KR100613038B1 - Diagnosis method and kit for CT disease caused by gene mutation except for overlapping chromosome 17ｐ11.2-ｐ12 region - Google Patents

Abstract

The present invention relates to a diagnostic method and kit for diagnosing a hereditary neurological disease caused by a genetic mutation excluding the overlap of the chromosome 17p11.2-p12 region. In the present invention, a novel CMT disease-causing mutation is identified, and a method for diagnosing hereditary neurological disease using the novel mutation is provided. In addition, the present invention provides a primer for detecting a variety of genetic mutations that cause hereditary neurological diseases and a diagnostic kit for CMT disease using the same. According to the present invention, accurate early diagnosis through genetic testing is possible for CMT disease caused by a single genetic defect with strong heritability, and can be treated early by applying a recently developed treatment method according to the accurate diagnosis, Furthermore, customized treatment according to the exact etiology is possible.

Hereditary neurological diseases, CMT, genetic mutations, diagnostic kits

Description

Diagnosis method and kits for CMT disease caused by mutations except for 17p11.2-p12 duplication}

1A and 1B are chromatograms of gene analysis results of normal (CTL) and patient (Patient) for CMT disease-causing gene mutations.

2 is a result of analyzing the family of patients with CMT disease using the diagnostic kit of the present invention.

The present invention relates to a method for diagnosing and diagnosing a disease of CMT caused by a genetic mutation excluding the overlap of the chromosome 17p11.2-p12 region.

Inherited neuropathy is a congenital disorder that has a family history and indicates dysfunction of motor and sensory nervous systems by mutations in genes. Hereditary neurological disorders include Charcot-Marie-Tooth disease (CMT), hereditary neuropathy with liability to pressure palsies (HNPP), Dejerine-Sottas syndrome (DSS) and congenital hypomyelination (CHS). They are caused by genetically heterogeneous factors and the highest incidence of hereditary neuropathy is about 1 in 2,500 people.

CMT disease is mainly characterized by slow muscle weakness and atrophy in the lower extremity fibrous muscles and is characterized by areflexia, distal sensory loss, pes cavus and deafness. Pray. The onset usually occurs around 10 years of age, but rarely occurs after the age of 30. However, the clinical phenotype and age of onset of CMT disease are very heterogeneous and neural biopsy is difficult to confirm, making accurate diagnosis and treatment difficult (Berger P., Young P., Suter U. Neurogenet. 4: 1-15 (2002)).

Hereditary neurological diseases have been classified as CMT1, a demyelinating neuron, and CMT2, axonal neuropathy, but the classification system has been newly established. CMT1 type is divided into CMT1A, 1B, 1C according to the genetic variation, CMT2 type is divided into 2A, 2B, 2C, 2D, 2E, 2F, 2G. CMT4, an autosomal recessive inheritance, is classified from CMT4A to 4F according to genetic variation. In addition, there are Dejerine-Sottas syndrome (DSS) and congenital hypomyelination (CH), which cause severe symptoms from birth. CMT1A accounts for the highest proportion of CMT diseases and is known to be due to duplication of chromosome 17p11.2-p12 containing the periphral myelin protein 22 (PMP22) gene of peripheral nerve myelin. The deletion of 17p11.2-p12 causes hereditary neuropathy with liability to pressure palsies. The next highest frequency after CMT1A is CMTX, which accounts for 10-20% of all hereditary neurological diseases and is caused by mutations in the Cx32 (Connexin 32) gene that form gap junctions. In addition, mutations in Myelin protein zero (MPZ), Early growth response 2 (EGR2), Neurofilament light (NEFL), and periaxin (PRX) genes also cause peripheral neuropathy including CMT. Table 1 shows the genetic patterns and phenotypes of important peripheral neuropathy causing genes known to date.

