WO2021107713A1

WO2021107713A1 - Method for diagnosing amyotrophic lateral sclerosis on basis of lats1 gene mutation marker

Info

Publication number: WO2021107713A1
Application number: PCT/KR2020/017169
Authority: WO
Inventors: 기창석; 김영은; 오기욱; 김승현
Original assignee: 주식회사 녹십자지놈
Priority date: 2019-11-29
Filing date: 2020-11-27
Publication date: 2021-06-03
Also published as: KR102294939B1; KR20210067530A

Abstract

The present invention relates to a method for diagnosing amyotrophic lateral sclerosis on the basis of a LATS1 gene mutation. The present invention provides an accurate and effective marker-based diagnosis method for diagnosing amyotrophic lateral sclerosis for which effective molecular diagnostic markers are insufficient. The marker of the present invention enables the diagnosis of amyotrophic lateral sclerosis even when none of the markers reported beforehand are observed, and suggests a novel diagnosis method for amyotrophic lateral sclerosis. The diagnosis method and the marker and a use thereof according to the present invention are very advantageous for diagnosis, prediction, treatment, and prevention of amyotrophic lateral sclerosis. Furthermore, the LATS1 gene of the present invention can be utilized in studying the pathological mechanism of amyotrophic lateral sclerosis and in developing a therapeutic drug for amyotrophic lateral sclerosis.

Description

LATS1 gene mutation marker-based diagnostic method for amyotrophic lateral sclerosis

The present invention relates to a method for diagnosing amyotrophic lateral sclerosis based on a LATS1 gene mutation, and more particularly, to a method and marker for diagnosing and predicting amyotrophic lateral sclerosis by detecting a LATS1 mutant gene or a LATS1 mutant protein encoded by it. it's about

Amyotrophic lateral sclerosis (ALS), also called Lou Gehrig disease, is a disease in which only motor neurons are selectively killed. cells), the brain stem (brainstem), and the lower motor neurons of the spinal cord are all gradually destroyed. Clinical symptoms start with slowly progressive weakness and atrophy of the extremities, and as the disease progresses, it is a fatal disease that eventually leads to death within a few years of respiratory muscle paralysis. It is known that about 1 to 2 people per 100,000 people a year develop Lou Gehrig's disease.

The onset of ALS increases from the late 50s, and the incidence rate is 1.4 to 2.5 times higher in men than in women. Amyotrophic lateral sclerosis is a fatal disease with the fastest symptom progression among motor neuron diseases (MND) and is known to have an average lifespan of less than 4-5 years, and as the disease progresses, death from respiratory failure and other complications due to respiratory muscle paralysis. will do Although there is no medical treatment that can cure it yet, drug treatment to slow the progression of symptoms, prevention and management of other complications, etc., have a great influence on the patient's prognosis, so early diagnosis is the most important for prevention and treatment ( Seoul National University Hospital medical information).

Although the cause of ALS is not yet known, several hypotheses have been proposed so far. About 5-10% of all ALS patients are known to have familial muscular atrophic familial sclerosis, and of these, mutations in the causative gene on chromosome 21 are confirmed in about 20% of families. So far, eight genes have been reported to cause familial ALS. In addition, in sporadic ALS, which is not hereditary, apoptosis (apoptosis, apoptosis, a phenomenon in which cells die by themselves under the control of genes) by excitotoxicity is thought to play an important role. Other hypotheses that a special virus is the cause and the action of environmental toxins have been suggested as the cause of ALS, but there is no direct evidence yet (Medical Information of Seoul National University Hospital).

Tests for the diagnosis of amyotrophic lateral sclerosis include blood tests, electromyography, brain and spinal cord imaging tests, and in some cases, muscle biopsy and genetic tests are additionally performed. After diagnosis, progress status and prognosis are evaluated, and appropriate drug treatment and rehabilitation treatment are performed according to the stage of the disease. As genes that cause ALS, superoxide dismutase 1 (SOD1), transactive response DNA-binding protein (TARDBP), fused in sarcoma (FUS), chromosome 9 open reading frame 72 (C9orf72), etc. are well known. With the recent discovery of a new gene associated with the onset of amyotrophic lateral sclerosis, it has been revealed that this disease is related to frontotemporal dementia and various types of muscular and degenerative diseases. is recognized as

Still, most diagnoses are based primarily on clinical symptoms. However, the diagnosis based on clinical symptoms has a problem in that the treatment time is missed because the diagnosis can be made only at a later stage when the symptoms appear prominent. In addition, although the exact cause of amyotrophic lateral sclerosis is not known, and it has a complex onset and progression mechanism, it is related to various types of diseases. The types of markers are limited, and a lot of effort and investment are being made to develop various biomarkers.

Under this technical background, the present inventors tried to develop a novel biomarker for diagnosis and prediction of amyotrophic lateral sclerosis. As a result, in the ALS trio study conducted on patients and parents with amyotrophic lateral sclerosis, parents and normal people The LATS1 gene, which is a causative gene for amyotrophic lateral sclerosis, was discovered through a novel mutation analysis not found in the control group but found only in the progenitor, and the present invention was completed.

The information described in the background section is only for improving the understanding of the background of the present invention, and it does not include information that forms the prior art known to those of ordinary skill in the art to which the present invention pertains. may not be

발명의 요약Summary of the invention

It is an object of the present invention to provide a method for diagnosing and predicting amyotrophic lateral sclerosis based on novel biomarkers.

Another object of the present invention is to provide a LATS1 mutant gene as a novel biomarker.

Another object of the present invention is to provide a LATS1 mutant protein as a novel biomarker.

Another object of the present invention is to provide a composition for diagnosis of amyotrophic lateral sclerosis.

Another object of the present invention is to provide a kit for diagnosis of amyotrophic lateral sclerosis.

In order to achieve the above object,

The present invention relates to amyotrophic lateral sclerosis (ALS) comprising detecting a LATS1 (Large Tumor Suppressor Kinase 1) mutant gene or a LATS1 mutant protein encoded by the LATS1 mutant gene in an isolated biological sample. A method of providing information for diagnosis is provided.

The present invention also provides a mutant LATS1 (Large tumor suppressor kinase 1) mutant gene comprising at least one of SNV, Indel and CNV as a diagnostic marker for amyotrophic lateral sclerosis, and a mutant LATS1 protein encoded by the gene.

