KR101981204B1 - Marker POLD1 for diagnosing sensorineural hearing loss and uses thereof - Google Patents

Abstract

The present invention relates to a marker POLD1 for diagnosing sensory nerve disorder and its use. More specifically, a polynucleotide comprising a consecutive nucleotide sequence selected from the nucleotide sequence of the exon region of the POLD1 gene or a complementary polynucleotide thereof And a method for detecting the mutation of the genomic DNA in order to provide information on diagnosis. The present invention relates to a method for detecting a mutation of the POLD1 gene using a kit or a composition according to one aspect, It can be analyzed with high sensitivity and accuracy. Based on these analysis results, it is possible to diagnose sensory nerve impairment efficiently.

Description

Marker POLD1 for diagnosis of sensory nerve impairment and its use

The present invention relates to a marker POLD1 for diagnosing sensory nerve disorder and its use. More specifically, a polynucleotide comprising a consecutive nucleotide sequence selected from the nucleotide sequence of the exon region of the POLD1 gene or a complementary polynucleotide thereof And a method for detecting the mutation of the genomic DNA to provide information on diagnosis. 2. Description of the Related Art

Congenital hearing loss occurs in about 3 out of 1,000 neonates, and more than 50% is known to be caused by genetic factors. Hereditary hearing loss is congenital and occurs after birth. There are many types of hearing loss, but sensorineural hearing loss is the most common.

Sensory nerve impairment refers to hearing loss caused by an abnormality in the function of detecting the sound of the cochlea or of an auditory nerve or a central nervous system that transmits a stimulation by sound to the brain. Sensory nerve impairment is classified into syndrome and non-syndrome according to the symptoms. Syndrome Sensory nerve impairment refers to the clinical symptoms or symptoms of other organs other than those caused by abnormal function of inner ear. It accounts for 30% of hereditary hearing loss, and over 300 types of syndromic hearing loss have been reported to date. Because it is easily classified as a characteristic symptom or anomaly, the causative gene can be easily traced in patients with the same cause. Non-syndrome Sensory neural deafness refers to a case in which there are no abnormal symptoms or symptoms of other organs other than internal dysfunction. It accounts for 70% of hereditary deafness, has a variety of causative genes, and strategic genetic diagnosis is required based on clinical information such as type of hearing loss, genetic type, and so on. Of the non-syndromic congenital hearing loss, 20% are caused by inheritance, 17% by dominant inheritance, 2-3% by X-linked and 1% by mitochondrial inheritance, Is known to be caused by mutation of GJB2 (Gap Junction Beta 2) gene.

The hearing loss genes may exhibit variable expressivity. One of the hearing loss genes may be expressed as a syndrome or non-syndrome deafness depending on the gene expression by the mutation type, and one mutation of the hearing loss gene may be a kind of mutation , Or they may be expressed by recessive inheritance or dominant inheritance.

On the other hand, genetic pleiotropy is an extreme form of various expressions, which refers to a phenomenon in which different mutations of one gene are not related to each other. For example, another mutation in one gene is expressed as a phenotype that represents an abnormality in another organ. The phenotypic mechanism of expression is the mechanism by which different specific mutations of a gene have different effects on the protein and the different mutations of one gene affect the total amount of functionally intact proteins to represent different phenotypes .

To date, some hearing loss genes have been studied, but the relationship between POLD1 mutations and sensory nerve deafness has never been reported. Under these circumstances , the present inventors have made efforts to find a marker for diagnosing sensory neuronal deafness, The present inventors have completed the present invention by confirming that it is possible to diagnose sensory nerve impairment by using mutations.

An aspect of the present invention is to provide a diagnostic kit for a sensory neuron deafness disorder comprising a polynucleotide comprising a consecutive nucleotide sequence selected from a nucleotide sequence of the exon region of the POLD1 gene or a complementary polynucleotide thereof to detect a mutation of the POLD1 gene.

Another aspect is to provide a composition for the diagnosis of sensory neural deafness comprising a polynucleotide comprising a consecutive nucleotide sequence selected from a nucleotide sequence of the exon region of the POLD1 gene or a complementary polynucleotide thereof to detect a mutation of the POLD1 gene.

