# Patent

## Title
Method for predicting risk of porencephaly or cerebral hemorrhage

## Abstract

As a result of intensive screening on mutations of the COL4A2 gene in 35 Japanese patients with porencephaly, it was found that the COL4A2 gene is a causative gene for familial and sporadic porencephalies. 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. It can be predicted that a living body having a COL4A2 gene mutation has a high risk of occurrence of porencephaly and/or cerebral hemorrhage.

## Background
REFERENCE TO SEQUENCE LISTING SUBMITTED VIA EFS-WEB
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.
TECHNICAL FIELD
The present invention relates to a method for predicting porencephaly and/or cerebral hemorrhage.
BACKGROUND ART
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.
PRIOR ART DOCUMENTS
Non-Patent Documents


    Non-patent Document 1: Berg, R. A., Aleck, K. A., and Kaplan, A. M. (1983). Familial porencephaly. Arch. Neurol. 40, 567-569.
    Non-patent Document 2: Govaert, P. (2009). Prenatal stroke. Semin Fetal Neonatal Med 14, 250-266.
    Non-patent Document 3: Hunter, A. (2006). Porencephaly. In Human Malformations and related Anomalies, S. R E and H. J G, eds. (New York, Oxford University Press), pp 645-654.
    Non-patent Document 4: Mancini, G. M., de Coo, I. F., Lequin, M. H., and Arts, W. F. (2004). Hereditary porencephaly: clinical and MRI findings in two Dutch families. Eur J Paediatr Neurol 8, 45-54.
    Non-patent Document 5: Vilain, C., Van Regemorter, N., Verloes, A., David, P., and Van Bogaert, P. (2002). Neuroimaging fails to identify asymptomatic carriers of familial porencephaly. Am J Med Genet 112, 198-202.
    Non-patent Document 6: Moinuddin, A., McKinstry, R. C., Martin, K. A., and Neil, J. J. (2003). Intracranial hemorrhage progressing to porencephaly as a result of congenitally acquired cytomegalovirus infection—an illustrative report. Prenat Diagn 23, 797-800.
    Non-patent Document 7: Gould, D. B., Phalan, F. C., Breedveld, G. J., van Mil, S. E., Smith, R. S., Schimenti, J. C., Aguglia, U., van der Knaap, M. S., Heutink, P., and John, S. W. (2005). Mutations in Col4a1 cause perinatal cerebral hemorrhage and porencephaly. Science 308, 1167-1171.
    Non-patent Document 8: Breedveld, G., de Coo, I. F., Lequin, M. H., Arts, W. F., Heutink, P., Gould, D. B., John, S. W., Oostra, B., and Mancini, G. M. (2006). Novel mutations in three families confirm a major role of COL4A1 in hereditary porencephaly. J Med Genet 43, 490-495.
    Non-patent Document 9: Lanfranconi, S., and Markus, H. S. (2010). COL4A1 mutations as a monogenic cause of cerebral small vessel disease: a systematic review. Stroke 41, e513-518.
    Non-patent Document 10: Meuwissen, M. E., de Vries, L. S., Verbeek, H. A., Lequin, M. H., Govaert, P. P., Schot, R., Cowan, F. M., Hennekam, R., Rizzu, P., Verheijen, F. W., et al. (2011). Sporadic COL4A1 mutations with extensive prenatal porencephaly resembling hydranencephaly. Neurology 76, 844-846.


