Patent Grant
9580753
This application includes an electronically submitted sequence listing in .txt format. The .txt file contains a sequence listing entitled “2016-10-14 0760-0440PUS1 ST25.txt” created on Oct. 14, 2016 and is 113,239 bytes in size. The sequence listing contained in this .txt file is part of the specification and is hereby incorporated by reference herein in its entirety.
The present invention relates to a method for predicting porencephaly and/or cerebral hemorrhage.
Porencephaly is a congenital disorder in which a cyst or cavity communicating with the cerebral ventricle is found in the cerebral hemisphere (Non-patent Document 1), and assumed to be caused by a disturbance of vascular supply such as infarction or hemorrhage during the fetal period (Non-patent Documents 2 and 3). Clinically, porencephaly causes hemiplegia (most often), quadriplegia, epilepsy, and intellectual disability (Non-patent Documents 4 and 5). Delivery of monozygous twins, cardiac arrest or abdominal trauma of the mother, a deficient protein C anticoagulant pathway, and cytomegalovirus infection are risk factors for sporadic porencephaly (Non-patent Documents 2 and 6).
In recent years, mutations in the gene encoding the α1 chain of type IV collagen (COL4A1, MIM 120130) were reported to be responsible for familial porencephaly (Non-patent Document 7). After that, de novo mutations in the COL4A1 gene were also reported in a sporadic case (Non-patent Documents 8 to 10), confirming involvement of abnormality of the COL4A1 gene in both sporadic and familial porencephalies. However, there still remain many cases in which no mutation in the COL4A1 gene can be identified.
The present invention aims to identify a novel causative gene for porencephaly and to provide a novel means that can be used for prevention of cerebral hemorrhage during the fetal period to perinatal period.
The present inventors focused on COL4A2 protein, which forms a heterotrimer with COL4A1 protein, and intensively screened for COL4A2 mutations in 35 Japanese patients with porencephaly. As a result, the present inventors successfully identified heterozygous mutations in 2 patients. Two mutations were not found in populations of healthy Japanese individuals, and their pathogenicity was strongly suggested by evaluation using pathogenicity prediction tools. One of the 2 patients represented a sporadic case, and the other represented a familial case. That is, the present inventors discovered that the COL4A2 gene is a causative gene for both familial and sporadic porencephalies, thereby completing the present invention.
That is, the present invention provides a method for predicting risk of porencephaly and/or cerebral hemorrhage, which is carried out for a sample separated from a living body, said method comprising investigating whether or not at least one mutation is present in the COL4A2 gene in a subject living body, wherein, in the case where at least one mutation is present in at least one allele of the COL4A2 gene, high risk of porencephaly and/or cerebral hemorrhage is predicted.
By the present invention, the COL4A2 gene was identified as a causative gene for porencephaly for the first time, and a novel method for predicting the risk of porencephaly and/or cerebral hemorrhage, especially porencephaly and/or cerebral hemorrhage during the fetal period to perinatal period, was provided. Since an identical heterozygous mutation of the COL4A2 gene was found in both a porencephaly patient and healthy individuals, this pathogenic mutation is considered to be dominantly inherited with incomplete penetrance. In cases where a COL4A2 mutation is found in at least one of the parents of a fetus, the COL4A2 mutation might be inherited to the fetus. It is also possible to investigate whether or not the COL4A2 mutation is present in the fetus itself by prenatal diagnosis. In cases where there is a concern about the risk of occurrence of porencephaly or cerebral hemorrhage during the fetal period to perinatal period, perinatal cerebral hemorrhage can be prevented by avoiding vaginal delivery, and positively selecting cesarean section, which is less likely to cause physical damages to the fetus. Further, since the COL4A2 gene is a gene associated with fragility of blood vessels, it is thought that healthy carriers have higher risk of hemorrhagic cerebrovascular diseases than healthy non-carriers. Therefore, healthy carriers should place emphasis on prevention of hemorrhagic cerebrovascular diseases. Thus, the present invention can also contribute to prevention of cerebral hemorrhage in adults.
