KR102333830B1 - Novel mitochondrial genomic markers for the diagnosis of macular degeneration - Google Patents

Abstract

The present invention relates to a novel marker for diagnosing macular degeneration using mutations in genes found in mitochondrial genes. According to the present invention, it is found that the N9a and M7b haplogroups are significantly associated with the risk of age-related macular degeneration, and these haplogroups are an important marker that can predict the risk of macular degeneration from mitochondrial genome analysis in Asians, so that when the haplogroups are used as the novel marker that can effectively diagnose macular degeneration in Asians in more detail in Koreans, there is an effect of predicting the risk and prognosis of macular degeneration before the onset or onset of macular degeneration.

Description

Novel MITOCHONDRIAL GENOMIC MARKERS FOR THE DIAGNOSIS OF MACULAR DEGENERATION

The present invention relates to novel markers for diagnosing macular degeneration, and more particularly, to mutations in genes found in mitochondrial genes.

In the cells of the human body, there are a nuclear genome that exists in the nucleus as genetic material and a mitochondrial genome that exists in mitochondria in the cytoplasm. In the process of inheritance from the upper generation to the next, the nuclear genome is inherited by receiving half of the nuclear genome of the male and the female, whereas in the mitochondrial genome, the mitochondrial genome in the fertilized egg of the female is transmitted to the next generation because the mitochondria exist in the cytoplasm. It has hereditary characteristics. The mitochondrial genome is a double-stranded circular structure consisting of 16569 base pairs, which is distinct from the double-helix nuclear genome and is also denoted as mtDNA (Homo sapiens mitochondrial complete genome, https://www.ncbi.nlm.nih.gov/nucleotide /NC_012920.1 , NCBI Reference Sequence: NC_012920.1). The mitochondrial genome can transcribe 37 genes, including 22 tRNA genes and 2 rRNA genes involved in protein synthesis, and 13 electron transport chain protein genes, which are instructions for making enzymes necessary for the ATP generation reaction. 1 shows the structure of the mitochondrial genome and the distribution of genes. A number of specific nucleotide polymorphisms exist in the mitochondrial genome as well. Various combinations of nucleotide variations are called haplogroups. Haplogroups are classified into uppercase English letters A to Z. Subdivided haplogroups include numbers and lowercase letters. It is indicated as an addition. In the mitochondrial genome, haplogroups are very distinct according to race, such as Africans, Westerners, and Asians. This is because, anthropologically, the origin of mankind is called African Eve, and as humans move from this to various regions such as Europe and Asia, mutations occurring in mitochondria over time form a phylogenetic tree in the process of settling in the region.

On the other hand, macular degeneration refers to a case in which the macula, which is the most important part of the retina, which is the neural tissue of the eye, changes the properties of its cells due to some cause, causing lesions such as decreased visual acuity and blindness. Macular degeneration is most commonly caused by aging. Age-related macular degeneration is called age-related macular degeneration (AMD or ARMD), and when the functions of cells and blood vessels in the macula decline due to aging, waste products from the retina accumulate under the retina without being processed. Changes such as the formation of blood vessels affect vision. Age-related yellowing is the most common cause of blindness in the population aged 65 years or older in the West, and the incidence rate is increasing in Korea due to the recent increase in the elderly population. According to the 2010 National Health and Nutrition Statistics, the prevalence of age-related macular degeneration is 1.4% for men in their 40s, 4.6% in their 50s, 10.2% in their 60s, and 14.8% in their 70s, and 1.1% for women in their 40s, 4.7% in their 50s, The prevalence was found to increase with the increase of age, with 11.8% in their 60s and 20.2% in their 70s and older. As such, age is the most important cause because macular degeneration occurs a lot in the elderly over 60 years old and does not appear well in young people. Another important cause is smoking. In addition, obesity, cardiovascular disease, high blood pressure, hyperlipidemia, and high fat intake are also causes of the disease. As environmental factors, exposure to ultraviolet light is mentioned as an important cause. The reason that macular degeneration is increasing among middle-aged people is that they are exposed to a lot of ultraviolet rays, which can cause macular degeneration due to active outdoor activities such as exercise and leisure. According to Statistics Korea, the proportion of the elderly population in Korea is expected to enter an aging society from 11% in 2010 to 14% in 2018 and 21% in 2025, which is expected to enter a super-aging society. see.