SUMMARY OF THE INVENTION
Problems to be Solved by the Invention
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.
Means for Solving the Problems
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.
EFFECT OF THE INVENTION
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.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A Pedigree of patient 1 with porencephaly, in whom the c.3455G>A (p.G1152D) mutation was identified in the COL4A2 gene. The arrow indicates the patient 1. A maternal uncle (III-1) of the patient had congenital left hemiplegia. The patient's mother (III-2) and maternal grandfather (II-7) were both healthy. The elder granduncle (II-5) was also afflicted by congenital hemiplegia and died in his 60s. FIG. 1B Pedigree of patient 2 with porencephaly, in whom the c.3110G>A (p.G1037E) mutation was identified in the COL4A2 gene. The arrow indicates the patient 2. His parents did not have this mutation, indicating that the mutation occurred de novo. FIG. 1C Electropherogram of the genomic sequence of the mutation. The left panel shows data from the patient 1 and his parents, and the right panel shows data from the patient 2 and his parents. FIG. 1D Amino acid sequence alignments of the COL4A2 protein.
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.
FIG. 2 (A-C) Brain MRIs of the patient 1 at 6 years old. (A) T2-weighted cross-sectional image. (B) Coronal image. The images in (A) and (B) show an enlarged right lateral ventricle and reduced volume of the right frontal white matter. (C) T1 weighted midline sagittal image showing atrophy of the body of corpus callosum (arrowheads). The lesion responsible for the left leg paresis is not evident in these images. (D-F) CT images of the patient 2 at two months of age. (D) Cross-sectional image. (E) Coronal image. (F) Sagittal image. The images in (D), (E) and (F) show bilateral enlargement of the lateral ventricle and extremely reduced volume of bilateral frontal white matter. The V-P shunt is also visible in the right lateral ventricle. The pontocerebellar structures seem to be normal.
FIG. 3 Amino acid sequence of the α2 chain of type IV collagen (SEQ ID NO: 2), which is encoded by the COL4A2 gene. The underlined portions are Gly-Xaa-Yaa repeat regions. Black boxes indicate the Gly residues involved in the 2 kinds of amino acid substitution mutations identified in Examples.



BEST MODE FOR CARRYING OUT THE INVENTION
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 FIG. 3. SEQ ID NOs: 1 and 2 in SEQUENCE LISTING are a sequence of the coding region in cDNA of the COL4A2 gene, and an amino acid sequence of COL4A2 protein, respectively. SEQ ID NO:3 is a mRNA sequence deposited in GenBank (accession number NM_001846). In SEQ ID NOs 4 to 38, sequences of exons and their proximal introns are shown as listed in Table 1.










TABLE 1







SEQ ID NO:
Exon




















Ex1
4
101-345
nt (UTR 101-345 nt)



Ex2
4
677-764
nt (UTR 677-720 nt)



Ex3
4
886-940
nt



Ex4
5
301-381
nt



Ex5
6
301-435
nt



Ex6
6
520-564
nt



Ex7
7
301-417
nt



Ex8
8
301-372
nt



Ex9
8
817-852
nt



Ex10
8
961-1023
nt



Ex11
9
301-336
nt



Ex12
10
301-342
nt



Ex13
11
301-399
nt



Ex14
12
301-336
nt



Ex15
12
917-967
nt



Ex16
13
301-345
nt



Ex17
14
301-354
nt



Ex18
15
301-367
nt



Ex19
16
301-411
nt



Ex20
16
927-1076
nt



Ex21
17
301-393
nt



Ex22
18
301-464
nt



Ex23
19
301-373
nt



Ex24
19
465-571
nt



Ex25
20
301-502
nt



Ex26
20
899-958
nt



Ex27
21
301-357
nt



Ex28
22
301-408
nt



Ex29
23
301-522
nt



Ex30
24
301-462
nt



Ex31
25
301-471
nt



Ex32
26
301-444
nt



Ex33
27
301-423
nt



Ex34
28
301-482
nt



Ex35
29
301-364
nt



Ex36
29
567-641
nt



Ex37
30
301-408
nt



Ex38
31
301-408
nt



Ex39
32
301-372
nt



Ex40
33
301-426
nt



Ex41
34
301-417
nt



Ex42
35
301-462
nt



Ex43
35
563-661
nt



Ex44
35
1027-1173
nt



Ex45
35
1328-1444
nt



Ex46
36
301-492
nt



Ex47
37
301-587
nt



Ex48
38
301-1163
nt (UTR 329-1163 nt)









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.