The present invention also provides a composition for diagnosis of amyotrophic lateral sclerosis comprising an agent capable of detecting a large tumor suppressor kinase 1 ( LATS1 ) mutant gene or a LATS1 mutant protein encoded by a mutant LATS1 gene.

The present invention also provides a kit for diagnosis of amyotrophic lateral sclerosis, comprising an agent capable of detecting a large tumor suppressor kinase 1 ( LATS1 ) mutant gene or a LATS1 mutant protein encoded by a mutant LATS1 gene.

The present invention also provides a diagnostic use for amyotrophic lateral sclerosis of a large tumor suppressor kinase 1 ( LATS1 ) mutant gene comprising at least one of SNV, Indel and CNV and a LATS1 mutant protein encoded by the gene.

The present invention also, in the isolated biological sample, LATS1 (Large tumor suppressor kinase 1) mutant gene or amyotrophic lateral sclerosis (ALS) comprising the step of detecting a mutant LATS1 protein encoded by the mutant LATS1 gene ) diagnostic methods are provided.

1 is a graph showing the expression level of the LATS1 gene for each tissue.

Figure 2 is the result of performing exome sequencing of amyotrophic lateral sclerosis patients and parents. Mutations not present in normal parents were found in the LATS1 gene.

3 is a result of reconfirming the mutation of the LATS1 gene discovered in exome sequencing of amyotrophic lateral sclerosis patients and parents by Sanger sequencing method.

발명의 상세한 설명 및 바람직한 구현예DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein and the experimental methods described below are well known and commonly used in the art.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that occurs in adults due to progressive loss of motor neurons in the cortex, cerebral cortex, and spinal cord. About 10% of patients have familial ALS with a family history, and about 90% of patients develop sporadically without a family history. To date, mutations in several genes, including SOD1, TARDBP, FUS, and C9orf72, have been reported as the cause of ALS, but the genetic cause is unknown in 1/3 of familial ALS patients and about 90% of sporadic ALS patients. Recently, with the discovery of a new gene related to the onset of amyotrophic lateral sclerosis, it has been revealed that this disease is related to frontotemporal dementia and various types of muscle diseases and degenerative diseases. There are problems in diagnosing or applying to treatment.

Amyotrophic lateral sclerosis is a disease that does not currently have an effective treatment method, and is rapidly invented and progressing with an average lifespan of less than 4-5 years. When diagnosed by clinical symptoms, treatment or improvement is almost impossible. Therefore, it is important to diagnose the disease at an early stage of onset through early diagnosis such as prenatal diagnosis.

Accordingly, the present inventors tried to identify a gene related to amyotrophic lateral sclerosis and the SNV of the gene for early diagnosis of amyotrophic lateral sclerosis. In an embodiment of the present invention, sequencing analysis was performed after extracting genomic DNA from blood samples for patients with sporadic amyotrophic lateral sclerosis and their parents. As a result of screening for mutations in the previously reported ALS-FTD causative gene or related gene for the differential diagnosis of amyotrophic lateral sclerosis, HSP and other disease genes for the differential diagnosis of amyotrophic lateral sclerosis, No previously reported mutations were found.

In another embodiment of the present invention, one novel mutation was found in the analysis through Sanger sequencing of the patient and parent, which was the deletion of the 2790th base from the nucleotide sequence of the LATS1 gene, and the 930th amino acid leucine was substituted with phenylalanine and it was confirmed that it is a frameshift mutation in which synthesis ends at the 27th (956th). It was confirmed that the mutation was a novel mutation not found in dbSNP, 1000 Genome Project, gnomAD, and KRGDB, which is the normal control group Korean data, and that the mutation could be used as a new diagnostic marker for amyotrophic lateral sclerosis.

Accordingly, in one aspect, the present invention provides, in an isolated biological sample, a large tumor suppressor kinase 1 ( LATS1 ) mutant gene or amyotrophic lateral sclerosis comprising the step of detecting a mutant LATS1 protein encoded by the mutant LATS1 gene. It relates to a method of providing information for the diagnosis of lateral sclerosis: ALS.

In another aspect, the present invention, in the isolated biological sample, LATS1 (Large tumor suppressor kinase 1) mutant gene or amyotrophic lateral sclerosis comprising the step of detecting a mutant LATS1 protein encoded by the mutant LATS1 gene : ALS) diagnosis method.

In the present invention, when the LATS1 mutant gene or the LATS1 mutant protein encoded by the LATS1 mutant gene is detected, amyotrophic lateral sclerosis may be diagnosed.

"Amyotrophic lateral sclerosis" is largely classified into two types depending on the cause, and is divided into a familial type and a sporadic type that occurs alone. Familial ALS accounts for about 10% of all cases, and mutations in one or more genes are the cause. About 15% of familial patients develop the disease due to a mutation in the SOD1 gene, and the disease can occur even if a defective gene is inherited from one parent in an autosomal dominant form. On the other hand, the cause of sporadic ALS has not been specifically identified. Heavy metals such as aluminum, mercury, and lead used in dental rods, which are environmental factors, are suggested as the cause, but it is not clear. Mutations in several genes have been reported as the cause of ALS, but the genetic cause is unknown in 1/3 of familial ALS and about 90% of sporadic ALS. In the present invention, amyotrophic lateral sclerosis may be characterized as familial or sporadic.

Diagnosis of amyotrophic lateral sclerosis is primarily based on clinical symptoms. Damage to both upper and lower motor nerves is characteristic. In the early stages, damage to only the upper motor nerve may appear, but generally, damage to the lower motor nerve occurs within 3 to 5 years after the onset. Diagnosis by clinical symptoms of amyotrophic lateral sclerosis is diagnosed by the condition exclusion method after the primary clinical findings because the causes and treatment methods can appear similar to those of completely different myopathy. In general, based on the patient's history and neurological examination, nerve conduction velocity test (NCV), muscle tissue biopsy, blood test, urine test, cerebrospinal fluid test, cervical spine X-ray and magnetic resonance imaging test, electromyography (EMG), etc. are performed to make a diagnosis For example, amyotrophic lateral sclerosis is excluded from the diagnosis if these symptoms are present because it is rarely accompanied by ocular movement disorders, sensory disorders, bladder and anal disorders, or intellectual disorders.