Although macular degeneration is not a disease determined by distinct genetic factors such as blonde hair and walleye, it shows a genetic link that increases the risk of occurrence if there is a specific genetic mutation. In a study by Haines et al. published in Science in 2005, it was known that complement factors related to the inflammatory response play an important role in the pathogenesis of age-related macular degeneration. It has been reported that the genes (CFH, CFB) encoding factors H and B, which are proteins of the complement system, cause age-related macular degeneration, followed by mutations in C3 and C2 genes, another protein of the complement system. The results were found to be closely related to Since then, genetic mutations related to the risk of age-related macular degeneration have been found in at least 34 genes until recently through genomic research. Through the studies so far, it is comprehensively understood that in individuals with these genetic abnormalities, triggering factors such as infection, smoking, and cardiovascular abnormalities act to trigger the pathological mechanism of this disease. It is known that 70% of the occurrence of progressive macular degeneration is determined by genetics, and the remaining 30% is determined by environmental factors such as smoking and eating habits.

In the treatment of macular degeneration, it can appear differently depending on the presence or absence of mutations in various genes such as CFH, HTRA1, and ARMS2. For example, if there is a mutation gene for CFH, the effect of delaying the progression of macular degeneration according to vitamin-mineral therapy or fish intake is different. It shows the need for customized management. For example, in patients with dry macular degeneration with specific genetic mutations, we provide genetic testing for personalized prescriptions based on research findings that long-term intake of zinc-containing eye health supplements is associated with aggravation of the disease into progressive wet macular degeneration. have. A diagnostic method aimed at predicting the risk of progressive macular degeneration, determining how to consume appropriate eye health supplements, and tailoring treatment for patients with macular degeneration. Arctic Dx's Macular Risk® there is This diagnostic method is a relatively accurate diagnostic method with a positive predictive value of 83%, which predicts the risk of developing progressive macular degeneration by the age of 80, and can be prevented and managed according to the individual risk. However, Arctic Dx's Macular Risk® introduces the mitochondrial haplogroup information found in the West into the risk calculation, so marker mutations analyzed in Macular Risk® are rarely found in Asians, so there is no meaning in diagnosis. there has been

The correlation between haplogroups in the mitochondrial genome and age-related macular degeneration has been studied in Westerners. A number of studies have found that the H haplogroup, which is found most often in Europeans, reduces the risk of age-related macular degeneration, and the J and T haplogroups are factors that increase the risk of age-related macular degeneration. . However, such haplogroups are not found in Asian races or have a very low frequency even if they are found.

This commercialized overseas genetic diagnosis method is not suitable for predicting the risk of age-related macular degeneration occurring in Korea because the genotype marker is specialized as a genotype suitable for Caucasians. Accordingly, the present inventors tried to discover a new genotype diagnostic marker to predict the age-related macular degeneration risk and treatment prognosis in Korea.

It is an object of the present invention to provide a composition for diagnosing mitochondrial haplogroups.

Another object of the present invention is to provide a composition for diagnosing macular degeneration or predicting prognosis.

Another object of the present invention is to provide a macular degeneration diagnostic kit.

In addition, it is an object of the present invention to provide an information providing method for diagnosing macular degeneration.

In order to solve the above problems, the present invention provides a composition for diagnosing mitochondrial haplogroup.

In addition, the present invention provides a composition for diagnosing macular degeneration or predicting prognosis.

The present invention also provides a macular degeneration diagnostic kit.

In addition, the present invention provides an information providing method for diagnosing macular degeneration.

According to the present invention, the N9a and M7b haplogroups were found to be significantly associated with the risk of age-related macular degeneration, and these haplogroups are important for predicting the risk of macular degeneration from mitochondrial genome analysis in Asian races. Since it is a marker, if it is used as a new marker that can effectively diagnose macular degeneration in Asians and more specifically in Koreans, it has the effect of predicting the risk and prognosis of macular degeneration before the onset or onset of macular degeneration.