TABLE 2







COL4A2 Gene Mutations










DNA
Amino acid

Specification of mutation site in


mutation
mutation
Exon
SEQUENCE LISTING





c.3455G > A
G→D at
38
Position 3455 in SEQ ID NO: 1



position 1152

Position 301 in SEQ ID NO: 31


c.3110G > A
G→E at
34
Position 3110 in SEQ ID NO: 1



position 1037

Position 385 in SEQ ID NO: 28









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.
EXAMPLES
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 (FIG. 1A, arrow). He was born at 36 weeks' gestation with a planned Caesarean section because, at 31 weeks' gestation, an antenatal ultrasound scan revealed an enlarged right lateral ventricle. Apgar scores were 9 at 1 minute and 10 at 5 minutes. He weighed 2,900 g (+1.09 SD) and had a head circumference of 32.5 cm (+0.05 SD). His early development was delayed with poor left hand use and abnormal leg movement. Brain MRI at 6 months showed an enlarged right lateral ventricle. Abrupt vomiting and nausea followed by motionless arrest developed at the age of 10 months. An electroencephalogram (EEG) showed focal spikes in the right frontal region, and carbamazepine treatment was initiated at the age of 12 months. Rehabilitation was started at 10 months of age. The patient started rolling at 12 months, crawling at 18 months, and walking alone at 3 years of age. He had spastic triplegia (diplegia and left hemiplegia) showing hemiplegic and diplegic gait with fluent speech and normal word comprehension. At the age of 5 years, he underwent orthopedic surgery for foot deformity due to spastic paresis. An EEG showed spikes in the right occipital to posterior temporal region and mid central region. Brain MRI at 6 years showed enlarged right lateral ventricle, reduced volume of the right frontal white matter and atrophic right cerebral peduncle and body of corpus callosum (FIG. 2A-C). His IQ score, evaluated at 6 years with WISC-III, was 74 (performance IQ: 69 and verbal IQ: 82). The patient 1 is now 7 years old and attending a local school. He can walk with ankle foot orthosis and hand assist. The epilepsy is well controlled with carbamazepine and clobazam. Of note, his maternal elder uncle showed congenital left hemiplegia with assisted walk, and his maternal granduncle had also been afflicted by congenital hemiplegia, suggesting a genetic predisposition in the family (FIG. 1A).
Patient 2 is one year and four months old and was born to non-consanguineous healthy parents (FIG. 1B, arrow). He was born at 35 weeks' gestation. His birth weight was 1,694 g ( ̃2.36 SD) and his head circumference was 29 cm (−1.77 SD). Mild asphyxia was observed with Apgar scores of 3 at 1 minute and 7 at 5 minutes. An ultrasound scan at 6 hours after birth revealed parenchymal hemorrhage of the right cerebral hemisphere with an enlarged left lateral ventricle. Because a blood test revealed no increase in D-dimer but revealed significant increases in prothrombin time (29.3 sec) and activated partial thromboplastin time (104.3 sec), he was treated with a daily infusion of fresh frozen plasma for 12 days. At 37 days after birth, he underwent a ventricular-peritoneal shunt (V-P shunt) operation because of progressive enlargement of the lateral ventricle. CT at two months of age showed an enlarged bilateral lateral ventricle and an extremely reduced volume of bilateral frontal white matter (FIG. 2D-F). Blood coagulation was normalized at 7 months of age. At the age of 7 months, the patient did not show any head control or rolling, and presented with abnormal posturing and spastic quadriplegia dominant on the left side of his body. With rehabilitation, he had full-range visual pursuit, social smile and incomplete head control. Although his spasticity improved, exaggerated deep tendon reflexes with synergic voluntary movement of the distal part of extremities were recognized. An EEG at one year of age showed no epileptic discharges. His present developmental quotient is below 20. The elder sister of patient 2 was found to have intraventricular hemorrhage two days after birth and underwent a V-P shunt. Her development was almost normal, and internal strabismus was noted. Unfortunately, she died in an accident at the age of four, and so her DNA sample was unavailable (FIG. 1B).
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.











TABLE 3






Primer sequence (5′ > 3′)
SEQ





Upper line, forward; lower line,
ID
Amplification
PCR


Exon
reverse
NO:
size (bp)
conditions**



















Ext
ATGGGCTGCCTCCCTCATCCT
39
202
KOD-FX



GAGAGTTACACCGAAGGGTCCATGC
40

2 step


 


Ex3
GCATGGACCCTTCGGTGTAACTCTC
41
198
KOD-FX



CCACTCAAACGTCCCAACCACTCTC
42

2 step


 


Ex4
TTGGAAGGATTCTCAACAGATG
43
230
HotStar



AGCGAGGCATGACTGTATGA
44




 


