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WO2018169333A1 - Pold1 présentant une pléiotropie génétique et méthode de diagnostic de maladies l'utilisant - Google Patents

Pold1 présentant une pléiotropie génétique et méthode de diagnostic de maladies l'utilisant Download PDF

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WO2018169333A1
WO2018169333A1 PCT/KR2018/003063 KR2018003063W WO2018169333A1 WO 2018169333 A1 WO2018169333 A1 WO 2018169333A1 KR 2018003063 W KR2018003063 W KR 2018003063W WO 2018169333 A1 WO2018169333 A1 WO 2018169333A1
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activity
dna polymerase
exonuclease
pold1
measuring
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Korean (ko)
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최병윤
오두이
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Seoul National University Hospital
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Seoul National University Hospital
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • G01N2333/91205—Phosphotransferases in general
    • G01N2333/91245—Nucleotidyltransferases (2.7.7)
    • G01N2333/9125—Nucleotidyltransferases (2.7.7) with a definite EC number (2.7.7.-)
    • G01N2333/9126—DNA-directed DNA polymerase (2.7.7.7)

Definitions

  • Congenital hearing loss occurs in about 3 out of 1,000 newborns, and more than 50% is known to be caused by genetic factors. Hereditary hearing loss is congenital and occurs after birth, but there are many types of hearing loss, but sensoryural hearing loss (Sensorine Hearing Loss) is the most common.
  • Sensorineural hearing loss refers to the hearing loss caused by abnormalities in the function of sensing the sound of the cochlea or abnormalities of the auditory nerve or central nervous system that transmits the stimulus to the brain.
  • Sensorineural hearing loss is classified into syndrome and non-syndrome according to symptoms, and syndrome sensory hearing loss refers to cases having clinical symptoms or symptoms of other organs other than those due to inner ear dysfunction. It accounts for 30% of hereditary deafness, and more than 300 types of syndrome hearing loss have been reported. Because it is easily classified into characteristic symptoms or malformations, it is easy to follow the cause gene in patients with the same cause.
  • Non-syndrome sensorineural hearing loss refers to a case in which abnormal symptoms or symptoms of organs other than the inner ear dysfunction are not shown, and only hearing impairment. It accounts for 70% of hereditary deafness, and because the genes are diverse, strategic genetic diagnosis is required based on clinical information such as the type of hearing loss and hereditary form. As for non-syndrome deafness, 80% of them are recessive, 17% of dominant, 2-3% of X-linked, and 1% of mitochondrial inheritance. Is known to be caused by mutations in the Gap Junction Beta 2 (GJB2) gene.
  • GJB2 Gap Junction Beta 2
  • Hearing loss genes may exhibit variable expressivity.Aberrations of one deafness gene may be expressed as syndrome deafness or non-syndrome deafness depending on the gene expression by the mutation type, and a mutation of one deafness gene is a type of mutation. For example, they may be expressed in recessive or dominant genetic manner.
  • Genetic pleiotropy refers to a phenomenon in which multiple mutations of one gene appear to be irrelevant at all, and are extreme forms of various expressions. For example, different mutations in one gene are expressed in phenotypes that indicate abnormalities in completely different organs. The mechanism of phenotypic pleiotropic expression suggests that different specific mutations of one gene affect the protein differently, and that different specific mutations of one gene affect the total amount of one protein that is functionally intact, resulting in different phenotypes. .
  • the GJB2, MYO7A, TMC1 genes and the like are known as hearing loss genes in which the same genes are expressed as autosomal recessive or dominant sensorineural hearing loss.
  • the present inventors have sought to find a new hearing gene expression indicating if the phenotypic results, POLD1
  • the present invention has been completed by confirming that the mutation causes mutually exclusion of three diseases that are completely different according to the position and genetic method of the mutation by a mechanism different from the mechanisms presented so far, and that the disease can be diagnosed using the same.
  • One aspect includes an agent for measuring the activity of DNA polymerase and exonuclease, the active domains of POLD1, and measuring the activity of exonuclease relative to the DNA polymerase, Or it provides a kit for diagnosing cancer, MDP syndrome, and sensorineural hearing loss, to measure the activity of the DNA polymerase compared to the normal control.
  • compositions for diagnosing cancer, MDP syndrome, and sensorineural hearing loss comprising an agent for measuring the activity of DNA polymerase and exonulease, the active domains of POLD1.
