first cousins III-1 and II-5 led respectively to the detection of 25,674 and 23,456 functional sequence variants in comparison to the human reference genome assembly (data not shown). After filtering out genetic variants reported with an MAF above 0.1% in the non-Finnish European (NFE) population from the ExAC database (24), we ended up with 29 rare variants shared between the first cousins, which were all manually reviewed by visual inspection of sequence reads using the Integrative Genomics Viewer (25).

[0106] In parallel, IBD analysis of the complete pedigree identified 12 haplotypes shared by the 4 affected relatives. Within these chromosomal intervals, individuals III.1 and III.5 shared 10 rare, non-synonymous variants (data not shown). By capillary sequencing, we determined that the 4 affected relatives share 8 of these variants (data not shown), among which one nonsense variant, c.1378A>T (p.Lys460Ter), in the ANGPTL6 gene.

[0107] Reduced ANGPTL6 Secretion Among Heterozygous Carriers

[0108] ANGPTL6 is one of the eight members of the secreted glycoprotein ANGPTL family, which share a common structure consisting of an amino-terminal coiled-coil domain, a linker region and a carboxy-terminal fibrinogen-like domain. The c.1378A>T ANGPTL6 variant leads to the occurrence of a premature stop codon in the last exon. The corresponding transcript may thus escape the nonsense-mediated mRNA decay and is predicted to result in a protein truncated by the last 11 amino acids (Lys460Ter-ANGPTL6). To analyze the functional properties of Lys460Ter-ANGPTL6, we established stable cell lines expressing similar levels of the wild-type (WT-ANGPTL6) and mutated (Lys460Ter-ANGPTL6) transcripts, respectively (FIG. 2A). Western blot using anti-flag antibody showed that WT-ANGPTL6 was secreted in the culture medium while Lys460Ter-ANGPTL6 was almost not detected in the supernatant of cells transfected with the variant (FIG. 2A). Quantification of ANGPTL6 concentration by ELISA confirmed the significant reduction of the secretion of Lys460Ter-ANGPTL6 compared to WT-ANGPTL6 (FIG. 2B). In agreement with this defective secretion, immunofluorescence labeling and quantification in permeabilized cells clearly showed the retention of Lys460Ter-ANGPTL6 in the cytoplasm (FIG. 2A). Altogether, these data strongly suggest that the c.1378A>T ANGPTL6 variant leads to effective expression of the truncated Lys460Ter-ANGPTL6 protein, which is not secreted. Accordingly, heterozygous carriers for the c.1378A>T ANGPTL6 variant are expected to present with decreased levels of circulating ANGPTL6. To assess this hypothesis, we performed ELISA to compare the serum concentration of ANGPTL6 in subjects from family A reported as homozygous for the WT-ANGPTL6 (n=5) versus heterozygous for the c.1378A>T ANGPTL6 (n=7), and found a 50% reduction in the serum level of ANGPTL6 in heterozygous carriers (FIG. 2B).

[0109] Enrichment in Rare Coding Variants within ANGPTL6 Among IA Carriers

[0110] We then extended genetic screening on the coding portion of ANGPTL6 to 94 additional index cases with familial IA. We identified 5 additional individuals carrying rare, non-synonymous variants in ANGPTL6 predicted as damaging in silico by PolyPhen-2 and/or SIFT (data not shown): two cases with the same missense mutation in exon 1 leading to the p.Glu131Val substitution in ANGPTL6, one case with a missense mutation in exon 4 leading to the p.Leu348Phe substitution, and two cases carrying the same CGCGCTGAGCCTCGGCGGA-bp (SEQ ID NO: 1) insertion leading to one premature STOP codon in exon 2 (p.Ala153ValfsTer66). By ELISA, we found no reduction in the serum concentration of ANGPTL6 between p.Glu131Val heterozygous carriers versus non carriers (data not shown).

[0111] Overall, from the 6 index cases, family screening led to the identification of 16 relatives with diagnosed IA (FIG. 1 and FIG. 3). Out of the 13 family members carrying IA and agreeing to participate in genetic research, 12 (92%) carry rare coding variants in ANGPTL6, versus 15 out of 41 (34%) unaffected ones. The only affected individual who does not carry any rare coding variant in ANGPTL6 is a 54 year-old male (III-5, family F, FIG. 3) presenting with an aneurysm on the anterior communicant artery, with no reported history of smoking, high blood pressure or any relevant associated disease.

[0112] The clinical characteristics of the remaining 12 cases are studied. Seven of them (58%) carry multiple IA (with a maximum of three). IA is located on the middle cerebral artery bifurcation in 7 cases (58%), on the anterior communicant artery, the anterior cerebral artery and the internal carotid artery in 3 cases (25%), and on the posterior communicant artery in 2 cases (17%).

[0113] To further test the association of ANGPTL6 rare variants with susceptibility to familial IA, we also compared the proportions of individuals carrying at least one rare, non-synonymous variant across this gene among the 95 index cases enrolled in the present study (6/95; 6.32%) versus 404 healthy individuals with French ancestry (8/404; 1.98%). We found a significant enrichment in carriers of non-synonymous variants with an MAF below 1% in the NFE reference population, among IA cases (SKAT, p=0.023). Similar results were found when comparing allele counts among the 95 index cases versus the 7,509 Non-Finnish European individuals with whole-genome sequences available in the gnomAD database (data not shown).

Example 2: Assessment of the Cerebral Vascular Phenotype of K447*Angplt6 Mice

[0114] Material & Methods

[0115] Angptl6 domains and sequence are highly conversed between humans and mice. K460 in the human Angptl6 sequence corresponds to K447 in the mouse sequence and the K447*Angplt6 mouse mutant also lacks the last 11 C-terminal residues. To assess the causal link between this Angptl6 variant and IA, inventors have generated a mouse model expressing the truncated form of Angptl6, analogue to the human mutation. The point mutation has been introduced into the Angptl6 gene sequence by homolog recombination. Mice express the K447*Angptl6 protein instead of the wild-type Angptl6 but the expression pattern and its regulation are not modified. Cerebral vasculature of heterozygous (Angpl6+/) and homozygous (Angptl6/) mice have been analyzed and compared with control mice (Angpl6+/+).

[0116] Results:

[0117] Inventors have observed: (i) an increased diameter of cerebral arteries of Angptl6 mutant mice compared to controls ex vivo (FIG. 4); (ii) with Micro-computed tomography (CT) imaging of cerebral arteries in Angptl6+/+ and Angptl6 mutant mice showing increased arterial diameter in the mutant mice at normal systolic blood pressure (SBP: 104.72.9 mm Hg in Angptl6/ mice and 103.14.2 mm Hg in Angpl6+/; FIG. 5 (left)); (iii) a defect in the adaptation to high blood pressure leading to a dilation of cerebral arteries (FIG. 5 (right); SBP: 127.64.4 mm Hg in Angptl6/ mice, 122.94.5 mm Hg in Angpl6+/ and 117.56.7 mm Hg in Angpl6 A/A), and (iv) reduced NO-dependent relaxation in response to flow (FIG. 6).

[0118] These results suggest that expression of the K447*Angptl6 variant leads to structural and functional defects of cerebral arteries, including endothelial dysfunction.

REFERENCES

[0119] Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

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