In another aspect, the method comprises contacting the composition with a genomic DNA isolated from an individual to obtain a hybridization product of the genomic DNA and the polynucleotide in the composition; Identifying a nucleotide sequence of the genomic DNA in the hybridization product; And comparing the confirmed nucleotide sequence of the genomic DNA with a standard nucleotide sequence to identify the mutation of the genomic DNA. A method for detecting the mutation of the genomic DNA to provide information on the diagnosis of the sensory neuron deafness disorder .

One aspect provides a diagnostic kit for a sensory neuron deafness disorder comprising a polynucleotide comprising a contiguous nucleotide sequence selected from a nucleotide sequence of the exon region of the POLD1 gene or a complementary polynucleotide thereof to detect a mutation of the POLD1 gene.

The term " polynucleotide " refers to a nucleotide polymer having an arbitrary length, wherein a polynucleotide can be used interchangeably with a nucleic acid, or an oligonucleotide.

The term " gene " refers to a structural unit that determines genetic information. The term " gene " refers to a structural unit that determines the nucleotide sequence of the protein's amino acid sequence or functional RNA (tRNA, rRNA, etc.) (E. G., A promoter, a repressor, an operative gene, etc.) that regulates gene expression. As used herein, the term " gene " is understood to mean a single stranded side comprising a nucleotide sequence that is transcribed to produce a product of the gene. For example, " nucleotide sequence of a gene " means a nucleotide sequence encoding a function contained in a single strand containing a nucleotide sequence to be transcribed to produce a product of the gene, and / or a nucleotide sequence controlling the expression of the structural gene It can mean.

The term " contiguous nucleotide sequence " refers to two or more contiguous nucleotide sequences.

The term " complementary " means having complementarity enough to selectively hybridize to the above-described nucleotide sequence under any particular hybridization or annealing conditions. The term " complementary " may encompass both substantially complementary and perfectly complementary, and may in particular be meant to be fully complementary.

The term " exon " refers to a region containing the synthesis information of a protein in a DNA sequence, for example, a nucleic acid molecule encoding part or all of the expressed protein.

The mutation may be one having a variation relative to the standard genomic DNA. Specifically, the mutation may include a mutation of the copy number of the gene or a mutation of the nucleotide sequence to the standard genomic DNA. The variation of the copy number of the gene may be, for example, Copy Number Variation (CNV). The variation of the nucleotide sequence may include substitution, insertion, deletion, or translocation of one or more nucleotide sequences relative to the standard genomic DNA. The substitution of the one or more nucleotide sequences may be, for example, Single Nucleotide Variation (SNV). Also, the mutation of the nucleotide sequence may comprise substitution, insertion, deletion, or translocation of one or more nucleotide sequences relative to the standard genomic DNA.

The term " Single Nucleotide Variation (SNV) " refers to a single nucleotide polymorphism (SNP) that refers to a single base difference in a large number of populations within a species, Refers to the difference of a single nucleotide in a population of nucleotide sequences, for example, a difference from a nucleotide sequence that appears in sequencing data.

The term " Copy Number Variation (CNV) " means a variation of a genomic DNA repeatedly appearing when a relatively large region of a specific chromosome is deleted or amplified. For example, It may be a variation where some are lost.

The mutation may not be a mutation of a DNA polymerase or an exonuclease, which is an active domain of POLD1, and may be a thermolabile mutation.

Such a mutation may be, for example, a p.S197HfsX54 or p.G1100R mutation. In the present invention refers to p.S197HfsX54 c.584_585delGA variation in POLD1, and is referred to p.G1100R c.G3298A variation. The c.584-585 dELGA mutation refers to a mutation in which guanine and adenine, which are 584 to 585 nucleotides in the POLD1 gene, are deleted, and the c.G3298A mutation refers to a mutation in which guanine, which is a 3298th nucleotide in the POLD1 gene, is substituted with adenine.