As described above, most diagnoses are made at the time when clinical symptoms appear after the onset. However, the diagnosis based on clinical symptoms has a problem in that the treatment time is missed because the diagnosis can be made only at a later stage when the symptoms appear prominent. Therefore, early diagnosis of amyotrophic lateral sclerosis is more important compared to other disease fields. Nevertheless, the cause of the onset is not clear, and a clear diagnostic marker is lacking due to the characteristics of the disease being caused by complex factors.

As used herein, the term “diagnosis” refers to determining a subject's susceptibility to a particular disease or disorder, determining whether a subject currently has a particular disease or disorder, or having a particular disease or disorder. determining a subject's prognosis, or therametrics (eg, monitoring a subject's condition to provide information about treatment efficacy). For the purposes of the present invention, diagnosis is to determine whether or not (risk) the development of amyotrophic lateral sclerosis.

In one embodiment of the present invention, LATS1 (large tumor suppressor kinase 1) gene was identified as a gene candidate causing amyotrophic lateral sclerosis. The present inventors discovered a novel frameshift mutation c.2790del (p.Leu930Phefs*27) of the LATS1 gene in the sALS trio. The mutation in the LATS1 gene was not present in 1722 same-race normal controls. And no corresponding mutations were reported in dbSNP, 1000 Genome Project (all and East Asians) and gnomAD.

In the present invention, the LATS1 mutant gene refers to a mutated LATS1 gene, for example, the mutation is a single nucleotide variant (Single Nucleotide variant: SNV), insertion or deletion (Indel) and gene copy number mutation (Copy-) number variation, CNV) may be any one or more selected from the group consisting of, but is not limited thereto, and refers to all genetic mutations recognized by those skilled in the art. Preferably, in the present invention, the mutation of the large tumor suppressor kinase 1 (LATS1) mutant gene may be a single nucleotide variant (SNV), insertion or deletion, and more preferably, may be a deletion.

Most preferably, in the present invention, the LATS1 (Large tumor suppressor kinase 1) mutant gene may be characterized in that the 2790th nucleotide is deleted from the nucleotide sequence of SEQ ID NO: 1.

In the present invention, the LATS1 (Large Tumor Suppressor Kinase 1) mutant gene may be characterized in that a frame shift occurs by insertion or deletion of one or more bases.

The " LATS1 gene" is located on chromosome 6q25.1, consists of 8 exons, and encodes 1,130 amino acids. LATS1 protein is a putative serine/threonine kinase that is involved in the initial meiosis process together with CDC2 kinase that regulates the cell division cycle, and is known to act as a tumor suppressor gene. LATS1 is expressed in various tissues, including the brain. The expression level for each tissue of the LATS1 gene is shown in FIG. 1 . The LATS1 gene includes all homologous genes found in all animals including humans, and the cDNA sequence of the normal human LATS1 gene confirmed in an embodiment of the present invention is the same as SEQ ID NO: 1 (NM_004690.4) Homo sapiens large tumor suppressor kinase 1 (LATS1), transcript variant 1, mRNA), confirmed in an embodiment of the present invention The amino acid sequence of the normal human LATS1 protein is the same as SEQ ID NO: 2 (NP_004681.1 serine/threonine-protein kinase LATS1) isoform 1 [Homo sapiens]), but not limited thereto.

In an embodiment of the present invention, as a result of analysis through gene sequencing and bioinformatics methods for patients with sporadic amyotrophic lateral sclerosis that did not develop in their parents, the previously reported cause of ALS-FTD or related genes, differential diagnosis of ALS No mutations were found in HSP and other disease genes for , and mutations in the LATS1 gene of the initiator were confirmed.

Therefore, in the present invention, the information providing method or the diagnostic method is characterized in that the gene is obtained and amplified from a sample isolated from an individual, and the occurrence of mutation in the LATS1 gene is analyzed using sequencing data of the amplified product. can be done with

As used herein, the term “amplification” refers to a reaction that amplifies a nucleic acid molecule. Various amplification reactions have been reported in the art, including polymerase chain reaction (PCR), reverse transcription-polymerase chain reaction (RT-PCR), ligase chain reaction (LCR) Gap-LCR, repair chain reaction , transcription-mediated amplification (TMA), self sustained sequence replication, selective amplification of target polynucleotide sequences, consensus sequence priming polymerase chain reaction (consensus) sequence primed polymerase chain reaction (CP-PCR), arbitrarily primed polymerase chain reaction (AP-PCR), nucleic acid sequence based amplification (NASBA), strand displacement amplification (strand displacement amplification) and loop-mediated isothermalamplification (LAMP).

PCR is the most well-known nucleic acid amplification method, and many modifications and applications thereof have been developed. For example, touchdown PCR, hot start PCR, nested PCR, and booster PCR have been developed by modifying traditional PCR procedures to enhance the specificity or sensitivity of PCR. In addition, real-time PCR, differential display PCR (DD-PCR), rapid amplification of cDNA ends (RACE), multiplex PCR, inverse polymerase chain reaction (inverse polymerase chain reaction) Chain reaction: IPCR), vectorette PCR and TAIL-PCR (thermal asymmetric interlaced PCR) have been developed for specific applications.

“Single Nucleotide Variant (SNV)” refers to a mutation that shows a difference in single nucleotide sequence among genomic mutations. Single nucleotide polymorphism (SNP) and point mutation are here can occur in somatic cells of any frequency Single nucleotide alterations in somatic cells (eg, caused by cancer) are also called single-nucleotide alterations Single nucleotide polymorphisms are identical in the genome of multiple individuals. It means that a specific nucleotide sequence at a position is changed to another nucleotide and expressed as a different trait, and it is the most common form of genetic mutation in the human genome.Single nucleotide polymorphisms generally occur with a frequency of 1% or more of the population, If it is less than 1%, it is classified as a mutation Point mutation is a substitution, insertion, or deletion of one base sequence, and can prevent or modify the production of a specific protein.

Single nucleotide mutations are classified according to their location and function in the genome. In addition, according to the presence or absence of amino acid sequence mutation, it is classified into synonymous SNV (sSNV) that does not cause amino acid sequence mutation and nonsynonymous SNV (nsSNV) that does not cause amino acid sequence mutation. Among single nucleotide mutations, indels can cause more serious mutations than substitutions. In the case of indel, a frame shift of the amino acid sequence sequence is induced, so that the amino acid translated after the SNV is changed.