Evolutionarily conserved amino acids are highlighted with gray or black boxes in the figure. Each black box indicates a Gly residue that showed a mutation. The respective amino acid sequences were obtained from the NCBI protein database: NP_001837.2 (Homo sapiens)(SEQ ID NOS: 131 and 132), NP_034062.3 (Mus musculus) (SEQ ID NOS: 133 and 134), NP_001155862.1 (Gallus gallus), (SEQ ID NOS: 135 and 136), XP_002933063.1 (Xenopus tropicalis) (SEQ ID NOS: 137 and 138), XP_687811.5 (Danio rerio) (SEQ ID NOS: 139 and 140), AAB64082.1 (Drosophila melanogaster) (SEQ ID NOS: 141 and 142), and CAA80537.1 (Caenorhabditis elegans) (SEQ ID NOS: 143 and 144). The alignment was performed with CLUSTAL W as shown in the website of clustalw.ddbj.nig.ac.jp.
The COL4A2 gene (MIM 120090), identified as a novel causative gene for porencephaly by the present inventors, encodes the α2 chain of type IV collagen. Type IV collagen is a basement membrane protein expressed in all tissues including the vasculature. Among type IV collagens, the most abundant collagens are COL4A1 (α1 chain) and COL4A2 (α2 chain), and these are known to form a heterotrimer (α1α1α2) at a ratio of 2:1 (Khoshnoodi, J., Pedchenko, V., and Hudson, B. G. (2008). Mammalian collagen IV. Microsc Res Tech 71, 357-370.). In the domain that forms the heterotrimer, there are Gly-Xaa-Yaa repeats (wherein Xaa and Yaa represent the same or different arbitrary amino acids), and a triple-helix structure is formed in this repeat region. The positions of the Gly-Xaa-Yaa repeats are indicated by underlines in
In the present invention, mutations in the COL4A2 gene are used as indices for predicting the risk of occurrence of porencephaly and/or cerebral hemorrhage in a subject living body. The subject living body is preferably a postnatal human (for example, human adult) or human fetus. The cerebral hemorrhage includes cerebral hemorrhage during the fetal period to perinatal period, and hemorrhagic cerebrovascular diseases that occur in adulthood (including old age). In cases where at least one mutation is present in at least one of the alleles of the COL4A2 gene, high risk of porencephaly and cerebral hemorrhage can be predicted. A heterozygous mutation has been found in both a porencephaly patient and healthy carriers, indicating that the mode of heredity is dominant inheritance with incomplete penetrance.
The mutations in the COL4A2 gene used as indices in the present invention include changes in the base sequence that cause changes in a very small number of amino acids in the α2 chain of type IV collagen, which is encoded by the COL4A2 gene, or those that cause deletion of at least a partial region in the α2 chain. The mutations also include mutations that cause deletion of all or part of the COL4A2 gene region. Specific examples of such mutations of the base sequence include missense mutations, nonsense mutations, frameshift mutations, in-frame deletion or insertion mutations (which causes deletion or insertion of one or more amino acids) due to substitution, deletion, insertion, duplication and/or the like of a base(s) in an exon and/or intron region(s); mutations that cause abnormal splicing; and microdeletions of the chromosomal region containing the COL4A2 gene.
Mutations in the COL4A2 gene can be detected by analyzing the base sequence using a nucleic acid sample such as genomic DNA or RNA. In particular, analysis of a genomic sequence using a genomic DNA sample is desirable since such analysis is most accurate. The nucleic acid sample such as genomic DNA can be easily prepared from peripheral blood, a swab of oral mucosa or the like by a conventional method. Various prenatal genetic testing methods are known, and it is also possible to investigate whether a fetus has a mutation in the COL4A2 gene or not. Examples of the various known methods include a method in which cells are collected from the fetus (using amniotic fluid, villi or cord blood), a noninvasive test method in which a genetic mutation of the fetus is tested using fetal cells present in maternal blood, and a method in which a single cell of the fertilized egg obtained by external fertilization is used (preimplantation diagnosis). In the noninvasive test method, the maternal blood sample containing fetal cells corresponds to the “sample separated from a living body”, and the fetus corresponds to the “subject living body”.