Each patient with CMT is caused by a single gene mutation, with varying degrees of symptoms, but the disease is very true to Mendel's law. Since most diseases currently undergoing genetic testing are those caused by a complex interaction of polygenic inheritance and environmental factors, genetic testing of them is merely suggesting a tendency to development. (E.g., MTHFR associated with hyperhomocysteinemia, BRC-A BRC-B genetic tests that cause breast cancer). However, CMT disease is almost directly related to genetic defects and the onset, as in the case of phenylketonuria (PKU), so the genetic test results are more than just a tendency or suggestion of onset. It is a very suitable genetic disease for the following.

Therefore, if the pathogenesis through genetic testing for CMT disease, which is highly heritable and is caused by a single gene defect, will be possible, more accurate diagnosis and fundamental treatment will be possible. In addition, until now, early diagnosis of CMT disease did not raise the necessity of early diagnosis because there is no appropriate treatment method, but ascorbic acid and progesterone antagonist are effective as treatments for CMT1A. This has been published in the paper (Sereda MW, Horste GM, Suter U., Uzma N., Nave K.-A. Nature Genet. 9: 1533-1537 (2003); Passage E., Norreel JC, Noack -Fraissignes P., Sanguedolce V., Pizant J., Thirion X., Robaglia-Schlupp A., Pellissier JF, Fontes M. Nature Genet. 10: 396-401 (2004)). Therefore, if the CMT disease can be correctly diagnosed early, early treatment will be possible according to the development of the treatment, and further customized treatment according to the genetic cause will be possible. The present invention relates to genetic analysis and accurate diagnosis of CMT disease caused by genetic mutations excluding duplication of 17p11.2-p12.

Since the 2000s, there has been considerable research on hereditary neurological diseases in Japan as well as in the US and Europe. Yoshihara et al. (Yoshihara T., Yamamoto M., Doyu M., Misu KI, Hattori N., Hasegawa Y., Mokuno K., Mitsuma T., Sobue G. Hum. Mutat. 16: 177-178 (2000) ) And Numakura et al. (Numakura C., Lin C., Ikegami T., Guldberg P., Hayasaka K. Human Mutat. 20: 392-398 (2002)) have studied CMT1A in over 100 Japanese patients with hereditary neurological diseases. Causal mutations in duplicates, PMP22, MPZ and Cx32 were detected.

Although there are no accurate statistics on the incidence of CMT disease in Koreans, it is estimated to be similar to Europeans. The present invention aims to accurately analyze the genetic causes of the onset of CMT disease in Korean CMT disease patients and their families, and to provide a diagnosis method accordingly.

The object of the present invention is to accurately diagnose CMT disease caused by a gene mutation except for overlapping regions of the chromosome 17p11.2-p12 region.

To this end, the present invention first correctly identifies the gene mutations that cause it. In the present invention, CMT-induced gene mutations are identified through SNP screens for the CMT disease causing genes, PMP22, Cx32, MPZ, EGR2, NEFL and PRX, which have been identified so far. Through this identification process, a novel CMT-novel mutation, which has not yet been reported worldwide, is disclosed in the present invention, and the present invention provides a method for diagnosing hereditary neurological disease using the novel mutation.

In addition, the present invention provides a primer for detecting a variety of gene mutations causing CMT disease, and a diagnostic kit for CMT disease using the same.

In addition, the present invention analyzes the CMT disease gene of Koreans and compares the results with foreign data to reveal the clinical characteristics, age of onset, progression of CMT disease in Korea.

Other objects and advantages of the invention will be described below, and will be better understood by practice of the invention.

In the present invention, seven newly identified CMT disease causing mutations are provided. The contents of the novel mutations identified in the present invention are as follows.

Ala106fs of PMP22: A mutation in which the frameshift occurs from amino acid sequence position 106 (alanine) by deletion of thymine at nucleotide sequence position 318 of the PMP22 gene.

Asp118Asn in MPZ: A mutation in which guanine is replaced with adenine at nucleotide sequence 352 of the MPZ gene, and asparagine is replaced with asparagine at amino acid sequence 118.

449-1G> T of MPZ: 3'-splice site mutation caused by substitution of thymine with guanine at nucleotide sequence 449-1 of the MPZ gene.