Depending on the position on the genome, the SNV present in the coding exon region is called the coding SNV (cSNV), and has the same ratio as the intron, 5' and 3' untranslated regions (UTR). The SNV present in the coding region is called non-coding SNV (ncSNV).

In the present invention, "SNP" and "SNV" may be used interchangeably, and may be used as the same meaning as "SNV of LATS1 gene" or " SNP of LATS1 gene".

“Single Nucleotide Polymorphism (SNP)” refers to a single nucleotide (A, T, C, or G) in the genome that differs between members of a species or between chromosomes of an individual. Refers to the diversity of DNA sequences Within a population, SNPs can be assigned to a minor allele frequency (MAF; the lowest allele frequency at a locus found in a particular population) Variations exist within the human population, and a single SNP allele common in a geological or ethnic group is very rare. A single base is a change (replacement), deletion (deletion) or addition ( The SNP may cause an inframe shift.

Various methods are used for SNV or SNP analysis. Most of the methods developed and used so far are based on the PCR method and mainly analyze a limited number of samples, but analyze a large number of SNPs at the same time using a DNA array or use ultra-precision analysis equipment such as MALDI-TOF. Analysis methods are also widely used. According to the principle of SNP genotyping, there are four types: Allele-Specific Hybridization, Primer Extension, Allele-Specific Oligonucleotide Ligation, and Cleavage depending on the difference between the sample preparation method and the search method.

As the main assays based on PCR, for example, SSCP (Single Strand Conformation Polymorphism), AFLP (Amplified Fragment Length Polymorphism), RFLP (Restriction Fragment Length Polymorphism), RAPD (Random Amplified Polymorphic DNA), AS-PCR (Allele-PCR) Specific PCR), but is not limited thereto.

"Insertion or deletion (Indel)" means that one or more nucleotides are inserted or deleted in a gene. Indel in SNV means that one nucleotide is inserted or deleted, and "insertion or deletion (Indel)" can be used to include it.

"Copy-number variation (CNV)" refers to a phenomenon in which the repetition of a certain section of a gene differs from one individual to another. The repetition may be repeated 0 times to n times. When it is repeated 0 times, the function of the gene is lost, and when it is repeated more than 1 time, the function of the gene is generally amplified, but is not limited thereto.

In the present invention, the sequencing may be characterized by Sanger sequencing or next generation sequencing (NGS).

In an embodiment of the present invention, novel mutations causing substitution of all amino acids according to the filtering criteria were evaluated through Sanger sequencing of DNA. All exon and exon-intron boundaries of the target gene were amplified by PCR using primers (Table 6).

The term “Sanger sequencing analysis” of the present invention is a method of analyzing a nucleotide sequence through DNA chain termination using dideoxynucleotide triphosphate (ddNTP). Sanger sequencing is very simple and less toxic, so it has spread rapidly compared to the Maxam-Gilbert method developed at the same time (Maxam and Gilbert, 1977), and other methods have been modified and developed in this method. . In the present invention, Sanger sequencing includes modifications and advanced analysis methods known to those skilled in the art, in addition to the first known method.

The term “next-generation sequencing (NGS)” in the present invention corresponds to the second-generation technology in terms of automation. NGS is a name called to distinguish it from the first automation device before and to distinguish it from the Next NGS device (also referred to as next-generation or third-generation NGS) that was created later. However, as competition for the development of efficient sequencing technology accelerates and sequencing technology continues to be developed based on the introduction of new technologies and the purpose of using the platform, the division of sequencing technology of each generation becomes vague, and NGS is used in a broad sense encompassing all of the sequencing technology after the automated Sanger sequencing technology. In the present invention, “next-generation sequencing” is used in a broad sense encompassing all sequencing techniques after Sanger sequencing technology.

With the development of sequencing technology, sequencing may be possible from a single molecule of DNA. Therefore, in the present invention, when analyzing a gene mutation using a next-generation sequencing method, the step of amplifying a gene can be omitted. For example, a sequencing method that does not amplify a gene includes, but is not limited to, a single molecule, real-time (SMRT), Oxford Nanopore sequencer, and the like.

In the present invention, the step of detecting the LATS1 mutant gene may LATS1 mutation using primers for the gene, it is also possible to use a pair (pair) or more primer sets. The primer can be used without limitation as long as it is a sequence capable of specifically binding to the LATS1 mutant gene, but is preferably a primer capable of amplifying a region containing a mutation at position 2970 of SEQ ID NO: 1. and, more preferably, the primer may include the sequence represented by SEQ ID NO: 4 or 5.

In the present invention, the step of detecting the LATS1 mutant gene is to use a probe that LATS1 mutant binding complementarily to the gene, wherein the probe is complementary to a region containing the mutation position of 2970-th positions in SEQ ID NO: 1 It may be characterized as a binding probe, but is not limited thereto.

In the present invention, a reporter may be attached to the 5' end of the probe, and another fluorescent material showing fluorescence may be attached, but is not limited thereto. For example, the reporter may be an enzyme, a fluorescent substance, a ligand, a luminescent substance, a microparticle, a redox molecule, a radioisotope, and the like, but is not limited thereto. As another example, the reporter is FAM, JOE, BHQ1, VIC, TAMRA, ROX, NED, HEX, TET, fluorescein, fluorescein chlorotriazinyl, rhodamine green ), rhodamine red, tetramethylrhodamine, FITC, Oregon green, Alexa Fluor, Texas Red, Cyanine-based dyes and cyanine It may be at least one selected from the group consisting of dicarbocyanine dyes. In addition, a black hole quencher-1 (BHQ-1) may be attached to the 3' end of the probe as a quencher, and other materials that can be used as a quencher may be attached thereto. Not limited. For example, the quencher may be selected from the group consisting of Dabcyl, TAMRA, Eclipse, DDQ, QSY, Blackberry Quencher, Qxl, Iowa black FQ, Iowa black RQ and IRDye QC-1. It may be one or more selected.