Ex5 &
TCGTGGAAATTGAACCTTTG
45
344
HotStar


6
CCTAGGATGCACGCAATGTT
46




 


Ex7
GCCGGGAACATGGCTTATGAGAATA
47
332
KOD-FX



GTTATGCTTCCGTTCTGGCCACAGT
48

2 step


 


Ex8
CTGCACCGAATGTTAATGGA
49
269
HotStar



GATTATGCCGCCATTCTAGG
50




 


Ex9 &
GGGCTGATCTGTTTGATATGC
51
343
HotStar


10
CCAGAGTGGGCACCTGTGT
52




 


Ex11
CAGAAACCTCCATGCATCCT
53
230
HotStar



CAAACAAACCCACAAACACCT
54




 


Ex12
TTGCCGATAAATAGGCCTTG
55
201
HotStar



TTTCCTGGCTGAGAAATGCT
56




 


Ex13
TTTCCTTTCGATTTAAAGACAACTGC
57
233
KOD-FX



TGGAATGTGGTTGAATACAATTGAAGA
58

3 step


 


Ex14
CATGTCATGAACCCTGATTGA
59
231
HotStar



ATGAGAGACTGGCGGTGTG
60




 


Ex15
AGTCCTGGAGCAGAGGATGA
61
186
HotStar



AAACCAAACCAAACCGACAA
62




 


Ex16
CGTAGTCAAGCCCTCTGGAA
63
197
HotStar



TGAGATGCCAAGGCCTATTT
64




 


Ex17
TTTGGAGTTATACATCAGAGACAAAAA
65
192
Hot Star



GTGGGCGAGACACCATAAGT
66




 


Ex18
CTCGGGTTTCTTCTTTGGAA
67
223
HotStar



GCTCTGTGTCCCTAACAG GAG
68




 


Ex19
CTCATCAGGCCGCATACAG
69
288
HotStar



GACCTGAGTGCAGGTGCTTT
70




 


Ex20
TCTGGACACGAACACAAAGG
71
277
HotStar



CGGGCTTCATCTGAACATTA
72




 


Ex21
CCTGCATCTGTGGTTGTCTC
73
199
HotStar



GGGGATGGATTTCACCTTCT
74




 


Ex22
GCTAAGAGGAATGCGGAACA
75
260
HotStar



GGAGGCCTCAGAGTGTCTTG
76




 


Ex23
GCCAGCTGTGTGAGATGAAA
77
270
HotStar



GTCCCCGCTCACCTAGAAAG
78




 


Ex24
TCCAGAACAATCACAACCAAAGGTGA
79
286
KOD-FX



GGGTGTTTGGAGAACCTGAAGGATG
80

2 step


 


Ex25
GGAAGTCGAGGCGATCTTTA
81
325
HotStar



CAAAGGAAAGCGTGGAATGT
82




 


Ex26
CCCAGACGAGCCAGTAACTC
83
215
HotStar



TTATCCCACGCATACTGCAA
84




 


Ex27
TAGGATTGCTTGGGCTCATC
85
235
HotStar



TTTGTGCTGAGATGCTGGAC
86




 


Ex28
TTATCCTCGTGGAGCCTGAT
87
300
HotStar



CTCCCAAGGACAAATGCAAA
88




 


Ex29
CCATGCTAACTTGTGGTTTGG
89
314
HotStar



CACTGTGCATCTGGGATGG
90




 


Ex30
AGTGTGTGGAGGGAGATGCT
91
279
HotStar



GTGAGGACCCCACTCGTTTA
92




 


Ex31
TGTTTGTCCACCCTGTTTGA
93
291
HotStar



CCAGCAGAGCTGTCTCAGGT
94




 


Ex32
CGAAATGTTACGGAGACGTG
95
297
HotStar



TGCCACCAAGAAAGGGTAAG
96




 


Ex33
CAGGCCTTCACCTGTGTTCT
97
280
HotStar



GTCTCTGGGGACGGAGAAG
98

Step down


 


Ex34
CAGCACGTAGGACAGCAAAA
99
321
HotStar



GCTCACAGAACAAGGGGAGT
100




 


Ex35
ACAGCTAAGCAAACCGCCTA
101
287
HotStar



TCTGAATTGTGGACTCCCTGT
102




 