The polynucleotide may be, for example, 10 to 100 nucleotides. When the polynucleotide has a size of 10 or less, the accuracy of capturing the target region is low, and when the polynucleotide has 100 or more sizes, the synthesis cost is increased. Therefore, the polynucleotide is economical and has a size optimized for detection of mutation of the genomic DNA.

The sensory neural deafness may be a non-syndromic sensorineural hearing loss (NS-SNHL). Sensory nerve impairment refers to hearing loss caused by an abnormality of the auditory nerve system or abnormality of the central nervous system, which causes abnormalities in the function of detecting the sound of the cochlea or the stimulation of the sound to the brain. Non-syndrome sensory nerve impairment Refers to hearing loss that does not show abnormal symptoms or symptoms of other organs.

The polynucleotide of the present invention can specifically bind to the sequence of the target gene. Using the specific binding characteristics of such polynucleotides, the target gene or fragment thereof can be effectively separated from the mixture from the mixed sample. Therefore, the polynucleotide may be referred to as a probe. The term " probe " means a substance that specifically detects a specific substance, site, state, and the like.

The kit may further comprise known materials required for hybridization of the polynucleotide with the genomic DNA. For example, it may further comprise reagents, buffers, buffers, cofactors, and / or substrates necessary for hybridization of the nucleic acid. In addition, when the kit is applied to a PCR amplification process, it may optionally include reagents necessary for PCR amplification, such as a buffer, a DNA polymerase, a DNA polymerase cofactor, and dNTPs. When the kit is applied for immunoassay , The kit of the present invention may optionally comprise a secondary antibody and a labeling substrate. The kit may further include instructions for use to amplify the target nucleic acid, and may be made from a number of separate packaging or compartments containing the reagent components described above.

Another aspect provides a composition for the diagnosis of sensory neural deafness comprising a polynucleotide comprising a contiguous nucleotide sequence selected from a nucleotide sequence of the exon region of the POLD1 gene or a complementary polynucleotide thereof to detect a mutation of the POLD1 gene.

The polynucleotide or its complementary polynucleotide is as described above.

In another aspect, the method comprises contacting the composition with a genomic DNA isolated from an individual to obtain a hybridization product of the genomic DNA and the polynucleotide in the composition; Identifying a nucleotide sequence of the genomic DNA in the hybridization product; And comparing the confirmed nucleotide sequence of the genomic DNA with a standard nucleotide sequence to identify the mutation of the genomic DNA. A method for detecting the mutation of the genomic DNA to provide information on the diagnosis of the sensory neuron deafness disorder .

In the above detection method, the composition is as described above.

The genetic DNA mutation may include a mutation of the copy number of the gene or a mutation of the nucleotide sequence with respect to the standard genomic DNA, as described above.

The genetic DNA mutation may specifically include at least one of a single nucleotide mutation, an insertion-deletion mutation, a copy number mutation, and a translocation, and more specifically, may include a single nucleotide mutation, an insertion- deletion mutation, have.

The term " insert-deletion mutation (Indel) " means that a nucleotide sequence capable of changing the number of nucleotides of a gene is inserted or deleted.

The term " translocation " refers to a phenomenon in which a cleavage occurs in a part of a chromosome, and the fragment binds to another part of the same chromosome or another chromosome, thereby changing the shape of the chromosome.

The genomic DNA isolated from an individual in the above detection method may be a genomic DNA separated from a biological sample or a fragment thereof. For example, the sample may be any one selected from the group consisting of blood, saliva, urine, feces, tissues, cells, and biopsies. The sample may be one that contains a stored biological sample or genomic DNA isolated therefrom. The storage may be stored by known methods. The genomic DNA may be DNA or RNA derived from tissues stored at room temperature in cryopreserved or formalin-fixed paraffin-embedded tissues. Methods for isolating genomic DNA from biological samples are well known. The cancer cells may be isolated from cancer patients. Thus, the sample may be isolated from cells, tissues, organs, body fluids of the hearing loss patient, in which case the sample may be subjected to biopsy using conventional methods, for example, methods well known to those skilled in the relevant medical arts ≪ / RTI >

In one embodiment, the genomic DNA contained in the sample may be fragmented to any size. The fragmentation can be performed by methods well known to those skilled in the art. For example, genomic DNA can be fragmented by using ultrasonic waves. The detection method may include ligation of a sequence for amplification at both ends of the fragmented genomic DNA after the fragmentation of the genomic DNA. The ligation method of the amplification sequence (for example, a paired-end tag or a universal tag) can be performed by appropriately selecting a known technique by a person skilled in the art.