Although the amino acid sequence of a protein may be influenced by mutations in not only exon regions but also intron regions, each exonic sequences and its adjacent ten to several hundred bases such as about 30 to 50 bases of the intron region are commonly tested in usual genetic testing. Also in the present invention, each exon and its adjacent intron may be sequenced. When detection of mutations is carried out by analysis of a genomic sequence, sequencing may be carried out by a normal method using a genomic DNA sample with primers designed as appropriate by reference to SEQ ID NOs:4 to 38 of the present application or genomic sequence of the COL4A2 gene available from known databases. By determining the base sequence of the COL4A2 gene on the genomic DNA of the subject living body and comparing the determined sequence with a wild-type sequence, a mutation(s) can be identified in detail. Detection of the mutation(s) and profiling of the determined base sequence can be easily carried out by analysis using known software such as SeqScape (registered trademark).
Whether a mutation is homozygous or heterozygous can be confirmed with the waveform data obtained by sequencing. In the case where a heterozygous mutation is present, 2 types of signals overlap with each other at the same position.
Since COL4A2 gene mutation(s) to be detected in the present invention is/are mainly heterozygous, the screening of COL4A2 gene mutations can be effectively carried out by detection of heteroduplexes. If a heterozygous mutation is present, heat denaturation of the genomic DNA sample followed by reassociation produces heteroduplexes by hybridization between the normal-type DNA and the mutant-type DNA. The heteroduplexes have properties including the followings: (1) heteroduplexes show a different mobility in nondenaturing polyacrylamide gel; (2) mismatched bases are more susceptible to cleavage by chemical substances and enzymes; (3) heteroduplexes show a different melting temperature upon denaturation. Methods for detecting heteroduplexes utilizing these properties are known in the art, and practically used as test methods for mutations. More specifically, examples of the known methods include a method in which heteroduplexes are detected by denaturing high-performance liquid chromatography (dHPLC), and the High Resolution Melt method.
The High Resolution Melt method is a method in which the process of melting of double-stranded DNA (heat denaturation) is detected as a change in the fluorescence intensity using a fluorescent dye that binds to double-stranded DNA at high density (e.g., SYTO (registered trademark) 9, LC Green (registered trademark), or EvaGreen (registered trademark)), thereby detecting heteroduplexes. That is, when double-stranded DNA stained with a fluorescent dye that binds to double-stranded DNA at high density is melted (heat-denatured), the fluorescent dye drops from the portion where dissociation of the double strand occurred, which results in a decrease in the fluorescence signal from the double-stranded DNA. Therefore, by using such a fluorescent dye, the process of heat denaturation of double-stranded DNA can be visually detected as a change in the fluorescence intensity. By obtaining and analyzing temperature-fluorescence data at high density, detection of heteroduplexes can be carried out rapidly and highly sensitively. This can be easily carried out using a commercially available device and kit and the like. The primers used can be designed as appropriate based on the sequence of each exon+adjacent intron region in the COL4A2 gene described in SEQUENCE LISTING of the present application. In the Examples below, examples of primers and reaction conditions that can be used for screening of COL4A2 gene mutations by the High Resolution Melt method are shown.
In the present invention, the presence/absence of a mutation may be determined by sequencing all the exon+adjacent intron regions in the COL4A2 gene. Alternatively, for example, detection of heteroduplexes may be carried out to narrow down the regions to be sequenced, and thereafter the target regions may be sequenced, thereby carrying out the testing more effectively.