  • Another aspect provides a method of diagnosing cancer, MDP syndrome, and sensorineural hearing loss in a composition comprising an agent for measuring the activity of DNA polymerase and exonuclease, the active domain of POLD1. to provide.
  • Another aspect includes measuring the activity of DNA polymerase and exonuclease, the active domains of POLD1; And measuring the activity of the exonuclease relative to the measured DNA polymerase activity, or measuring the activity of the DNA polymerase relative to the normal control group, to diagnose cancer, MDP syndrome, and sensorineural hearing loss. It provides a method of providing information for.
  • Another aspect includes measuring the activity of DNA polymerase and exonuclease, the active domains of POLD1; And measuring the activity of the exonuclease relative to the measured DNA polymerase activity, or measuring the activity of the DNA polymerase relative to the normal control group to diagnose cancer, MDP syndrome, and sensorineural hearing loss. Provide a method of treatment.
  • One aspect includes an agent for measuring the activity of DNA polymerase and exonuclease, the active domains of POLD1, and measuring the activity of exonuclease relative to the DNA polymerase, Or it provides a kit for diagnosing cancer, MDP syndrome, and sensorineural hearing loss, to measure the activity of the DNA polymerase compared to the normal control.
  • Diagnosis is intended to identify the presence or characteristic of pathophysiology. Diagnosis in the present invention is to identify the presence and / or extent of cancer, MDP syndrome, and sensorineural hearing loss.
  • POLD1 is a DNA polymerase delta 1, whose gene and protein information can be easily identified through a known database such as the National Center for Biotechnical Information (NCBI) NCBI gene bank. Accession No. of Gene Bank It may be POLD1 which is NG_033800, but is not limited thereto.
  • MDP syndrome means a metabolic disease indicating mandibular dysplasia, deafness and premature ejaculation.
  • the cancer may be at least one selected from the group consisting of liver cancer, glioblastoma, ovarian cancer, colon cancer, head and neck cancer, bladder cancer, renal cell cancer, gastric cancer, breast cancer, metastatic cancer, prostate cancer, pancreatic cancer and lung cancer, specifically It may be colon cancer.
  • POLD1 since the phenotypes are very different from sensory nerve deafness, cancer, and MDP syndrome according to genotype of c.584_585delGA nucleotide or c.G3298A nucleotide variation in POLD1, POLD1 exhibits phenotypic pleiotropic expression including non-syndrome deafness. It was first identified as a gene. It was first identified that phenotypic pleiotropic expression is indicated by the activity ratio of the functions of two domains in the POLD1 protein, such as the DNA polymerase active domain and the exonuclease domain.
  • the present invention includes an agent for measuring the activity of DNA polymerase and exonuclease, which are active domains of POLD1, and measures the activity of exonuclease relative to the DNA polymerase, or Cancer, MDP syndrome, and sensorineural hearing loss can be diagnosed by measuring the activity of the DNA polymerase relative to the normal control. Specifically, when the activity of the exonuclease relative to the DNA polymerase is less than 0.7, it is diagnosed as cancer, and when the activity of the DNA polymerase is greater than 25% and less than 50% as compared to the normal control group, it is diagnosed as sensorineural hearing loss, Less than 25% can be diagnosed with MDP syndrome and greater than 50% can be diagnosed as normal.
  • the pol index and the error index are calculated. If the error index is greater than 0, the cancer and the error index are 0 and the pol index is 0.5. If the abnormality is normal, if the error index is 0 and the pol index is more than 0.33, the MDP syndrome can be diagnosed if the non-syndrome sensorineural hearing loss and the error index are 0 and the pol index is 0.33 or less.
  • the error index can be expressed as 'relative primer extension efficiency x (1-relative exonuclease activity)', and the error index and pol index are the results of experiments measuring relative primer extension efficiency and relative exonuclease activity. Can be calculated based on
  • the term "agent for measuring the activity of DNA polymerase and exonuclease, the active domain of POLD1,” means DNA polymerase and exonude, the active domain of POLD1 that exhibits phenotypic pleiotropic expression in an individual. Means a molecule that can be used for the detection and / or quantification of the marker by confirming the mRNA level of the clease gene, according to one embodiment primer pair specifically binding to the DNA polymerase and exonuclease gene, Probe, oligonucleotide or antisense oligonucleotide.