In the above detection method, the hybridization may be performed by a known method. For example, by incubating the polynucleotide and the genomic DNA in a buffer known to be appropriate for the hybridization of the nucleic acid. Hybridization can be performed at an appropriate temperature. Suitable temperatures for hybridization may be, for example, 40 to 80 占 폚, 50 to 75 占 폚, 60 to 70 占 폚, or 62 to 67 占 폚. The hybridization temperature is not limited thereto, and can be appropriately selected depending on the sequence and the length of the polynucleotide contained in the composition. The hybridization time can be, for example, from 1 hour to 12 hours (overnight). In one embodiment, the polynucleotide contained in the composition is hybridized with a fragment of a genomic DNA having a sequence of the target genes.

The method may further comprise separating the hybridization product of the genomic DNA and the polynucleotide from the contact product obtained in the contacting step, before confirming the nucleotide sequence of the genomic DNA in the hybridization product. The separation may be by using a moiety for separation or purification attached to the polynucleotide. The separation or purification may be carried out by a substance or a magnetic field that specifically binds to the moiety.

The method may further include amplifying the genomic DNA by PCR using the isolated hybridization product or the genomic DNA as a template and using a universal primer complementary to the sequence for amplification attached to each of the genomic DNAs as a primer, As shown in FIG. The nucleotide sequence can be confirmed using the amplified genomic DNA.

Identification of the nucleotide sequence can be confirmed by, for example, a sequencing method. Specifically, the nucleotide sequence can be confirmed by a next-generation sequencing method. The term " next generation sequencing " (NGS) sculpts a full-length genome in a chip-based and PCR-based paired end format, . Next-generation sequencing can generate a large amount of sequence data for a sample to be analyzed within a short time.

The method comprises comparing the identified nucleotide sequence of the genomic DNA with a standard sequence. The term " reference neucleotide sequence " can refer to a human genomic sequence that does not contain a mutation that is referenced for mutation identification. For example, a human nucleotide sequence, specifically NCBI37.1 or UCSC hg19 (GRCh37), published in the database of the National Institute of Bioscience and Biotechnology Information (NCBI) under the National Institutes of Health may be used as the standard sequence. A comparison between the nucleotide sequence of the genomic DNA and the standard nucleotide sequence can be performed using various known sequence comparative analysis programs such as Maq, Bowtie, SOAP and GSNAP.

Refers to any animal classified as a mammal susceptible to, or suspected of having, hearing loss, and refers to any animal classified as a mammal, including, , Pigs, sheep, goats, dogs, cats or rodents. Specifically, the subject is a human male or female of any age or race. &Quot; Subject " and " patient " are used interchangeably herein.

In the step of obtaining the genomic DNA from the subject, the genetic DNA may be obtained from the blood of the subject, or oral epithelial cells. The method of obtaining can utilize methods known to those skilled in the art of separating genomic DNA from tissues or cells.

When the mutation is confirmed in the POLD1 gene through the above method, the individual can be diagnosed as having a sensory neural deafness disorder. The method can be used to diagnose sensory nerve impairment in an individual, without further experimentation in in vitro and in vivo conditions.

All technical terms used in the present invention are used in the sense that they are generally understood by those of ordinary skill in the relevant field of the present invention unless otherwise defined.

Using the kits or compositions according to one aspect, the mutation of the POLD1 gene can be analyzed with high sensitivity and accuracy, and the sensory neuron deafness can be efficiently diagnosed based on the analysis result.