Lys236Glu of MPZ: A mutation in which adenine is replaced with guanine at nucleotide sequence 706 of the MPZ gene and lysine is replaced by glutamic acid at amino acid sequence 236.

Val136Ala of Cx32: A mutation in which thymine is substituted for cytosine at nucleotide sequence position 407 of the Cx32 gene so that valine is replaced with alanine at amino acid sequence position 136.

Cys168Arg of Cx32: A mutation in which thymine is replaced with cytosine at nucleotide sequence position 502 of the Cx32 gene, and cysteine is replaced with arginine at amino acid sequence position 168.

Leu334Pro of NEFL: A mutation in which thymine is replaced by cytosine at nucleotide sequence position 1001 of the NEFL gene and leucine is replaced by proline at amino acid sequence position 334.

In addition, the present invention provides a diagnostic method for wheat neuropathy detecting each mutation on the basis of the mutation identification results.

That is, the present invention provides a method for diagnosing CMT1 characterized by detecting a mutation in which thymine is deleted at nucleotide sequence position 318 of the PMP22 gene and a frameshift occurs from amino acid sequence position 106 (alanine).

The present invention also provides a method for diagnosing CMT2, characterized by detecting a mutation in which guanine is substituted with adenine at nucleotide sequence position 352 of the MPZ gene, and aspartic acid is replaced with asparagine at amino acid sequence position 118.

In the present invention, there is provided a method for diagnosing CMT1 characterized by detecting a 3'-splice site mutation caused by guanine substitution with thymine at nucleotide sequence 449-1 of the MPZ gene.

In the present invention, there is provided a diagnostic method of CMT2 characterized by detecting a mutation in which adenine is substituted with guanine at nucleotide sequence position 706 of the MPZ gene and lysine is replaced with glutamic acid at amino acid sequence position 236.

In the present invention, there is provided a diagnostic method of CMT1 characterized by detecting a mutation in which thymine is replaced by cytosine at nucleotide sequence position 407 of the Cx32 gene and valine is replaced with alanine at amino acid sequence position 136.

In the present invention, there is provided a diagnostic method of CMT1 characterized by detecting a mutation in which thymine is replaced by cytosine at nucleotide sequence position 502 of the Cx32 gene and cysteine is replaced by arginine at amino acid sequence position 168.

In the present invention, there is provided a diagnostic method of CMT2 characterized by detecting a mutation in which thymine is replaced with cytosine at nucleotide sequence position 1001 of the NEFL gene and leucine is replaced with proline at amino acid sequence position 334.

In addition, the present invention provides a diagnostic kit for hereditary neurological diseases capable of accurately detecting CMT disease-causing gene mutations, including genetic mutations newly identified in the present invention and previously reported gene mutations.

That is, the present invention provides a diagnostic kit comprising 17 primer pairs of SEQ ID NOS: 1 to 34 for detecting mutations of myelin Cx32, PMP22, and MPZ among CMT disease causing genes.

In another aspect, the present invention provides a diagnostic kit comprising 22 primer pairs of SEQ ID NOs: 35 to 78 for detecting mutations in EGR2, NEFL, and PRX, which are nonmycotic genes, which provide a relatively low pathogen among CMT disease causing genes. .

In addition, the present invention provides a diagnostic kit comprising 39 primer pairs of SEQ ID NO: 1 to 78 so as to be able to diagnose the myelin gene and non-myelin gene mutations simultaneously or sequentially.

Hereinafter, the present invention will be described in more detail with reference to examples such as specific experiments and analytical examples. However, the scope of the present invention is not limited by the following examples, and those skilled in the art to which the present invention pertains should be within the equivalent scope of the technical concept of the present invention and the claims to be described below. Of course, various modifications and variations are possible.

Mutation Detection in CMT Disease-causing Genes

CMT disease-causing mutations were identified through SNP screening for the CMT disease-causing genes identified so far, PMP22, Cx32, MPZ, EGR2 and NEFL.