  • the agent for measuring mRNA expression levels of the DNA polymerase and exonuclease is a primer or probe specifically binding to the mRNA of the gene, since the mRNA nucleic acid sequence of the POLD1 gene is known, Can design primers or probes that specifically bind to mRANA of these genes based on the sequence. It is also possible to modify the design to some extent. Those skilled in the art can also use sequences that maintain at least 80%, specifically at least 90%, more specifically at least 95%, and even more specifically 98% homology by such artificial modifications.
  • mRNA expression level measurement is a process for determining the presence and expression level of mRNA of the POLD1 gene, the amount of mRNA, and according to one embodiment of the present invention can be measured using a primer or probe for mRNA.
  • Analytical methods for this include reverse transcription polymerase chain reaction (RT-PCR), competitive RT-PCR (real-time RT-PCR), DNA chips, and enzyme-linked immunosorbent (ELISA). assay), protein chips and the like, but is not limited thereto.
  • primer refers to a nucleic acid sequence having a short free 3 'hydroxyl group, which forms a complementary template and base pair and serves as a starting point for template strand copying. Refers to the sequence. Primers can initiate DNA synthesis in the presence of four different nucleoside triphosphates and reagents for polymerization (ie, DNA polymerase or reverse transcriptase) at appropriate buffers and temperatures.
  • probe refers to a nucleic acid fragment such as RNA or DNA, which may be short to several bases to hundreds of bases that can specifically bind to mRNA, and is labeled so that the presence or absence of a specific mRNA and its amount (expression amount) )can confirm.
  • Probes may be made in the form of oligonucleotide probes, single strand DNA probes, double strand DNA probes, RNA probes and the like.
  • hybridization may be performed using a probe complementary to the mRNA of the POLD1 polynucleotide, and the radiation exposure may be determined by measuring the expression level of mRNA through the degree of hybridization. Selection of appropriate probes and hybridization conditions may be appropriately selected according to techniques known in the art.
  • Primers or probes of the invention can be chemically synthesized using phosphoramidite solid support methods, or other well known methods. Such nucleic acid sequences can also be modified using many means known in the art. Non-limiting examples of such modifications include methylation, capping, substitution of one or more homologs of natural nucleotides, and modifications between nucleotides, eg, uncharged linkages such as methyl phosphonate, phosphotriester, phosph Modifications to poroamidates, carbamates, etc.) or charged linkers (eg, phosphorothioates, phosphorodithioates, etc.).
  • Nucleic acids may be selected from one or more additional covalently linked residues, such as proteins (eg, nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), inserts (eg, acridine, psoralene, etc.). ), Chelating agents (eg, metals, radioactive metals, iron, oxidizing metals, etc.), and alkylating agents. Nucleic acid sequences of the invention can also be modified using a label that can provide a detectable signal directly or indirectly. Examples of labels include radioisotopes, fluorescent molecules, biotin, and the like.
  • the "measurement of the activity of DNA polymerase and exonuclease, the active domain of POLD1 exhibiting phenotypic pleiotropic expression,” can be measured by primer extension assay.
  • the kit is used to diagnose hearing loss, cancer, and MDP syndrome by measuring the activity levels of DNA polymerase and exonuclease, the active domains of PODL1, from samples of individuals suspected of developing deafness, cancer, or MDP syndrome.
  • the present invention may include, but is not limited to, a primer or a probe for measuring the activity of the active domain of PODL1, as well as one or more other component compositions, solutions or devices suitable for analytical methods.
  • the kit may be a reverse transcription polymerase chain reaction (RT-PCR) kit, a DNA chip kit, an enzyme-linked immunosorbent assay (ELISA) kit, a protein chip kit, a rapid kit, or a multiple reaction monitoring (MRM) kit. It is not limited to this.
  • RT-PCR reverse transcription polymerase chain reaction
  • DNA chip kit a DNA chip kit
  • ELISA enzyme-linked immunosorbent assay
  • MRM multiple reaction monitoring
  • the sensorineural hearing loss may be Non Syndromal sensorineural hearing loss (NS-SNHL).
  • Sensorineural hearing loss refers to hearing loss caused by abnormalities in the function of sensing the sound of the cochlea or abnormalities of the auditory nerve or the central nervous system that transmits the stimulus to the brain. Hearing loss that does not show abnormal symptoms or symptoms of other organs.
  • compositions for diagnosing cancer, MDP syndrome, and sensorineural hearing loss comprising an agent for measuring the activity of DNA polymerase and exonuclease, the active domain of POLD1.