1 is a schematic diagram showing the structure of the mitochondrial genome.
2 is a schematic diagram showing the difference in the distribution of mitochondrial haplogroups between species.
3 is a schematic diagram illustrating a hybridization site of a primer set for amplifying a mitochondrial control region genome.
4 is a graph showing the frequency of mtDNA haplogroups.

Hereinafter, the present invention will be described in detail by way of embodiments of the present invention with reference to the accompanying drawings. However, the following embodiments are presented as examples of the present invention, and when it is determined that detailed descriptions of well-known techniques or configurations known to those skilled in the art may unnecessarily obscure the gist of the present invention, the detailed description may be omitted, and , the present invention is not limited thereby. Various modifications and applications of the present invention are possible within the scope of equivalents interpreted therefrom and the description of the claims to be described later.

In addition, the terms used in this specification are terms used to properly express the preferred embodiment of the present invention, which may vary according to the intention of a user or operator, or customs in the field to which the present invention belongs. Accordingly, definitions of these terms should be made based on the content throughout this specification. Throughout the specification, when a part "includes" a certain component, it means that other components may be further included, rather than excluding other components, unless otherwise stated.

As used herein, the term "subject" or "patient" means any single individual in need of treatment, including humans, apes, monkeys, cattle, dogs, guinea pigs, rabbits, chickens, insects, and the like. Also included in the subject are any subjects who participated in a clinical study trial without any clinical manifestations of any disease, or subjects who participated in epidemiological studies or subjects used as controls.

As used herein, the term “sample (sample)” refers to a biological sample obtained from a subject or patient. Sources of biological samples may include fresh, frozen and/or preserved organ or tissue samples or solid tissue from biopsies or aspirates; blood or any blood component; The cells may be at any point in the pregnancy or development of the subject.

All technical terms used in the present invention, unless otherwise defined, have the meaning as commonly understood by one of ordinary skill in the art of the present invention. In addition, although preferred methods and samples are described herein, similar or equivalent ones are also included in the scope of the present invention. The contents of all publications herein incorporated by reference are incorporated herein by reference.

In one aspect, the present invention relates to a mitochondrial haplogroup diagnostic composition comprising the primers of SEQ ID NOs: 1 to 6.

In one embodiment, the composition comprises a pair of primers of SEQ ID NOs: 1 and 2; and a primer of SEQ ID NO: 1 and a primer of SEQ ID NO: 3 to 6.

In one embodiment, the composition may be for diagnosis of N9a or M7b haplogroup.

In one embodiment, after PCR amplification using the primer pair of SEQ ID NOs: 1 and 2, the haplogroup is diagnosed/determined by identifying the mitochondrial genome of 1237bp using the primers of SEQ ID NO: 1 and primers of SEQ ID NOs: 3 to 6 can

In one aspect, the present invention provides a composition for diagnosing one or more single nucleotide polymorphisms (SNP) present on the mitochondrial genome.

In one aspect, the present invention relates to a composition for diagnosing macular degeneration or predicting a prognosis, comprising an agent for determining a haplogroup of a mitochondrial genome.

In one embodiment, the composition may be for diagnosis of N9a or M7b haplogroup.

In one embodiment, the agent for determining the N9a or M7b haplogroup specifically recognizes a specific nucleic acid sequence of the haplogroup, a nucleic acid sequence complementary to the specific nucleic acid sequence, the specific nucleic acid sequence and a fragment of the complementary sequence may include a primer pair, a probe, or a primer pair and a probe that (Competitive RT-PCR), nuclease protection assay (RNase, S1 nuclease assay), in situ hybridization, nucleic acid microarray, fish-reverse blot hybridization assay, Northern blot, performed by a method selected from the group consisting of DNA chip can be

In one embodiment, the primer pair of SEQ ID NOs: 1 and 2; and a primer of SEQ ID NO: 1 and a primer of SEQ ID NO: 3 to 6.

In one embodiment, the macular degeneration may be Age-related macular degeneration (AMD or ARMD).

As used herein, the term "detection" or "determining" refers to a single nucleotide polymorphism (SNP) mutation of a target that shares a common ancestor to detect or determine a group of similar haplotypes. means that

As used herein, the term "primer" is a nucleic acid sequence having a short free 3 hydroxyl group, which can form a complementary template and base pair, and serves as a starting point for template strand copying. It refers to a short nucleic acid sequence that functions. The primers can initiate DNA synthesis in the presence of reagents for polymerization (ie, DNA polymerate or reverse transcriptase) and the four different nucleoside triphosphates in an appropriate buffer and temperature.