The cDNA sequence and genomic sequence of the COL4A2 gene and the amino acid sequence of the COL4A2 protein encoded thereby shown in SEQUENCE LISTING are typical examples of normal COL4A2 sequences. In the present invention, the presence/absence of a mutation can be judged by using the COL4A2 gene sequence shown in SEQUENCE LISTING as a reference and performing comparison with this reference sequence. A mutation of the COL4A2 gene that causes alteration of the amino acid sequence can be regarded as a pathogenic mutation for porencephaly and cerebral hemorrhage. In particular, a gene mutation that alters an amino acid that is evolutionarily highly conserved is highly likely to produce a COL4A2 protein whose normal function is deteriorated, for example, a COL4A2 protein that cannot qualitatively or quantitatively form the normal α1α1α2 heterotrimer, and such a gene mutation is therefore a typical example of the pathogenic mutation for porencephaly and cerebral hemorrhage. Sequences of COL4A2 protein (type IV collagen α2 chain) of various animals are known, and deposited in databases such as GenBank. Therefore, those skilled in the art can easily obtain the sequence information, and investigate evolutionary conservation of each amino acid by a conventional method. Representative examples of the mutation of an evolutionarily conserved amino acid residue include mutations that substitute Gly in the Gly-Xaa-Yaa repeats (wherein Xaa and Yaa represent the same or different arbitrary amino acids), which are the triple helix domain of the heterotrimer. Further, also in cases where the detected base mutation is a mutation that is not found in populations of many healthy individuals or a mutation that has not been deposited in well-known databases related to diversity of base sequences such as dbSNP by NCBI or 1000 Genomes Project, the mutation can be regarded as a pathogenic mutation that can be used as an index in the present invention.
Various prediction tools with which whether a mutation in a gene is a pathogenic mutation or not can be investigated are known. Examples of such tools include SIFT (http://sift.jcvi.org/), PolyPhen (http://genetics.bwh.harvard.edu/pph/), PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/), Mutation Taster (http://neurocore.charite.de/MutationTaster/index.html) and Align GVGD (http://agvgd.iarc.fr/agvgd_input.php). In cases where a mutation of the COL4A2 gene has been detected by carrying out the method of the present invention and whether the mutation is pathogenic or not is uncertain, such a prediction tool may be used to judge whether the mutation is pathogenic or not. In SIFT, a substitution is predicted to be intolerant (having influence on a protein functional change) when the score is less than 0.05. In PolyPhen, pathogenicity is predicted when the score exceeds 2.0. In PolyPhen-2, the score ranges from 0.000 (most probably benign) to 0.999 (most probably damaging), and when the judgment based on the score is possibly or probably damaging, the mutation is strongly suggested to be pathogenic. In Align GVGD, the class score is evaluated within the range of Class C0 (less likely) to Class C65 (most likely), and a COL4A2 mutation with a class score of C55 or higher is suggested to be a pathogenic mutation.
The mutations shown in Table 2 are two kinds of pathogenic mutations for porencephaly and cerebral hemorrhage, which were identified in two unrelated pedigrees in Examples. All of these mutations are substitution mutations in evolutionarily conserved Gly residues in the Gly-Xaa-Yaa repeats, and not found in the population of many Japanese healthy individuals. These mutations were strongly suggested to be pathogenic based on evaluation using the above-described prediction tools. However, these two kinds of mutations are mere examples of COL4A2 gene mutations that can be used as indices in the present invention, and, of course, pedigrees other than these two pedigrees may have different pathogenic mutations. Therefore, the scope of the present invention is not limited to these specific examples.
In cases where one or more COL4A2 gene mutations are found in at least one of the parents, the mutation might be inherited to the fetus. Therefore, the risk of occurrence of porencephaly and/or cerebral hemorrhage during the fetal period to perinatal period in the fetus can be predicted to be higher than usual. Since a method of prenatal diagnosis in which a gene of a fetus is investigated is known and already being practically used, whether or not the fetus itself actually has a COL4A2 gene mutation that has been inherited from a parent or occurred de novo may be investigated, if desired. In cases where there is a concern about the risk of occurrence of porencephaly and/or cerebral hemorrhage during the fetal period to perinatal period, vaginal delivery may give physical damage to the fetus to cause cerebral hemorrhage. Therefore, positive selection of cesarean section is effective for avoiding cerebral hemorrhage during the perinatal period. Thus, the present invention can be utilized for selecting a safe delivery method.