Figure 1 is a family diagram of SB127-497, SB127-277, SB127-235, SB127-247, and SB127-219, which are members of the cohort (SB127) subject of the present invention. Also, it is a graph showing the results of hearing tests of SB127-235, SB127-247, SB-127-277, and SB127-219.
Figure 2a shows the result of singer sequencing tracing for p.S197HfsX54 and c.G3298A in normal subjects SB127-247.
FIG. 2B is a diagram showing the result of singer sequencing tracking for p.S197HfsX54 and c.G3298A in the initiator SB127-219.
FIG. 2C is a view showing the result of confirming the preservation in the oligo logarithm and para log of POLD1.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example  1: Select experiment object

In the present invention, the experiment was approved by the Institutional Review Committee of SNUBH (IRB-B-1007-105-402) and SNUH (SNUH) of Seoul National University Bundang Hospital (IRBYH-0905-041-281) Respectively.

In order to measure the prevalence of mutations in Korean individuals with hearing impairment and to find individuals with autosomal recessive or sporadically isolated nonsyndromic sensinaural hearing loss (NS-SNHL) Syndrome features or radiographic markers. Next, sequencing of GJB2 (gap junction protein beta 2) was performed by a known method. Finally, from May 2010 to April 2012, 51 foot terminals (cohort) with negative GJB2 mutations were selected from 51 unrelated households in the SNUH and SNUBH otolaryngology departments.

The cohort was categorized as hearing impairment before language acquisition and after hearing impairment according to the timing of hearing loss. In the present invention, hearing impairment prior to language acquisition refers to the onset of hearing loss prior to the age of 3 years of speech development. On the other hand, hearing impairment before language acquisition refers to hearing loss after the onset of speech development. In the present invention, attention has been paid to foot terminals having hearing impairment after language acquisition. As a result of the classification, it was confirmed that the cohort consisted of 37 participants who were classified as hearing impaired before language acquisition and 14 participants who were classified as hearing impaired after language acquisition. Patients / caregivers and their relatives were interviewed to exclude perinatal, neurological, infectious, and traumatic factors that could lead to inhaled hearing loss from comprehensive clinical records.

Example  2: Molecular genetic screening

To screen for candidate variants associated with sensory nerve impairment by molecular genetic screening, the following was performed.

Whole blood was extracted from SB127-219, a rudimentary sensorineural hearing loss (SNHL) with no developmental disturbance. Whole blood was extracted from the whole exomnia using the SureSelect Human All Exon Kit V3 (Agilent, Santa Clara, CA, USA) Sequencing (Whole exome sequencing: WES) was performed. In addition, sequencing was performed using HiSeq 2000 (Illumina, San Diego, Calif., USA) with a 101 base pair reading and bioinformatics analysis was performed on whole-exome sequencing reveals diverse modes of inheritance in sporadic mild to moderate sensorineural hearing loss in a pediatric population. Genet Med 17: 901-911. Sequencing readings 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 alignment, indexing, rearrangement, and duplicate readout. The mutation was named using the GATK Unified Genotyper and the mutation was re-measured. Variations were annotated using ANNOVAR.

After selecting 918991 mutations from the WES data, 24279 exon and splicing mutations (SNVs and InDels) were selected. Of these, the same SNV was deleted. We then identified 970 rare mutations that showed an allele frequency of <1% in a normal Korean database of NHLBI-ESP 6500, 1000 Genome Project, and 1020 Koreans. 407 mutations were screened by excluding previously reported single nucleotide polymorphism (dbSNP) mutations that did not cause disease later, but excluding flagged SNPs reported to be clinically significant. Then, 12 mutations were screened according to the genetic pattern, which is shown in Table 1 below.