  • compositions comprising an agent for measuring the activity of DNA polymerase and exonuclease, the active domain of POLD1, for diagnosing cancer, MDP syndrome, and sensorineural hearing loss.
  • Another aspect includes measuring the activity of DNA polymerase and exonuclease, the active domains of POLD1, in a biological sample isolated from an individual; And measuring the activity of the exonuclease relative to the measured DNA polymerase activity, or measuring the activity of the DNA polymerase relative to the normal control group, to diagnose cancer, MDP syndrome, and sensorineural hearing loss. It provides a method of providing information for.
  • the activity of the DNA polymerase and exonuclease which are the active domains of POLD1, is measured, and the activity of the DNA polymerase relative to the normal control is as described above.
  • the method is determined to be cancer when the activity of the exonuclease relative to the DNA polymerase is less than 0.7, and to the sensory nerve deafness when the activity of the DNA polymerase is greater than 25% and less than 50% relative to the normal control group. If less than 25% may be determined to be MDP syndrome, if more than 50% may further comprise the step of determining normal.
  • the method calculates the pol index and the error index by measuring the relative activity of the polymerase / exonuclease, and when the error index is greater than zero, the cancer and error index is 0. If the pol index is 0.5 or more, normal, the error index is 0 and if the pol index is more than 0.33 non-syndrome sensorineural hearing loss, if the error index is 0 and the pol index is 0.33 or less may further include diagnosing MDP syndrome Can be.
  • the error index can be expressed as 'relative primer extension efficiency x (1-relative exonuclease activity)', and the error index and pol index are the results of experiments measuring relative primer extension efficiency and relative exonuclease activity. Can be calculated based on
  • Another aspect includes measuring the activity of DNA polymerase and exonuclease, the active domains of POLD1; And measuring the activity of the exonuclease relative to the measured DNA polymerase activity, or measuring the activity of the DNA polymerase relative to the normal control group to diagnose cancer, MDP syndrome, and sensorineural hearing loss. Provide a method of treatment.
  • the phenotype is represented by cancer, MDP syndrome, and sensorineural hearing loss, and according to one aspect, cancer, MDP syndrome, and by measuring the activity of DNA polymerase and exonuclease, the active domains of POLD1, and Sensory neuropathy can be discriminated at the same time for early diagnosis and customized treatment.
  • SB127 is a family diagram to which SB127-497, SB127-277, SB127-235, SB127-247, and SB127-219 belong to a cohort (SB127), which is a test subject of the present invention. Moreover, it is a graph which shows the result of having confirmed the hearing of SB127-235, SB127-247, SB-127-277, and SB127-219.
  • Figure 2b is a diagram showing Sanger sequencing trace results for p.S197HfsX54 and c.G3298A in the initiator SB127-219.
  • Figure 2c is a diagram showing the results of confirming the preservation in the allolog and paralog of POLD1.
  • Figure 3a is a diagram showing the results confirmed by the primer extension assay polymerase activity of wild type (WT) Pol ⁇ or mutant Pol ⁇ .
  • 3B is a diagram showing primer extension results of Pol ⁇ wild type or p.G1100R mutants from two independent samples.
  • FIG. 3C is a diagram showing the results of polymerization activity identification as a percentage of fully elongated product in total primer substrate at 50, 100, and 200 fmol.
  • Figure 3d is a diagram showing the results of confirming the exonuclease activity of wild type (WT) Pol ⁇ or mutant Pol ⁇ .
  • Figure 4 shows the results confirmed by immunoblotting the adhesion of p.G1100R mutant Pol ⁇ and wild type Pol ⁇ to the primer / template DNA.
  • FIG. 5 is a diagram showing the results of confirming the cell level of POLD1 in lymphocytes derived from patients (SB127-235, SB127-219, SB127-284, and SB127-291).
  • Fig. 7 shows the results of confirming the expression of POLD1 in the mouse inner ear by fluorescence microscopy (scale bar, 50 ⁇ m).
  • FIG. 8 is a schematic diagram of a disease diagnosis method using the POLD1 gene showing phenotypic pleiotropic expression.
  • the cohorts were classified into hearing impairment before language acquisition and hearing impairment after language acquisition.
  • deafness before language acquisition refers to the beginning of hearing loss before 3 years of age, which is the age at which speech is usually developed.
  • hearing impairment before language acquisition refers to hearing loss after the beginning of speech development.