As used herein, the term "probe" refers to a nucleic acid fragment such as RNA or DNA corresponding to several bases to several hundred bases as short as possible to achieve specific binding to mRNA, and is labeled to check the presence or absence of a specific nucleic acid. can The probe may be manufactured in the form of an oligonucleotide probe, a single stranded DNA probe, a double stranded DNA probe, an RNA probe, or the like. The selection of suitable probes and hybridization conditions may be modified based on those known in the art, and therefore, the present invention is not particularly limited thereto.

The primers or probes of the present invention can be chemically synthesized using the phosphoramidite solid support method, or other well-known methods. Such nucleic acid sequences may also be modified using a number of means known in the art. Non-limiting examples of such modifications include methylation, encapsulation, substitution with one or more homologues of natural nucleotides, and modifications between nucleotides, such as uncharged linkages such as methyl phosphonates, phossotriesters, phosphoro amidates, carbamates, etc.) or charged linkages (eg phosphorothioates, phosphorodithioates, etc.).

In the present invention, suitable conditions for hybridizing a probe with a cDNA molecule can be determined in a series of processes by an optimization procedure. These procedures are carried out as a series of procedures by those skilled in the art to establish protocols for use in the laboratory. For example, conditions such as temperature, concentration of components, hybridization and washing times, buffer components and their pH and ionic strength depend on various factors such as probe length and GC amount and target nucleotide sequence. Detailed conditions for hybridization are described in Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001); and M.L.M. Anderson, NucleicAcidHybridization, Springer-Verlag New York Inc. N.Y. (1999). For example, the high stringency conditions among the stringent conditions include hybridization in 0.5 M NaHPO4, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C., and 0.1×SSC (standard saline citrate)/0.1% SDS at 68° C. It means washing under conditions. Alternatively, high stringency conditions mean washing at 48° C. in 6×SSC/0.05% sodium pyrophosphate. Low stringency conditions mean, for example, washing at 42° C. in 0.2×SSC/0.1% SDS.

In one aspect, the present invention relates to a macular degeneration diagnostic kit comprising the composition of the present invention.

In one embodiment, the kit may further include tools and/or reagents for collecting a biological sample from a subject or patient, as well as tools and/or reagents for preparing genomic DNA, cDNA, RNA or protein from the sample. have. For example, it may include PCR primers for amplifying a relevant region of genomic DNA. The kit may include probes of genetic factors useful for pharmacogenomic profiling. In addition, in the use of such a kit, the labeled oligonucleotide can be easily identified during analysis.

In one embodiment, the kit may further contain a labeling material such as a DNA polymerase and dNTPs (dGTP, dCTP, dATP and dTTP), a fluorescent material, and the like.

As used herein, the term “macular degeneration diagnostic kit” refers to a kit including the composition for diagnosing macular degeneration of the present invention. Therefore, the expression “macular degeneration diagnostic kit” can be used interchangeably or mixed with “a composition for diagnosing macular degeneration”. As used herein, the term “diagnosis” refers to determining a subject's susceptibility to a specific disease or disorder, determining whether an object currently has a specific disease or disorder, or having a specific disease or disorder. Determining a subject's prognosis (e.g., identifying a macular degeneration condition, determining the staging of macular degeneration, or determining responsiveness to treatment), or therametrics (e.g., to provide information about the efficacy of treatment) monitoring the state of the object).

In one embodiment, the kit may be for a fishial-reverse blot hybridization assay.

In one aspect, the present invention comprises the steps of isolating mtDNA from a specimen; PCR amplifying the control region of mtDNA; And it relates to a method of providing information for diagnosing macular degeneration, comprising the step of determining a haplogroup.

In one embodiment, the primer pair of SEQ ID NOs: 1 and 2; and a primer of SEQ ID NO: 1 and a primer of SEQ ID NO: 3 to 6.

In one embodiment, PCR amplification may be performed using a pair of primers of SEQ ID NOs: 1 and 2, and haplogroups may be determined using the primers of SEQ ID NO: 1 and primers of SEQ ID NOs: 3 to 6.