Ex36
TCCCAGTGGAAAGTCCTGTT
103
205
HotStar



TTGATCTGTTTGGCAAGTCG
104




 


Ex37
GAAGGAGCAGCAGTGTGGTT
105
285
HotStar



AATGTTGACCGCCTTTGTTC
106




 


Ex38
CCAGGACCTCACCACACAG
107
216
HotStar



ACTCTGGGTCTGGGTGACCA
108




 


Ex39
GCTGTCCCACACATGAAATAA
109
314
HotStar



ACACCTCTGCGTGGGACTC
110




 


Ex40
GCTGCCTCTGTTTCTTTGCT
111
295
HotStar



CTCTGGGTGGGTTCTGGTTA
112




 


Ex41
GCACCTCCCATCACTGTCTC
113
316
HotStar



CTACATTAAGCGGGCCATTG
114




 


Ex42
AGAGACTGTCGCCTGAATGGGTGAC
115
343
KOD-FX



GACGTTAGGGACACGAAAGTCTGTGG
116

2 step


 


Ex43
CTGGCCACAGTGAGAGGAG
117
272
HotStar



GACCCATGCCAGAGAGGAT
118

Step down


 


Ex44
ACTCGGAGCAAGAGAGTGGA
119
293
HotStar



GAACACAAGAGGACGCAATG
120




 


Ex45
CATTGCGTCCTCTTGTGTTC
121
248
HotStar



AGCACTAGGACCTGGGAAGG
122




 


Ex46
GGGCTGCTCTCTCTCTCTTT
123
586
KOD-FX



AACTTACCAGCCGTGGAGGGTTTG
124

2 step


 


Ex47-1*
GGCCCTCCAGTAGGTGGCTAAACTC
125
310
KOD-FX



GGCTGATGTAGGGCTTGATCTCGTC
126

2 step


 


Ex47-2*
TCCTGTACTGCAACCCTGGTGATGT
127
317
KOD-FX



CAAAGGCAGCTGTTCTTGCTGTGTC
128

2 step


 


Ex48
CAGGCTGTGATTCCTAACCCTGTCC
129
341
KOD-FX



GAATAAGCACCAAAATGGCCCTTC
130

Step down





*HRM and sequencing of exon 47 were carried out for 2 separate regions.


**The number of cycles of PCR was 35, and the reaction was carried out under the following conditions:


HotStar: 94° C. for 30 seconds-55 or 57° C. (exon 38) for 30 seconds-72° C. for 60 seconds


HotStar Step down: the annealing temperature was decreased by 1° C./cycle in the first 5 cycles (58° C.→54° C.)


KOD-FX 2 step: 98° C. for 10 seconds-68° C. for 30 seconds


KOD-FX 3 step: 98° C. for 10 seconds-64° C. for 30 seconds-68° C. for 30 seconds


KOD-FX Step down: the annealing temperature was decreased by 2° C. every 5 steps (72° C.→68° C.)


Enzyme used for PCR amplification: “KOD-FX” indicates use of KOD-Fx DNA polymerase (Toyobo Co., Ltd., Osaka, Japan), and ”HotStar⇄ indicates use of HotStarTaq (Qiagen).






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 (FIG. 1D), suggesting that the two mutations might alter the collagen IV α1α1α2 heterotrimers. These mutations were absent in 200 Japanese normal controls, and evaluation using web-based prediction tools strongly suggested that these substitutions are pathogenic (Table 4).













TABLE 4










Muta-



Pa-
Muta-



tion
Align


tient
tion
SIFT
PolyPhen
PolyPhen-2
taster
GVGD







1
c.3455G > A
0.00
probably
probably
Disease
C65



p.G1152D

damaging
damaging
causing






2.142
1.00




2
c.3110G > A
0.00
probably
probably
Disease
C65



p.G1037E

damaging
damaging
causing






2.367
1.00









The following tools were used for the prediction.

    (1) SIFT (http://sift.jcvi.org/)


Scores less than 0.05 indicate substitutions are considered to be intolerant (a protein functional change is affected).

    (2) PolyPhen (http://genetics.bwh.harvard.edu/pph/)


Scores more than 2.0 are considered to be pathogenic.