  • the cohort was composed of 37 individuals classified as deaf before language acquisition and 14 as classified as deaf after language acquisition. Patients / guardians and their relatives were interviewed to exclude perinatal, otonic neurotoxicity, infectious, and traumatic factors that could lead to non-genetic hearing loss from a comprehensive clinical record.
  • Sequencing reads were aligned with the human genome reference (hg19) using BWA v 0.7.5 and SAMTOOLS v 0.1.18 and CATK v 2.4-7 and Picard v 1.93 were used for sorting, indexing, rearrangement, and duplicate read marks. Variations were named using the GATK Unified Genotyper, and the variations were remeasured. Variations were annotated using ANNOVAR.
  • 918991 variants were selected from the WES data, of which 24279 exon and splicing variants (SNVs and InDels) were selected. The same SNV was deleted. Since then, 970 rare variants with an allele frequency of ⁇ 1% were identified in a normal Korean database of NHLBI-ESP 6500, 1000 Genome Project, and 1020 Korean individuals. The 407 mutations were then screened by excluding the previously reported monobasic dysplasia (dbSNP) variants that did not cause disease but without the flagged SNPs that were reported to be clinically significant. Then 12 mutations were screened according to the genetic pattern, which is shown in Table 1 below.
  • dbSNP monobasic dysplasia
  • the p.S197HfsX54 mutation was inherited from mother (SB127-497), and the p.G1100R mutation was assumed to be inherited from the deceased father. In addition, it was confirmed that these two mutations were not detected in the 2040 control chromosome of the Korean control group with normal hearing.
  • SB127 non-synthetic hearing loss household
  • the genotype of SB127-497 is p.S197HfsX54 / +
  • the genotype of SB127-277 is p.S197HfsX54 / p.G1100R
  • the genotype of SB127-235 is + / p.G1100R
  • the genotype of SB127-247 is + / +
  • SB127- The genotype of 219 is p.S197HfsX54 / p.G1100R.
  • Figure 2b is a diagram showing Sanger sequencing trace results for p.S197HfsX54 and c.G3298A in the initiator SB127-219.
  • Figure 2c is a diagram showing the results of confirming the preservation in the allolog and paralog of POLD1.
  • the two amino acid residues of POLD1, p.S197 and p.G1100 are highly conserved from the olsolog of POLD1 to human zebrafish, and only some of the p.G1100 residues in the paralog It was confirmed that it was preserved.
  • Blood tests were also performed on the subjects. Blood tests include a panel of lipids to measure serum triglycerol and cholesterol levels. In addition, basal and postprandial glucose, insulin resistance, serum HbA1c levels, thyroid function (meaning serum thyroid stimulating hormone and free thyroxine [free T4]), and liver function (aspartate aminotransferase and alanine aminotransferase) Mean).
  • the lipid panel can identify fat dysplasia characterized by a deficiency of fat cells and hypertriglyceridemia in MDP syndrome.
  • Metabolic disorders, insulin resistance, and other possible metabolic defects in MDP syndrome were screened using glucose and insulin resistance, HbA1c levels for confirming hypoxic function, and liver function tests. Table 3 shows the results of the blood tests performed on this characteristic test for SB127-219.
  • SB127-219 and SB127-277 exhibited normal skeletal morphology, facial morphology, fat distribution and reproductive function without exceptional features.
  • SB127-219 showed a normal serum lipid profile and normal glucose and insulin levels.
  • the results of thyroid and liver function tests were also confirmed normal. Normal fasting insulin and triglyceride levels in SB127-219 were also clearly observed to be normal.
  • Pol ⁇ with mutated p.P327L, p.S478N, p.S605del, and p.G1100R was used.
  • primer extension assays were performed in the presence of replication factor C (RFC) and PCNA (processivity clamp proliferating cell nuclear antigen).
  • the p.P327L and p.S478N mutations are mutations present in the exonuclease domain, both of which reduce exonuclease activity of Pol ⁇ and are known to be associated with colorectal or endometrial cancer.
  • the p.S605del mutation is known to be associated with MDP syndrome.
  • Pol ⁇ holoenzyme has four subunits: p125 (Pol3), p55 (Pol31), p40 (Pol32), and p18 (Cdm1).
  • the largest subunit, p125 is encoded by POLD1, is highly conserved in eukaryotes, and contains a polymerase active site, an exonuclease active site, a protein-protein interaction site such as a C-terminal zinc finger, PCNA ( It has several domains, such as binding motifs for processivity clamp proliferating cell nuclear antigens. PCNA has been reported to be essential for high productivity of pol ⁇ .