In cases where a COL4A2 gene mutation was found in a postnatal healthy subject living body, the subject living body is considered to have higher risk of cerebral hemorrhage than a healthy individual who does not have the COL4A2 gene mutation, since the COL4A2 gene is a gene associated with fragility of blood vessels. In such cases, emphasis should be placed on prevention of hemorrhagic cerebrovascular diseases by, for example, paying sufficient attention to the lifestyle and the dietary life. The present invention can also be utilized for prevention of cerebral hemorrhage in adults.
The present invention is described below in more detail by way of Examples. However, the present invention is not limited to the Examples below.
As a result of screening of COL4A2 gene mutations in 35 Japanese patients with porencephaly, substitution of a Gly residue in the Gly-Xaa-Yaa repeat was identified in two patients (patients 1 and 2). Clinical information and peripheral blood samples were obtained from their family members after obtaining written informed consent. Experimental protocols were approved by the Institutional Review Board of Yokohama City University School of Medicine.
Patient 1 is seven years old and was born to non-consanguineous healthy parents (
Patient 2 is one year and four months old and was born to non-consanguineous healthy parents (
Genomic DNA was isolated from peripheral blood leukocytes according to standard methods. DNA for mutation screening was amplified with illustra GenomiPhi V2 DNA Amplification Kit (GE Healthcare, Buckinghamshire, UK). DNA of familial members of patient 1 was isolated from saliva samples using Oragene (DNA Genotek Inc., Ontario, Canada).
Exons 2 to 48 covering the entire COL4A2 gene coding region (GenBank accession number NM_001846.2) were examined by high-resolution melting curve (HRM) analysis or direct sequencing (for exon 46). Real-time PCR and subsequent High resolution melting analysis were carried out in a 12-μl reaction system using RoterGene-6000 (Corbett Life Science). The composition of the reaction liquid was as follows: for exons 2/3/7/13/24/42/46/47/48, 30 ng of DNA, 0.3 μM each primer, 0.4 mM each dNTP, 1.5 μM SYTO9, 1×PCR Buffer for KOD FX, and 0.3 U KOD FX polymerase; and for the other exons, 30 ng of DNA, 0.25 μM each primer, 1.5 μM SYTO9, and 1×HotStarTaq-plus mastermix. The PCR primers and reaction conditions used for the HRM and sequencing are shown in Table 3.
Samples showing aberrant melting curve patterns in the HRM analysis were sequenced. The PCR products were purified with ExoSAP-IT (GE healthcare), and cycle sequencing reaction was carried out using BigDye Terminator chemistry version 3 (Applied Biosystems). The reaction products were purified by gel filtration using Sephadex G-50 (GE healthcare) and Multiscreen-96 (Millipore), and sequences were obtained with ABI Genetic Analyzer 3100 (Applied Biosystems). The obtained sequences were subjected to analysis of the presence/absence of a mutation using SeqScape version 2.1.1 software (Applied Biosystems). The sequences of samples in which a mutation was found were subjected to sequence analysis again using the genomic DNA as a template to confirm the mutation in the genomic DNA.
As a result, two heterozygous mutations, c.3455G>A (p.G1152D) in the patient 1 and c.3110G>A (p.G1037E) in the patients 2, were identified. Both mutations were found at evolutionarily conserved Gly residues in the Gly-X-Y repeats (
The following tools were used for the prediction.
Scores less than 0.05 indicate substitutions are considered to be intolerant (a protein functional change is affected).
Scores more than 2.0 are considered to be pathogenic.
The score ranges from 0.000 (most probably benign) to 0.999 (most probably damaging).
From Class C0 (less likely) to Class C65 (most likely).
The c.3455G>A mutation of the patient 1 was also found in the patient's mother and maternal grandfather, both of whom are asymptomatic, and in his maternal uncle who showed congenital left hemiplegia (
| Number | Date | Country | Kind |
|---|---|---|---|
| 2011-247457 | Nov 2011 | JP | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2012/077903 | 10/29/2012 | WO | 00 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2013/069495 | 5/16/2013 | WO | A |
| Entry |
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| Number | Date | Country | |
|---|---|---|---|
| 20140315208 A1 | Oct 2014 | US |