It is easy for those skilled in the art to design a primer set capable of specifically amplifying the mutation site of the LATS1 gene and a probe that complementarily binds to the region including the mutation site of the gene. The primer set and probe can be used for real time polymerase chain reaction (PCR), and more preferably, can be used for multiplex real time PCR. The nucleic acid sequence contained in the probe or primer may also be modified using a number of means known in the art. Non-limiting examples of such modifications include methylation, "encapsulation", substitution of one or more homologues of natural nucleotides, and modifications between nucleotides, such as uncharged linkages (eg, methyl phosphonates, phosphotriesters, phosphoroamidates, carbamates, etc.) or charged linkages (eg phosphorothioates, phosphorodithioates, etc.).

In the present invention, the step of detecting the LATS1 mutant gene is a polymerase chain reaction, nucleic acid digestion (nuclease digestion), hybridization (hybridization), Southern blotting (Southern blotting), restriction enzyme fragment polymorphism (restriction) Enzyme fragment polymorphism), primer extension (primer extension), single stranded conformation polymorphism, DNA chip (DNA chip), microarray (Microarray) and a method selected from the group consisting of a combination thereof to analyze using It may be characterized, but is not limited thereto, and in addition to the above method, it may be analyzed using a known molecular biological method together.

In the present invention, the "simultaneous multiplex PCR" means that two or more sets of primers used for PCR are used in one amplification reaction.

A “primer” is a short single-stranded oligonucleotide that serves as a starting point for DNA synthesis. A primer binds specifically to a polynucleotide as a template under suitable buffer and temperature conditions, and DNA polymerase adds a nucleoside triphosphate having a base complementary to the template DNA to the primer to link it. is synthesized A primer generally consists of a sequence of 15 to 30 bases, but is not limited thereto, and the temperature (melting temperature, Tm) at which it binds to the template strand varies depending on the base composition and length.

"Probe" refers to a fragment of a polynucleotide such as RNA or DNA with a length of several to several hundred base pairs that can specifically bind to mRNA or cDNA (complementary DNA) of a specific gene. and the presence or absence and expression level of the target mRNA or cDNA to be bound by labeling can be confirmed. In the present invention, the probe specifically binds to the LATS1 mutant gene, preferably, it is characterized in that it specifically binds to a site containing the mutation of the LATS1 gene, most preferably the LATS1 gene of SEQ ID NO: 1 It is characterized in that it specifically binds to a site including a site in which the 2790th base is deleted.

In one embodiment of the present invention, as a result of analyzing the protein encoded by the deletion at the 2790th base of the LATS1 gene of SEQ ID NO: 1, this is a frame shift mutation, 930 of the LATS1 protein of SEQ ID NO: 2 when transcribed and translated The th amino acid was substituted with phenylalanine (Phe) from leucine (Leu), and it was confirmed that protein synthesis was terminated at the 27 th amino acid (956 th) from this (SEQ ID NO: 3). Therefore, when a LATS1 mutant protein encoded by the LATS1 mutant gene is detected from a sample isolated from an individual, amyotrophic lateral sclerosis can be diagnosed.

In the present invention, in the mutant LATS1 protein encoded by the mutant LATS1 gene, in the amino acid sequence of SEQ ID NO: 2, the 930th amino acid is substituted from leucine (Leu) to phenylalanine (Phe), and from this, the 27th amino acid (956) It may be characterized in that protein synthesis is terminated in the second), and preferably it may be characterized as a protein represented by the amino acid sequence of SEQ ID NO: 3.

In the present invention, the detecting of the LATS1 mutant protein encoded by the LATS1 mutant gene may be characterized in that it is detected using an antibody or aptamer specific for the protein.

In the present invention, the step of detecting the LATS1 mutant protein encoded by the LATS1 mutant gene is Western blotting, ELISA, radioimmunoassay, radioimmunodiffusion method, Ouchterlony immunodiffusion method, rocket immunoelectrophoresis, immunization The detection may be characterized by using a method selected from the group consisting of histochemical staining, immunoprecipitation analysis, complement fixation analysis, FACS, protein chip, and combinations thereof.

As used herein, the term “antibody” refers to an immunoglobulin that specifically binds to an antigenic site. Antibodies for use in the present invention include monoclonal or polyclonal antibodies, immunologically active fragments (eg, Fab or (Fab)2 fragments), antibody heavy chains, humanized antibodies, antibody light chains, genetically engineered single chain Fv molecules, a chimeric antibody or the like. All immunoglobulin antibodies, antigen-binding sites and fragments thereof that specifically bind to the LATS1 mutant protein encoded by the LATS1 mutant gene are included. In the present invention, the antibody specifically binds to the mutant LATS1 protein encoded by the mutant LATS1 gene, and preferably, in the amino acid sequence of SEQ ID NO: 2, amino acid at position 930 is substituted from leucine (Leu) to phenylalanine (Phe) From this, it may be an antibody that specifically binds to a protein whose protein synthesis is terminated at the 27th amino acid (956th) or a protein represented by the amino acid sequence of SEQ ID NO: 3.

"Aptamer" refers to a small single-stranded oligonucleic acid capable of specifically recognizing a target material with high affinity as a single-stranded DNA or RNA molecule. Aptamers can be used as one element of a biosensor capable of recognizing molecules in a detection and analysis system, and thus has been recognized as an antibody substitute. In particular, unlike antibodies, aptamers can be used as target molecules of various organic and inorganic substances, including toxins, and once an aptamer that specifically binds to a specific substance is isolated, it is reproduced at low cost and consistency by an automated oligomer synthesis method. This was possible and economical. Therefore, since the first aptamer-based biosensor that measured a target protein using a fluorescently-labeled aptamer was developed in 1996, various aptamer biosensors have been developed based on the advantages and structural characteristics of the aptamer. Yes (Kim Yeon-seok & Koo Man-bok, NICE, 26(6): 690, 2008). In the present invention, the aptamer specifically binds to the LATS1 mutant protein encoded by the LATS1 mutant gene, and preferably, in the amino acid sequence of SEQ ID NO: 2, the 930th amino acid is substituted from leucine (Leu) to phenylalanine (Phe) and, from this, it may be an aptamer that specifically binds to a protein whose protein synthesis is terminated at the 27th amino acid (956th) or a protein represented by the amino acid sequence of SEQ ID NO:3.

In the present invention, the biological sample is a nucleic acid sample isolated from a sample selected from the group consisting of blood, hair, saliva, urine, semen, vaginal cells, oral cells, placental cells or amniotic fluid containing fetal cells and mixtures thereof. can be characterized as The nucleic acid may be genomic DNA, cell free DNA (cfDNA), RNA or micro RNA, but is not limited thereto.