  • the p.G1100R mutant Pol ⁇ was prepared by expressing c.G3298A (p.G1100R), another variation detected in bacteria.
  • site specific mutation induction was performed by PCR using appropriate primers.
  • Expression and purification of recombinant human Pol ⁇ was performed by known methods.
  • Primer extension assay was performed as follows. DNA polymerization activity was measured by 32 P-labeled 41 nt primers annealed to 93 nt templates. All primer extension reactions were performed at 37 ° C. for 15 minutes in the presence of 200 fmol of trimer PCNA and 100 fmol of RFC. After the reaction, samples were subjected to sequencing gel electrophoresis and detected using a phosphoimager. Polymerization activity is expressed as percent of fully elongated product of the total primers used in the reaction.
  • Figure 3a is a diagram showing the results confirmed by the primer extension assay to confirm the polymerase activity of wild type (WT) Pol ⁇ or mutant Pol ⁇ .
  • the recombinant mutant Pol ⁇ with p.P327L and p.S478N mutations had similar levels of polymerization activity as the wild type, whereas Pol ⁇ with p.G1100R mutations had wild type and p.P327L and p.S478N mutations. Compared to the recombinant mutant Pol ⁇ showed a low level of DNA polymerization activity.
  • 3B is a diagram showing primer extension results of Pol ⁇ wild type or p.G1100R mutants from two independent samples.
  • FIG. 3C is a diagram showing the results of polymerization activity identification as a percentage of fully elongated product in total primer substrate at 50, 100, and 200 fmol.
  • the G1100R mutant Pol ⁇ has similar levels of exonuclease activity as the wild type, whereas the exonuclease activity of the p.P327L, p.S478N, and p.S605del mutant Pol ⁇ are wild type, respectively. Of ⁇ 30%, -25%, and -40%.
  • the p.G1100R mutant Pol ⁇ binds to the DNA substrate with an efficiency similar to that of the wild type.
  • the decrease in polymerization activity observed in the p.G1100R mutant Pol ⁇ is not due to the reduced accessibility of the mutant enzyme to the DNA substrate.
  • the p.G1100R mutant Pol ⁇ which maintains normal binding activity to the DNA substrate, competes with the wild type Pol ⁇ , potentially leading to further reduction of Pol ⁇ polymerization activity in a predominantly negative manner in the normal carrier state.
  • FIG. 5 is a diagram showing the results of confirming the cell level of POLD1 in patients (SB127-235, SB127-219, SB127-284, and SB127-291) and control (cont1, cont2) -derived lymphoblast cell lines.
  • DNA synthesis by all three extracts was inhibited by apidicholine but not by ddTTP, meaning that DNA synthesis activity was mostly catalyzed by replication enzymes.
  • mice 3 weeks old C57BL / 6J mice were purchased from SLC, Inc. (Shizuoka, Japan). Animal experiments for histological studies were performed according to the regulations on animal experiments at Kyoto University.
  • Rabbit anti-POLD1 polyclonal antibody (pAb) and mouse anti- ⁇ -tubulin monoclonal antibody (mAb) were purchased from ATLAS ANTIBODIES (# HPA046524) and Covance (# MMS-435P), respectively.
  • DAPI # D1306)
  • Alexa Fluor 546-phalloidin # A22283
  • Alexa Fluor 488 goat anti-rabbit IgG pAb # A11008
  • Alexa Fluor 546 goat anti-mouse IgG pAb # A11030
  • the inner ear was dissected from the mice and fixed with 4% (w / v) formaldehyde in PBS for 2 hours at room temperature (RT). After fixation, the tissues were washed with PBS and delimed at 4 ° C. for 36 hours with 10% (w / v) EDTA and incubated for 4 hours at 4 ° C. with 10-30% sucrose / PBS solution respectively.
  • the sample was cryopreserved with OCT compound (Sakura Finetek Japan, Inc.) as liquid nitrogen. The frozen sections were cut into 10-12 ⁇ m thick and fixed on slide glass, followed by air drying for 30 minutes.
  • Fig. 7 shows the results of confirming the expression of POLD1 in the mouse inner ear by fluorescence microscopy (scale bar, 50 ⁇ m).