(1) Collecting household samples and clinical data of CMT patients

① Sample and clinical data

In the present invention, a sample of about 100 households including Korean CMT patients and their families was collected and analyzed. Samples of patients and their families were collected from hospitals for more than 10 years since 1993, which obtained clinical characteristics and family history of patients, and examined the correlation between clinical characteristics and genetic variation of each patient.

② Electrophysiological examination of patients

Median, ulnar and motor nerves and sensory nerves, fibula nerve, posterior tibial nerve, and gastrocnemius nerve were examined in all subjects. In addition, brainstem auditory evoked potential (BAEP) was performed in all patients suspected of hearing impairment. Facial neural conduction and blink reflex were performed on patients with facial paralysis.

③ Classification of functional disability scale (FDS) due to CMT disease

In order to measure the severity of hereditary neurological disease, a nine-stage hypofunction scale was used. 0: normal, 1: normal but with cramps and fatigability, 2: inability to run, 3: difficult to walk but able to walk without help, 4 : Walking with cane, 5: walking with crutches, 6: walking with a walker, 7: walking a wheelchair If you must ride (wheelchair bound), 8: if you are lying down (bedridden).

(2) Sampling and DNA Extraction

Blood or saliva was sampled from members of CMT households and normal subjects (control). The blood is collected in a tube treated with EDTA, followed by extraction of genome DNA using a DNA purification device, and DNA extraction from hair roots or saliva is treated with proteinhrase K (Proteinase K) at 55 ° C for 3hr. : Extracted with chloroform method.

(3) search for the cause gene

(A) Gene screen

The causative mutation is searched for in patients with peripheral neuropathy who are not HNPP patients with type CMT1A and deletion due to overlap of 17p11.2-p12. Mutations such as SNPs were searched in the following order according to the ratio of genetic cause provision.

① Mutation screen of Cx32 that induces CMTX (except for male-to-male inheritance in family tree analysis and female symptoms are milder than male)

② MPZ mutant screens that induce autosomal dominant CMT1 and CMT2

③ PMP22 screen causing CMT1 and HNPP

④ EGR2 mutant screens causing CMT4, DSS, CH

⑤ NEFL mutant screens that induce dominant and recessive CMT1 and CMT2

⑥ PRX mutant screen to induce autosomal recessive CMT1 and DSS

(B) sequencing

The base sequences of candidate genes considered as causal genes were examined around promoter regions (about 1 kb), exons, and adjacent intron regions. PCR was performed by preparing primers for PCR of each DNA fragment, and the nucleotide sequence was immediately determined by using an automatic base sequencer (ABI 3100 automatic sequencer) after purifying amplified DNA.

(4) results

A search for causative mutations in the PMP22, MPZ, Cx32, EGR2, and NEFL genes of 42 hereditary neurological disease families with no CMT1A duplication revealed a total of 10 causative mutations from the 9 families, 8 of which are still global. It was a novel mutation that was not reported. The contents and clinical characteristics of the mutations are summarized in Table 2 below. Marked as "New" in Table 2 is the new mutation identified in the present invention.

As a result of comparing the analysis result with foreign data, it was found that Arg359Trp missense mutation of EGR2 was a symptomatic DSS patient in European group, but CMT1 patient in Korean, and there was a difference in race.

In addition, all the variations of the nucleotide sequence found through the genetic analysis including the mutations are summarized in Table 3 below. Marked as 'This study' in the table is a genetic variation of the Korean population first revealed in the present invention has not yet been reported worldwide.

The chromatograms obtained by the automatic sequencing by analyzing the genes of normal (CTL) and patient (Patient) for the gene mutations summarized in Table 3 are shown in FIGS. 1A and 1B.