The nucleic acid can be obtained through a conventional method known in the art. For example, the tissue is treated with a DNA lysis buffer (e.g., tris-HCl, EDTA, EGTA, SDS, deoxycholate, and tritonX and/or NP-40) to isolate DNA. However, the present invention is not limited thereto.

The information providing method or diagnostic method can be used alone for the diagnosis of amyotrophic lateral sclerosis, and in addition to the detection of the LATS1 gene of the present invention, it can be used in combination with the detection of other genes or proteins known as diagnostic markers of amyotrophic lateral sclerosis. have. In addition, based on clinical symptoms, patient history, and neurological examination, nerve conduction velocity test (NCV), muscle tissue biopsy, blood test, urine test, cerebrospinal fluid test, cervical spine X-ray and magnetic resonance imaging test, electromyography (EMG) ) can also be used with non-genetic diagnostic methods such as

In another aspect, the present invention provides a diagnostic marker for amyotrophic lateral sclerosis, a single nucleotide variant (SNV), an insertion or deletion (Indel), and any one or more of a copy-number variation (CNV). It relates to a LATS1 (Large Tumor Suppressor Kinase 1) mutant gene, including.

In the present invention, the LATS1 (Large Tumor Suppressor Kinase 1) mutant gene may be characterized in that insertion or deletion occurs.

In the present invention, the LATS1 (Large tumor suppressor kinase 1) mutant gene may be characterized in that one or more of the nucleotides of the gene are deleted.

In the present invention, the LATS1 gene may be characterized in that it comprises a nucleotide sequence represented by SEQ ID NO: 1.

In the present invention, the LATS1 (Large tumor suppressor kinase 1) mutant gene may be characterized in that the mutation occurs at the 2790th base in the nucleotide sequence of SEQ ID NO: 1.

In the present invention, the LATS1 (Large Tumor Suppressor Kinase 1) mutant gene may be characterized in that the 2790th nucleotide is deleted from the nucleotide sequence of SEQ ID NO: 1.

In the present invention, the LATS1 (Large Tumor Suppressor Kinase 1) mutant gene may be characterized in that a frame shift occurs due to mutation.

In the present invention, the LATS1 (Large Tumor Suppressor Kinase 1) mutant gene may be characterized in that a frame shift mutation occurs by deletion of the 2790 th nucleotide in the nucleotide sequence of SEQ ID NO: 1.

In another aspect, the present invention provides a diagnostic marker for amyotrophic lateral sclerosis, a single nucleotide variant (SNV), an insertion or deletion (Indel), and a copy-number variation (CNV) any one It relates to a LATS1 mutant protein encoded by a large tumor suppressor kinase 1 ( LATS1 ) mutant gene including the above.

In the present invention, the LATS1 mutant protein is that in the amino acid sequence of SEQ ID NO: 2, the 930th amino acid is substituted from leucine (Leu) to phenylalanine (Phe), and protein synthesis is terminated at the 27th amino acid (956th) therefrom. It may be characterized, and preferably, it may be characterized as represented by the amino acid sequence of SEQ ID NO: 3.

As used herein, the term "diagnostic marker" refers to an organic biomolecule that is a substance that can distinguish cancer patients from healthy people and diagnose them, and shows an increase in cancer-causing individuals compared to healthy people. Examples of the organic biomolecules include, but are not limited to, polypeptides, proteins, nucleic acids, lipids, glycolipids, glycoproteins, and sugars. In the present invention, preferably, the organic biomolecule refers to DNA, RNA, polypeptide or protein. In the present invention, the diagnostic marker is a LATS1 mutant gene or a LATS1 mutant protein encoded by the gene.

In another aspect, the present invention relates to a diagnostic composition for amyotrophic lateral sclerosis comprising an agent capable of detecting a large tumor suppressor kinase 1 ( LATS1 ) mutant gene or a LATS1 mutant protein encoded by a mutant LATS1 gene.

In the present invention, the preparation may include a primer capable of specifically amplifying the mutation site of the LATS1 gene or a probe that complementarily binds to the region including the mutation site of the LATS1 gene. have.

In the present invention, the preparation may include an antibody or aptamer specific for the LATS1 mutant protein.

In the present invention, one or more pairs of primer sets may be used. The primer can be used without limitation as long as it is a sequence that can specifically bind to the LATS1 mutant gene, but is preferably a primer capable of amplifying a site containing a mutation at position 2970 of SEQ ID NO: 1. . More preferably, it may be characterized in that it is capable of amplifying a site containing a deletion at position 2970 of SEQ ID NO: 1, and most preferably, the primer comprises the nucleotide sequence represented by SEQ ID NO: 4 or 5 can be characterized as

In the present invention, the probe specifically binds to the LATS1 mutant gene, preferably, it is characterized in that it specifically binds to a site containing the mutation of the LATS1 gene, most preferably the LATS1 gene of SEQ ID NO: 1 It may be characterized in that it specifically binds to a site including a site in which the 2790th base of the is deleted.

In the present invention, the antibody specifically binds to the LATS1 mutant protein encoded by the LATS1 mutant gene, and preferably, in the amino acid sequence of SEQ ID NO: 2, the 930th amino acid is leucine (Leu) to phenylalanine ( Phe), and from which protein synthesis is terminated at the 27th amino acid (956th), it may be an antibody that specifically binds to a protein represented by the amino acid sequence of SEQ ID NO:3.

In the present invention, the aptamer specifically binds to the LATS1 mutant protein encoded by the LATS1 mutant gene, and preferably, in the amino acid sequence of SEQ ID NO: 2, the 930th amino acid is leucine (Leu) to phenylalanine (Phe) It may be an aptamer that specifically binds to a protein in which protein synthesis is terminated at the 27th amino acid (956th) or a protein represented by the amino acid sequence of SEQ ID NO:3.

In the present invention, the composition may include all of the diagnostic primer, probe, antibody, and aptamer, and may include any combination thereof.

The diagnostic composition of the present invention may include a reagent known in the art used for detecting a mutated LATS1 gene or mutated protein for specific diagnosis of amyotrophic lateral sclerosis.