    (3) PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/)


The score ranges from 0.000 (most probably benign) to 0.999 (most probably damaging).

    (4) Mutation Taster (http://neurocore.charite.de/MutationTaster/index.html)
    (5) Align GVGD (http://agvgd.iarc.fr/agvgd_input.php)


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 (FIGS. 1A and B). Therefore, the c.3455G>A mutation can be considered to be a dominant pathogenic mutation with incomplete penetrance. The c.3110G>A mutation in the patient 2 was not found in his parents, indicating that this mutation occurred de novo (FIG. 1C).

## Claims

1. A method for diagnosing a risk of porencephaly and/or cerebral hemorrhage and treating porencephaly and/or cerebral hemorrhage, said method comprising:
(a) obtaining a sample from a in utero human fetus;
(b) detecting at least one mutation present in at least one allele of a COL4A2 gene in the sample;
(c) diagnosing the human fetus as having a high risk of porencephaly and/or cerebral hemorrhage after detecting the presence of at least one mutation in at least one allele of the COL4A2 gene; and
(d) administering cesarean section to the woman carrying the in utero human fetus diagnosed with a high risk of porencephaly and/or cerebral hemorrhage,
wherein said at least one mutation comprises at least one selected from the group consisting of (1) and (2) below:
(1) mutation of G at position 3455 of the COL4A2 gene coding region (position 301 of SEQ ID NO:31) to A; and
(2) mutation of G at position 3110 of the COL4A2 gene coding region (position 385 of SEQ ID NO:28) to A.

2. The method according to claim 1, wherein the detecting step is in a genomic sequence using a genomic DNA sample.

3. The method according to claim 1, wherein DNA in said sample is amplified prior to said detecting.

4. The method according to claim 3, wherein said DNA is amplified by PCR.

5. The method according to claim 3, wherein said DNA is amplified by PCR using at least one primer pair selected from the group consisting of primer pair SEQ ID NO: 99 and SEQ ID NO: 100 and primer pair SEQ ID NO: 107 and SEQ ID NO: 108.

6. A method for diagnosing a risk of porencephaly and/or cerebral hemorrhage, said method comprising:
(a) obtaining a sample separated from a subject;
(b) amplifying DNA using primer pairs;
(c) detecting the amplified sample of at least one mutation present in at least one allele of a COL4A2 gene in the sample; and
(d) diagnosing the subject as having a high risk of porencephaly and/or cerebral hemorrhage after detecting the presence of at least one mutation in at least one allele of the COL4A2 gene is detected,
wherein said at least one mutation comprises at least one selected from the group consisting of (1) and (2) below:
(1) mutation of G at position 3455 of the COL4A2 gene coding region (position 301 of SEQ ID NO:31) to A; and
(2) mutation of G at position 3110 of the COL4A2 gene coding region (position 385 of SEQ ID NO:28) to A, and
wherein said primer pair is at least one selected from the group consisting of primer pair SEQ ID NO: 99 and SEQ ID NO: 100 and primer pair SEQ ID NO: 107 and SEQ ID NO: 108.

7. The method according to claim 6, wherein said detecting is by high-resolution melting curve (HRM) analysis or direct sequencing.

8. The method according to claim 6, wherein said subject is a postnatal human or human fetus.

9. The method according to claim 6, wherein the diagnosing the risk of porencephaly and/or cerebral hemorrhage occurs during the fetal period to perinatal period in a human fetus.

10. The method according to claim 6, wherein the detecting step is in a genomic sequence using a genomic DNA sample.

11. The method according to claim 6, wherein said DNA is amplified by PCR.

12. A method for detecting at least one mutation in at least one allele of a COL4A2 gene in a human subject, comprising:
(a) obtaining a sample separated from the subject; and
(b) detecting at least one mutation present in at least one allele of a COL4A2 gene in the sample by high-resolution melting curve (HRM) analysis or direct sequencing, wherein said at least one mutation comprises at least one selected from the group consisting of (1) and (2) below:
(1) mutation of G at position 3455 of the COL4A2 gene coding region (position 301 of SEQ ID NO:31) to A; and
(2) mutation of G at position 3110 of the COL4A2 gene coding region (position 385 of SEQ ID NO:28) to A.