gene ExonicFu nc Genbank No Exon Nucleotides protein Sanger Sequencing SB127-219 (onset) SB127-235 (normal) SB127-247 (normal) SB127-277
(attack) LYSMD3 nonsynonymous SNV NM_198273 3 c.C569G p.T190R Confirmed + + + LYSMD3 nonsynonymous SNV NM_198273 3 c.T415G p.S139A Confirmed + + + HIVEP1 nonsynonymous SNV NM_002114 4 c.A778G p.T260A Confirmed + - + HIVEP1 nonsynonymous SNV NM_002114 4 c.A1570G p.N524D Confirmed + - + OR2W1 nonframeshift deletion NM_030903 One c.747_749delTAT p.M250del Confirmed + + - OR2W1 nonsynonymous SNV NM_030903 One c.C746A p.S249Y Confirmed + + - RHOBTB 2 nonsynonymous SNV NM_001160037 5 c.G751T p.V251L Confirmed + + - RHOBTB 2 nonsynonymous SNV NM_001160037 6 c.G1597A p.A533T Confirmed + + - ATAD2 nonframeshift deletion NM_014109 7 c.828_833delTGATGA p.D276_D277del Confirmed ATAD2 nonframeshift deletion NM_014109 7 c.807_818del p.D269_D272del Failed POLD1 frameshift deletion NM_001256849 5 c.584_585delga p.S197HfsX54 Confirmed + - - + POLD1 nonsynonymous SNV NM_001256849 27 c.G3298A p.G1100R Confirmed + + - +

Twelve mutations in LYSMD3 , HIVEP1 , OR2W1 , RHOBTB2 , ATAD2 , and POLD1 were detected as complex heterozygous zygotes after the step I filtering process based on the dbSNP database, allele frequency, and genetic pattern.

Among the above 12 mutations, other mutations were confirmed by ginger sequencing, but ATAD2 was excluded because it was confirmed that a false positive reaction was observed for the mutation of ATAD2 . Next, we identified family phase and segregation of mutations in two uninfected brothers (SB127-235 and 247) and one affected sister (SB127-277), and when they matched, selected them as mutation candidates, and finally P.S197HfsX54 with c.584_585delGA mutation and p.G1100R with c.G3298A mutation were selected in POLD1.

The selection process of p.S197HfsX54 and p.G1100R, which are two mutations in POLD1 described above, is shown in Table 2 below.

No step Number of mutations One Raw SNV 918991 2 Annotated variants 918991 3 Exonic and splicing variants 24279 4 After elimination of synonymous SNV 12567 5 With allele frequency of <1% 970 6 Not registered in dbSNP + Flagged SNP 407 (404 + 3) 7 Inheritance pattern matched 12 8 Phase or segregation checked 2 9 No detection in normal hearing control subjects 2

The p.S197HfsX54 mutation was inherited from the mother (SB127-497) and the p.G1100R mutation was presumed to have been inherited from the deceased father. Also, it was confirmed that these two mutations were not detected in the 2040 control chromosome of the control group of normal hearing Korean.

FIG. 1 is a block diagram of SB127-497, SB127-277, SB127-235, SB127-247, and SB127-219, which are members of the non-syndromic hearing loss household (SB127) of the present invention. Also, it is a graph showing the results of hearing tests of SB127-235, SB127-247, SB-127-277, and SB127-219.

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 + / + The genotype of 219 is p.S197HfsX54 / p.G1100R.

Sanger sequencing was performed on the selected p.S197HfsX54 and p.G1100R mutations and the conservation of their conservation between oligo and para-log of POLD1 was confirmed. The results are shown in Figs. 2A to 2C.

Figure 2a shows the result of singer sequencing tracing for p.S197HfsX54 and c.G3298A in normal subjects SB127-247.

FIG. 2B is a diagram showing the result of singer sequencing tracking for p.S197HfsX54 and c.G3298A in the initiator SB127-219.

FIG. 2C is a view showing the result of confirming the preservation in the oligo logarithm and para log of POLD1.

As shown in Fig. 2C, it was confirmed that p.S197 and p.G1100, two amino acid residues of POLD1 from humans to zebrafish, were highly conserved in the oligo of POLD1, and only p.G1100 residues It is confirmed that it is preserved.

Example  3: Metabolic  Property Inspection

To exclude the syndromic characteristics of individuals with POLD1 mutations, metabolic characteristics were examined in the affected foot (SB127-219: 27 year old male) and another patient (SB127-277: 40 year old female). In order to rule out the possibility of syndrome hearing loss and MPD syndrome, physical examination and several types of blood tests were performed.