In one embodiment, when belonging to the N9a haplogroup, it can be determined that the risk of occurrence of macular degeneration increases by 1.5 to 3 times.

In one embodiment, when belonging to the M7b haplogroup, it can be determined that the risk of occurrence of macular degeneration is reduced by 0.1 to 0.7 times.

In one embodiment, the macular degeneration may be Age-related macular degeneration (AMD or ARMD).

In one embodiment, the biological sample may include a sample such as tissue, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid or urine, and the like.

In one aspect, the present invention relates to the use of haplogroup N9a as a diagnostic marker for macular degeneration.

A mitochondrial haplogroup is determined by combining all variations on the mitochondrial genome. Although the mitochondrial genome is 16569 bp in length, which is not as large as the nuclear genome, it is not short enough to be analyzed by Sanger sequencing method. It was found that the haplogroup can be determined by accumulating the mitochondrial nucleotide sequence into a database, and analyzing a specific part without analyzing all the nucleotide sequences for haplogroup analysis through their analysis. Among them, a representative part is a part named D-loop or control region (FIG. 1). This region does not code for a gene, but contains a region where mycotondrial replication is initiated and has three hypervariable regions. The fact that a haplogroup can be determined only by sequencing of this part is widely accepted in the art. Numerous mutations are found in mitochondria, and haplogroups are determined according to a specific combination of mutations (FIG. 2). Therefore, in one embodiment, the present invention analyzes mitochondrial haplogroups for age-related macular degeneration patients and controls for Koreans in one embodiment. This is the first study in Asians where the mitochondrial haplogroup distribution is completely different from that of Westerners and Africans.

As a result of the study, it was found that the N9a haplogroup found in Koreans increased the risk of age-related macular degeneration by 2.19 times, and the M7b haplogroup reduced the risk by 0.43 times, and these results were also statistically significant. Since these haplogroups are important markers for predicting the risk of macular degeneration from mitochondrial genome analysis in Asians, if the information on the N9a and M7b haplogroups discovered through the present invention is used, in more detail in Asians, macular degeneration in Koreans As a novel marker that can effectively diagnose sex, it can be usefully used for early diagnosis of macular degeneration.

The present invention will be described in more detail through the following examples. However, the following examples are only intended to embody the contents of the present invention, and the present invention is not limited thereto.

Example 1. Determination of age-related macular degeneration-related mitochondrial haplogroups

After receiving IRB approval from Busan Paik Hospital as a clinical trial, age-related macular degeneration patients and normal control samples were collected through a multicenter clinical trial, and then the control region of the mitochondrial genome was hybridized in the direction of FIG. 3 to analyze the haplogroup. It was analyzed by the method described in Int J Legal Med., 2006, 120(1):5-14 using the primers of Table 1 below. After amplifying the mitochondrial control region genome using the following primers, the nucleotide sequence was analyzed by Sanger sequencing method. Haplogroups were determined by using the mtDNA manager program (http://mtmanager.yonsei.ac.kr/) after collecting variants compared to the standard human mitochondrial genome in the mitochondrial sequence of the analyzed patient group and control group. After comparing the frequency of haplogroups between the patient group and the control group, statistical analysis was performed using methods such as Fisher'exact test, Chi-square test, and Odds ratio.

purpose start position Primer sequence (5'→3') Primer length (bp) Amplification product length (bp) SEQ ID NO: for PCR 15971→ TTA ACT CCA CCA TTA GCA CC 20 1237 One ←638 GGT GAT GTG AGC CCG TCT AAA 21 2 for sequencing 15971→ TTA ACT CCA CCA TTA GCA CC 20 One 0→ CTT AAA TAA GAC ATC ACG ATG 21 3 ←16549 CAT CGT GAT GTC TTA TTT AAG 21 4 314→ CCG CTT CTG GCC ACA GCA CT 20 5 ←569 GGT GTC TTT GGG GTT TGG TTG 21 6

As a result of comparing the distribution of mitochondrial haplogroups in the age-related macular degeneration patient group and the control group, the N9a haplogroup and the M7b haplogroup showed a difference in frequency at a statistically significant level (Table 2 and FIG. 4). As a result of analyzing how these two haplogroups were related to the risk of disease, the N9a haplogroup increased the risk of age-related macular degeneration by 2.19 times, and the M7b haplogroup increased the risk of age-related macular degeneration. It was shown to decrease by 0.43 times (Table 3).