Genetically, one PMP22 mutation (2.4%), three MPZs (7.1%), three Cx32s (7.1%), one EGR2 (2.4%) and two NEFLs (4.8%) were detected. Eight of the ten mutations were missense mutations, one frameshift and one 3'-splice site mutation. Seven of these mutations (Ala106fs of PMP22, Asp118Asn of MPZ, 449-1G> T, Lys236Glu, Val136Ala of Cx32, Leu334Pro of Cys168Arg and NEFL) were newly identified in the present invention and showed Korean-specificity ("Table 2"). New "). Arg359Trp in EGR2 has been reported worldwide in four patients, all of whom were DSS patients with respiratory damage and early onset, but the patients observed in the present invention showed no respiratory compromise and had an onset of 8 years of age. It appeared relatively late. In addition, median NCV showed symptoms of CMT1, not DSS, at 25 m / sec, indicating a different clinical phenotype for the same genetic cause (Taroni F., Pareyson D., Botti S., Sghirlanzoni A.). , Nemni R., Riva D. Neurology 52 [Suppl 2]: 258-259 (1999)). The mutation rates of MPZ (7.1%) and PMP22 (2.4%) investigated in the present invention were similar to those of 4.8-10.6% of MPZ and 1.6-2.8% of PMP22 reported in the European population. The mutation rate of Cx32 was 7.1%, significantly lower than the European population (Spain 21.3%, Finland 19.0%, Russia 13.0%, Italy 16.7%, Germany 11.9%), but similar to 5.6-5.7% of Japanese (Yoshihara T). , Yamamoto M., Doyu M., Misu KI, Hattori N., Hasegawa Y., Mokuno K., Mitsuma T., Sobue G. Hum.Mutat. 16: 177-178 (2000); Numakura C., Lin C., Ikegami T., Guldberg P., Hayasaka K. Human Mutat. 20: 392-398 (2002)). These results suggest that Cx32 is less likely to cause CMT disease than Europeans in the Asian population. There are few population genetic studies of the NEFL and EGR2 genes. In the present invention, the NEFL mutation rate was 4.8%, whereas Jordanova et al. Searched for 7 causative mutations from 323 white CMT populations and reported a mutation rate of 2.2% (Jordanova A., De Jonghe P., Boerkoel CF, Takashima H., De Vriendt E., Ceuterick C., Martin JJ et al. Brain 126: 590-597 (2003)). These findings suggest that the mutation rate of NEFL may be similar to or higher than that of PMP22.

Very specificly, Cx32 Val136Ala and EGR2 Arg164Gln mutations were found in one CMT1 patient. Val136Ala of Cx32 is a de novo mutation, and the daughter of two mutants has more severe symptoms than a father with only EGR2 mutation. To date, few mutations have been reported in the same gene from one patient (Silander K., Meretoja P., Juvonen V., Ignatius J., Pihko H., Saarinen A., Wallden T., Herrgard E., Aula P., Savontaus M.-L. Hum.Mutat. 12: 59-68 (1998); Mersiyanova IV, Ismailov SM, Polyakov AV, Dadali EL, Fedotov VP, Nelis E., et al. Human Mutat. 15: 340-347 (2000)), the first time a double mutation has been reported in another gene.

Diagnostic Kit

(1) Myelin Gene Detection Kit

① Composition of Kit

The kit is designed to detect mutations in the Cx32, PMP22, and MPZ genes that produce the peripheral myelin family, a transmembrane protein that plays an important role in communication between exons and Schwann cells. To do this, prepare primers to accurately amplify all the mutations identified above. The primer has an amplification length of about 300-500bp and a Tm value of 60 ° C. or more to minimize amplification of nonspecific DNA.

The amplification units for each gene are PMP22 6, Cx32 5, and MPZ 6, and the kit consists of a total of 17 PCR amplification units, and establishes the exact amplification conditions for each amplification unit.

Primers constituting the myelin gene detection kit (MyelinGene Detection Kit) are summarized in Table 4 below.

(2) Nonmyelin Gene Detection Kit

Kits are constructed to detect mutations in the EGR2, NEFL, and PRX genes that have been recently discovered and provide a relatively low cause of onset.

EGR2 and NEFL utilized the gene mutation data explored above and the amplification conditions established in the present invention. The PRX gene was obtained by amplifying each exon and using SNP variability in Korean CMT patients.