  • RM Reissner's membrane
  • SV stria vascularis
  • SL spiral ligament
  • OC organ of Corti
  • TM technical membrane Surrounded by TM.
  • POLD1 immune responses were found in the cell portions (arrows) in RMs, nuclei of cells in SL, and OC.
  • strong nonnuclear signals of SV and corti rods appear to be false positive (indicated by an asterisk in FIG. 7A).
  • FIG. 7C to 7E are photographs observed at higher magnification of the cochlear side shown in squares in FIG. 7A.
  • FIG. 7C shows POLD1
  • FIG. 7D shows DAPI
  • FIG. 7E shows POLD1 / DAPI.
  • FIG. 7F to 7H are photographs of the primary sensory neurons of the spiral ganglion (SG), cochlea at higher magnification.
  • FIG. 7F shows POLD1
  • FIG. 7G shows ⁇ III-tubulin
  • FIG. 7H shows DAPI / POLD1 / ⁇ IIII-tubulin.
  • Example 8 for the prediction of phenotypes associated with mutations Polymerase And Proofing Proposed Hypothesis Model Through Modulation of Activity
  • MDP syndrome multi-system disease with mandibular dysplasia, deafness and premature ejaculation
  • the error index and the pol index are calculated by calculating the average of the two alleles after calculating the error index and the pol index for the two alleles of POLD1, respectively.
  • POLD1 is a gene expressing phenotypic polymorphism because the phenotype appears very differently to sensorineural hearing loss, cancer, and MDP syndrome depending on the genotype of c.584_585delGA nucleotide or c.G3298A nucleotide variation in POLD1.

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Abstract

La présente invention concerne un kit pour diagnostiquer le cancer, le syndrome de MDP et la surdité de perception à l'aide d'un POLD1 présentant une pléiotropie génétique, et un procédé pour obtenir des informations pour le diagnostic. Les phénotypes de la mutation de POLD1 sont exprimés dans le cancer, le syndrome de MDP et la surdité de perception en fonction des génotypes, et par conséquent selon un aspect de la présente invention, l'activité des ADN polymérases et des exonucléases des domaines actifs de POLD1 est mesurée de façon que le cancer, le syndrome de MDP et la surdité de perception puissent être simultanément identifiés, ce qui permet un diagnostic précoce des maladies et un traitement personnalisé.
PCT/KR2018/003063 2017-03-15 2018-03-15 Pold1 présentant une pléiotropie génétique et méthode de diagnostic de maladies l'utilisant Ceased WO2018169333A1 (fr)

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US20090226920A1 (en) * 2008-02-19 2009-09-10 Med-El Elektromedizinische Geraete Gmbh Mutation In The Regulatory Region Of GJB2 Mediates Neonatal Hearing Loss Within DFNB1
US20120219944A1 (en) * 2011-02-24 2012-08-30 Kongju National University Industry-University Coopertion Foundation Myh14 as causative gene responsible for complex phenotype of peripheral neuropathy, myopathy, hearing loss and hoarseness, and diagnostic method and kit for the complex phenotype using the same
US20160090597A1 (en) * 2014-09-30 2016-03-31 University Of Iowa Research Foundation Methods to prevent and treat autosomal dominant non-syndromic hearing loss

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US20090226920A1 (en) * 2008-02-19 2009-09-10 Med-El Elektromedizinische Geraete Gmbh Mutation In The Regulatory Region Of GJB2 Mediates Neonatal Hearing Loss Within DFNB1
US20120219944A1 (en) * 2011-02-24 2012-08-30 Kongju National University Industry-University Coopertion Foundation Myh14 as causative gene responsible for complex phenotype of peripheral neuropathy, myopathy, hearing loss and hoarseness, and diagnostic method and kit for the complex phenotype using the same
US20160090597A1 (en) * 2014-09-30 2016-03-31 University Of Iowa Research Foundation Methods to prevent and treat autosomal dominant non-syndromic hearing loss

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LESSEL, DAVOR ET AL.: "POLD1 Germline Mutations in Patients Initially Diagnosed with Werner Syndrome", HUMAN MUTATION, vol. 36, no. 11, 2015, pages 1070 - 1079, XP055560193 *
WEEDON, MICHAEL N. ET AL.: "An In-frame Deletion at the Polymerase Active Site of FOLD 1 Causes a Multisystem Disorder with Lipodystrophy", NATURE GENETICS, vol. 45, no. 8, 2013, pages 947 - 950, XP055560190 *

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