The diagnostic composition of the present invention may be provided in a state of being fixed on a suitable carrier or support using various known methods in order to increase the speed and convenience of diagnosis. Non-limiting examples of suitable carriers or supports include agarose, cellulose, nitrocellulose, dextran, sephadex, sepharose, liposome, carboxymethylcellulose, polyacrylamide, polyesterin, gabbro, filter paper, ion exchange resin, plastic. films, plastic tubes, glass, polyamine-methyl vinyl-ether-maleic acid copolymers, amino acid copolymers, ethylene-maleic acid copolymers, nylon, cups, flat packs and the like. Other solid substrates include cell culture plates, ELISA plates, tubes, and polymeric membranes. The support may have any possible shape, for example spherical (bead), cylindrical (test tube or well inner surface), planar (sheet, test strip).

The diagnostic composition of the present invention can be used alone for the diagnosis of amyotrophic lateral sclerosis, and other genes or proteins known as diagnostic markers for amyotrophic lateral sclerosis in addition to the detection of the LATS1 gene of the present invention can be used in combination with a composition for detection can In addition, based on clinical symptoms, patient history, and neurological examination, nerve conduction velocity test (NCV), muscle tissue biopsy, blood test, urine test, cerebrospinal fluid test, cervical spine X-ray and magnetic resonance imaging test, electromyography (EMG) ) can also be used with compositions for non-genetic diagnosis such as

In another aspect, the present invention relates to a kit for diagnosis of amyotrophic lateral sclerosis, comprising an agent capable of detecting a mutant LATS1 (Large tumor suppressor kinase 1) gene or a LATS1 mutant protein encoded by a mutant LATS1 gene.

In the present invention, the kit may include the composition for diagnosis of amyotrophic lateral sclerosis.

In the present invention, one or more pairs of primer sets may be used. The primer can be used without limitation as long as it is a sequence capable of specifically binding to the LATS1 mutant gene, but preferably it may be characterized as a primer capable of amplifying the mutation at position 2970 of SEQ ID NO: 1. Most preferably, the primer may be characterized in that it comprises a nucleotide sequence represented by SEQ ID NO: 4 or 5.

In the present invention, the antibody specifically binds to the LATS1 mutant protein encoded by the LATS1 mutant gene, and preferably, in the amino acid sequence of SEQ ID NO: 2, the 930th amino acid is leucine (Leu) to phenylalanine ( Phe), from which protein synthesis is terminated at the 27th amino acid (956th); Or it may be an antibody that specifically binds to the protein represented by the amino acid sequence of SEQ ID NO: 3.

In the present invention, the aptamer specifically binds to the LATS1 mutant protein encoded by the LATS1 mutant gene, and preferably, in the amino acid sequence of SEQ ID NO: 2, the 930th amino acid is leucine (Leu) to phenylalanine (Phe) a protein from which protein synthesis is terminated at the 27th amino acid (956th); Or it may be an aptamer that specifically binds to the protein represented by the amino acid sequence of SEQ ID NO: 3.

In the present invention, the kit may further include one or more other component compositions, solutions, or devices suitable for the analysis method as well as primers, probes, antibodies, etc. for diagnosing amyotrophic lateral sclerosis, preferably RT - It may be in the form of a PCR kit, a microarray chip kit, a DNA chip kit, or a protein chip kit, but is not limited thereto.

As a specific example, the kit for measuring the mRNA expression level of the marker genes in the present invention may be a kit including essential elements necessary for performing RT-PCR. In addition to each primer pair specific for a marker gene, the RT-PCR kit includes a test tube or other suitable container, reaction buffer, deoxynucleotides (dNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNase, RNase inhibitors, DEPC -Water (DEPC-water), sterile water, etc. may be included.

In addition, the kit of the present invention may be a kit including essential elements necessary for performing a microarray. The microarray kit includes a substrate to which cDNA corresponding to a gene or fragment thereof is attached as a probe, and the substrate may include cDNA corresponding to a quantitative control gene or fragment thereof. It can be easily prepared by a manufacturing method commonly used in In order to fabricate a microarray, a micropipetting method using a piezoelectric method or a pin type to immobilize the detected marker on a substrate of a DNA chip using the probe DNA molecule It is preferable to use a method using a spotter, but is not limited thereto. The substrate of the microarray chip is preferably coated with an active group selected from the group consisting of amino-silane, poly-L-lysine and aldehyde, but is not limited thereto. In addition, the substrate is preferably selected from the group consisting of slide glass, plastic, metal, silicon, nylon membrane and nitrocellulose membrane, but is not limited thereto.

In addition, the kit of the present invention may be a kit including essential elements necessary for performing a DNA chip. The DNA chip kit may include a substrate to which cDNA or oligonucleotide corresponding to a gene or fragment thereof is attached, and reagents, agents, enzymes, etc. for preparing a fluorescent probe. The substrate may also contain cDNA or oligonucleotides corresponding to control genes or fragments thereof.

In the present invention, the kit may further include, without limitation, an agent for detecting a diagnostic marker of amyotrophic lateral sclerosis known to those of ordinary skill in the art, in addition to the agent.

The diagnostic kit of the present invention can be used alone for the diagnosis of amyotrophic lateral sclerosis, and in addition to the detection of the LATS1 gene of the present invention, other genes or proteins known as diagnostic markers for amyotrophic lateral sclerosis can be used in combination with a kit for detection can In addition, based on clinical symptoms, patient history, and neurological examination, nerve conduction velocity test (NCV), muscle tissue biopsy, blood test, urine test, cerebrospinal fluid test, cervical spine X-ray and magnetic resonance imaging test, electromyography (EMG) ) can also be used with kits for non-genetic diagnostics such as

In another aspect, the present invention relates to a diagnostic use of amyotrophic lateral sclerosis of a large tumor suppressor kinase 1 ( LATS1 ) mutant gene and a LATS1 mutant protein encoded by the gene, including any one or more of SNV, Indel and CNV. will be.

In another aspect, the present invention, in the isolated biological sample, LATS1 (Large tumor suppressor kinase 1) mutant gene or amyotrophic lateral sclerosis comprising the step of detecting a mutant LATS1 protein encoded by the mutant LATS1 gene It relates to a diagnostic method for sclerosis: ALS.

Example

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not to be construed as being limited by these examples.