Primers constituting the non-myelinating gene diagnostic kit (MyelinGene Detection Kit) are summarized in Table 5 below.

(3) Comparative clinical trial and diagnosis rate

Analytical data on DNA mutations, including SNPs, were obtained from normal (100 patients) and family (50 patients) patients. Based on these findings, the probability of diagnosis was determined by the isolation of pathogenic mutations and the rate of disease-caused genes.

Genetic Testing Using Diagnostic Kits

Six families of patients with actual CMT disease (FC53, FC26, FC19, FC2) using the myelinGene Detection Kit and MyelinGene Detection Kit of the present invention configured as shown in Tables 4 and 5 above , FC22, FC7) were tested genetically.

(1) Analyzed patient line

(a) FC53: Patient family with Ala106fs mutations in PMP22

(b) FC26: Patient family with Asp118Asn mutation of MPZ

(c) FC19: Patient family with 449-1G> T mutation of MPZ

(d) FC2: Patient family with V136A of Cx32, R359W mutation of EGR2

(e) FC22: patient family with C168R mutation of Cx32

(f) FC7: Patient family with E397K mutation of NEFL

(2) PCR conditions

Primers and PCR conditions for each gene are shown in Tables 6 to 11 below.

Table 6 shows the PCR of PMP22, Table 7 shows the PCR of MPZ, Table 8 shows the PCR of Cx32, Table 9 shows the PCR of EGR2, Table 10 shows the PCR of NEFL, and Table 11 shows the PCR conditions of PRX. .

(3) analysis and results

PCR amplification products were analyzed with an autobase sequencer (ABI 3100) and genotypes were analyzed using Chromas program (Ver. 2.23).

Pedigree analysis results are shown in FIG. 2. In each family tree of FIG. 2, the members with numbers written in the boxes represent persons with the corresponding mutations, and arrows ↗ indicate probands. In the FC2 family, III-2 showed two mutations, V136A of Cx32 and R359W of EGR2, whereas II-3 showed only R359W of EGR2.

According to the present invention, a new gene mutation is identified as a etiology in CMT disease patients in Korea, and using this, an accurate diagnosis of hereditary neuropathy caused by the gene mutation as a pathogen is made. When using the myelin gene diagnosis kit of the present invention in combination with the kit for detecting overlap of the 17p11.2-p12 region of another inventor of the present invention, the confirmation rate for the inherited neurological disease is 80% or more, and the non-number of the present invention When used together with the hereditary genetic diagnostic kit, the rate of diagnosis of hereditary neurological disease is more than 90%. This has the surprising effect that it is possible to make accurate diagnosis using only the genetic diagnostic kit for anatomical disorders due to the nature of neurological disease, which is difficult to accurately diagnose anatomical and electrophysiological diagnosis. The diagnosis will have the effect of reducing the burden of medical expenses and treatment costs.

Therefore, according to the present invention, accurate early diagnosis through genetic testing is possible for CMT disease caused by a single genetic defect with strong heredity, and can be treated early by applying a recently developed treatment method according to the accurate diagnosis. In addition, customized treatment according to the exact etiology is possible. In addition, the present invention is to analyze the genetic causes of CMT disease in Korean first and develop a genetic diagnostic kit based on the data in the state of the development of genetic diagnosis system for hereditary neurological disease in the world yet, It can be the foundation for the provision of quality health care, taking into account ethnic characteristics.

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Claims (10)

delete delete delete delete delete delete delete A diagnostic kit for hereditary neurological diseases comprising 17 primer pairs of SEQ ID NOs: 1 to 34 for detecting mutations in the Cx32, PMP22 and MPZ genes. A diagnostic kit for hereditary neurological diseases comprising 22 primer pairs of SEQ ID NOs: 35 to 78 for detecting mutations in the EGR2, NEFL and PRX genes. A diagnostic kit for hereditary neurological diseases comprising 39 primer pairs of SEQ ID NOs: 1 to 78 to detect mutations of Cx32, PMP22, MPZ, EGR2, NEFL, and PRX genes simultaneously or sequentially.

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