Haplogroup control
(n=254) age-related macular degeneration
(n=526) p-value N* 9 12 0.308 F 19 52 0.274 B 40 78 0.737 A 26 35 0.081 N9a 8 35 0.045 ^* N9b One 0 0.150 M* 26 56 0.861 M7a 6 10 0.670 M7b 12 11 0.042 ^* M7c 5 6 0.358 D4/G 21 54 0.375 G* 2 10 0.236 G1 3 17 0.090 G2 9 17 0.820 D4 57 97 0.189 D5 10 36 0.106

N* haplogroup includes a Y sub-haplogroup

M* haplogroup includes the sub-haplogroup as M8,M10,M11

G* haplogroup includes a G3 sub-haplogroup.

haplogroup or genetic mutation frequency (%) Odds ratio
(95% CI) p-value macular degeneration control N9a 6.7 3.1 2.19 (1.00-4.80) 0.045 C16611T 3.6 0.8 4.72 (1.09-20.43) 0.038 C16257A 7.4 3.1 2.46 (1.13-5.35) 0.023 M7b 2.1 4.7 0.43 (0.19-0.99) 0.042 T199C 5.9 10.6 0.43 (0.19-0.96) 0.040 T16297C 2.3 5.1 0.51 (0.29-0.88) 0.015

Therefore, if the information of the N9a and M7b haplogroups discovered through the present invention is used, it can be used to more accurately predict the risk in Asians, especially in Koreans.

<110> inje university industry-academic cooperation foundation <120> NOVEL MITOCHONDRIAL GENOMIC MARKERS FOR THE DIAGNOSIS OF MACULAR DEGENERATION <130> P20200101KR-00 <160> 6 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 15971 forward primer <400> 1 ttaactccac cattagcacc 20 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> 638 reverse primer <400> 2 ggtgatgtga gcccgtctaa a 21 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> 0 forward primer <400> 3 cttaaataag acatcacgat g 21 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> 16549 reverse primer <400> 4 catcgtgatg tctttattaa g 21 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 314 forward primer <400> 5 ccgcttctgg ccacagcact 20 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> 569 reverse primer <400> 6 ggtgtctttg gggtttggtt g 21

Claims (14)

delete delete As a composition for diagnosing macular degeneration or predicting prognosis, comprising an agent for determining a haplogroup of the mitochondrial genome comprising the primers of SEQ ID NOs: 1 to 6,
The haplogroup is a N9a and M7b haplogroup, a composition for diagnosing or predicting macular degeneration. delete delete According to claim 3, Age-related macular degeneration (Age-related macular degeneration, AMD or ARMD), the composition for diagnosing or predicting prognosis of macular degeneration. A macular degeneration diagnostic kit comprising the composition of claim 3 . The macular degeneration diagnostic kit according to claim 7, which is for a fish-reverse blot hybridization assay. a) isolating mtDNA from the specimen;
b) PCR amplifying the control region of mtDNA; and
c) as an information providing method for diagnosing macular degeneration, comprising the step of determining a haplogroup,
The haplogroup in step c) is an N9a and M7b haplogroup, an information providing method for diagnosing macular degeneration. The method for providing information for diagnosing macular degeneration according to claim 9, wherein it is determined that the risk of macular degeneration increases by 1.5 to 3 times when belonging to the N9a haplogroup. The method for providing information for diagnosing macular degeneration according to claim 9, wherein the risk of occurrence of macular degeneration is determined to be reduced by 0.1 to 0.7 times when belonging to the M7b haplogroup. The method for providing information for diagnosing macular degeneration according to claim 9, which is age-related macular degeneration. The method of claim 9, wherein PCR amplification is performed using the primer pair of SEQ ID NOs: 1 and 2. The method of claim 9, wherein the haplogroup is determined using the primers of SEQ ID NO: 1 and the primers of SEQ ID NOs: 3 to 6, information providing method for diagnosing macular degeneration.

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