In particular, the general appearance of individuals with POLD1 mutations was assessed by the presence of lipodystrophies, skeletal anomalies, and any other pathological features. The subjects were also subjected to blood tests. Blood tests include serum triglycerol and a lipid panel to measure cholesterol levels. In addition, there is a strong correlation between 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 ). Lipid panel can identify lipodystrophy which is a characteristic of deficiency of adipocytes and hypertriglyceridemia in MDP syndrome. Metabolic disorders, insulin resistance, and other possible metabolic defects were screened in MDP syndrome using glucose and insulin resistance, HbA1c levels for ascertaining hepatic function, and liver function tests. Table 3 shows the results of blood tests performed for SB127-219.

inspection shame Reference value Lipid panel Triglyceride 90 <150 mg / dL cholesterol 140 <200 mg / dL HDL 36 &Gt; 40 mg / dL (M), > 5040 mg / dL (F) Insulin / glucose resistance insulin spandrel 6.8 4-16 ul / ml glucose spandrel 70 <100 mg / dL After meals (2 hours) 86 HbAlC 5.1 3.5-6.4% Thyroid function test TSH 4.14 9-30 ul / ml T4 1.13 0.7-1.9 ng / dl Liver function test AST 24 ALT 23 0-40 U / L

SB127-219 and SB127-277 showed normal skeletal morphology, facial shape, fat distribution and reproductive function without exceptional characteristics. Also, as shown in Table 3 above, SB127-219 showed normal serum lipid profile and normal glucose and insulin levels. Results of thyroid and liver function tests were also normal. Normal fasting insulin and triglyceride levels were also clearly observed in SB127-219.

Therefore, it was confirmed that the diagnosis of non-syndrome sensory neural deafness, which does not exhibit the syndrome characteristic, can be diagnosed when the POLD1 gene mutation, specifically the p.S197HfsX54 or p.G1100R mutation, is present.

Claims (14)

A diagnostic kit for a sensory neuron deafness disorder comprising a polynucleotide comprising a contiguous nucleotide sequence selected from the nucleotide sequence of the exon region of the POLD1 gene or a complementary polynucleotide thereof for detecting the p.S197HfsX54 or p.G1100R mutation of the POLD1 gene. The diagnostic kit of claim 1, wherein the mutation comprises a variation of a nucleotide sequence relative to a standard genomic DNA. 3. The diagnostic kit of claim 2, wherein the mutation of the nucleotide sequence comprises substitution, or deletion, of one or more nucleotide sequences relative to the standard genomic DNA. The diagnostic kit according to claim 1, wherein the mutation is not a mutation of a DNA polymerase and an exonuclease, which are active domains of POLD1. The diagnostic kit according to claim 1, wherein the mutation is a thermal mutation. delete The diagnostic kit of claim 1, wherein the polynucleotide is from 10 to 100 nucleotides in length. The diagnostic kit according to claim 1, wherein the sensory nerve impairment is non-syndromic sensorineural hearing loss (NS-SNHL). A polynucleotide comprising a contiguous nucleotide sequence selected from the nucleotide sequence of the exon region of the POLD1 gene or a complementary polynucleotide thereof for detecting the p.S197HfsX54 or p.G1100R mutation of the POLD1 gene. Contacting the composition of claim 9 with a genomic DNA isolated from an individual to obtain a hybridization product of the genomic DNA and the polynucleotide in the composition;
Identifying a nucleotide sequence of the genomic DNA in the hybridization product; And
Comparing the identified nucleotide sequence of the genomic DNA with a standard nucleotide sequence to identify the p.S197HfsX54 or p.G1100R variant of the genomic DNA, / RTI &gt; 11. The method of claim 10, wherein the p.S197HfsX54 or p.G1100R mutation of the genomic DNA comprises a variation of a nucleotide sequence relative to a standard genomic DNA. 11. The method of claim 10, wherein the pS197HfsX54 or p.G1100R mutation of the genomic DNA is a single nucleotide mutation or an insert-deletion mutation. delete The method according to claim 10, further comprising a step of separating the hybridization product of the genomic DNA and the polynucleotide from the contact product obtained in the contacting step, before confirming the nucleotide sequence of the genomic DNA in the hybridization product .

Images