Materials and Methods

Sporadic ALS Trio (sALS Trio) Selection

Patients and parents with sporadic ALS who visited Hanyang University Neurology ALS Clinic were included in the ALS trio study. The patient was born in Korea. For clinical diagnosis, a neurological evaluation including electromyography, clinical examination, and appropriate imaging methods was performed on the patient. All ALS patients were diagnosed by a neurologist specializing in neuromuscular disease, and it was confirmed that they met the revised El Escorial diagnostic criteria (reference required). Such as spinal muscular atrophy, Kennedy syndrome, monomelic atrophy, Hirayama syndrome and multifocal motor neuropathy not considered in the ALS spectrum Patients diagnosed with the condition were excluded from the study.

To objectively evaluate response to treatment or disease progression during follow-up, ALSFRS-R was used to determine the extent of functional impairment for diagnosis by physicians. ALSFRS-R includes language skills, salivation, swallowing, handwriting, food cutting and instrumentation (with or without gastrostomy), dressing and hygiene, bed rotation and futon adjustment, walking, stair climbing, dyspnea, and orthepnea. ) and 12 questions assessing respiratory failure. Scores for each item were summed to give a score between 0 and 48. Progression rate was calculated as ΔFS (48-ALSFRS-R at diagnosis/time from onset to diagnosis), and patients were divided into three groups [slow (cut-off value < 0.66), medium (0.66-1.00), and fast (>1.00)]. Clinical reports included indications of upper and lower motor neuron damage at the spinal and medulla oblongata. Sporadic ALS was defined as cases in which the initiator exhibited signs such as progressive superior or inferior motor neuron damage and had no clinically affected family history of the same lineage.

It was conducted with approval from the institutional ethics review committee of Hanyang University Hospital (#HYI-10-01-3) and Samsung Seoul Hospital (#2013-04-131-002), and participants filled out a consent form for the approved research protocol did. The present inventors previously screened genes known to cause ALS, and no pathological mutations were found in the participants.

Whole exome sequencing

Genomic DNA (gDNA) was isolated from peripheral blood leukocytes using the Wizard Genomic DNA Purification Kit (Promega, Madison, WI) according to the manufacturer's instructions. DNA was confirmed by 1% agarose gel electrophoresis and PicoGreen® dsDNA Assay (Invitrogen, Life Technologies, Waltham, Mass.). As far as possible, the DNA should be intact with an OD 260/280 ratio of 1.8-2.0. The SureSelect sequencing library was prepared according to the manufacturer's instructions using the Agilent SureSelect all Exon kit 50Mb (Agilent, Santa Clara, CA) with The Bravo automated liquid handler. Dissolved in 120 μl EB buffer using a Covaris-S2 instrument (Covaris, Woburn, Mass.) set to 10% utilization, intensity 5, cycles per burst 200, and mode frequency sweeping at 4° C. for 360 sec. 3 μg of genomic DNA was cut to a size of 150 bp. The cleavage efficiency was evaluated by capillary electrophoresis of a DNA1000 chip (Bioanalyzer, Agilent).

The sequencing adapter was ligated to the DNA fragment according to the manufacturer's protocol (Agilent). The adapter bound to the DNA was amplified using PCR. The quality of the PCR product was evaluated by capillary electrophoresis (Bioanalyzer, Agilent). SureSelect hyb #1, #2, #3, and #4 reagents (Agilent) were mixed to make the hybridization buffer. The amplified DNA fragment was concentrated to 750 ng in 3.4 μl. SureSelect block #1, #2, and #3 reagents (Agilent) were added to 750 ng of DNA. Hybridization buffer and DNA blocker mix were incubated at 95°C for 5 minutes and then at 65°C for 10 minutes using a gene amplifier. The RNase block (Agilent) was added to the SureSelect oligo capture library (Agilent). The capture library was incubated at 65° C. for 2 minutes.

First, hybridization buffer and then DNA blocker mix were added to the capture library, and the mixture was incubated at 65° C. for 24 hours using a gene amplifier. Dynal MyOne Streptavidin T1 (Invitrogen) coated with 50 ml streptavidin was washed three times with 200 ml SureSelect binding buffer (Agilent), and then resuspended in 200 μl binding buffer. The hybridization mixture was added to the bead suspension and incubated for 30 minutes while mixing at room temperature. Beads were washed with 500 μl SureSelect Wash Buffer #1 (Agilent) for 15 minutes at room temperature, and then washed 3 times with 500 μl SureSelect Wash Buffer #2 (Agilent) at 65° C. for 10 minutes. DNA was eluted with 30 μl water at room temperature for 5 minutes. The reaction was purified with AMPure XP beads (Beckman Coulter, Brea, CA).

Captured libraries were amplified to add index tags using Herculase II Fusion DNA Polymerase (Finnzymes, Life Technologies). The quality of the amplified library was evaluated by capillary electrophoresis (Bioanalyzer, Agilent). QPCR was performed using SYBR Green PCR Master Mix (Applied Biosystems, Life Technologies), and then 6 libraries tagged at equimolar amounts in the pool were combined. Cluster formation was performed using a cBot automated cluster generation system (illumine, San Diego, CA) and sequencing was performed with a unit length of 2x100 bp using a HiSeq 2500 sequencing system (illumina).

Bioinformatics analysis

The reads were mapped to the GRCh37/hg19 build using a Burrows-Wheeler Aligner (BWA) 0.7.10. Picard-tools 1.114 was used to display duplicate reads (http://picard.sourceforge.net/). A GATK (v3.2-2) IndelRealigner was used to reconcile reads around insertion or deletion (indel) positions. The quality of the read results was measured using the GATK BaseRecalibrator. Genotypes were generated simultaneously for all samples by the GATK HaplotypeCaller. Variant quality score adjustment was performed using GATK VariantRecalibrator, and was filtered with 99.7 truth sensitivity level. To identify rare mutations, dbSNP, 1000 Genomes Project (http://www.1000genomes.org/), and gnomAD (http://http://gnomad.broadinstitute.org/) were identified. Annotation of mutations was performed using an in-house custom-made script (Table 1).

Filter by

For the initiator, for the differential diagnosis of ALS, previously known ALS and FTD causative genes or related genes, HSP, and mutations in other disease genes requiring differentiation were first screened (Table 2-5). In order to identify previously known pathogenic mutations, a list of mutations against the human gene mutation database (HGMD®2014.1Proversion) was compared.

The novel mutation was confirmed that the parent was homozygous for the reference sequence, and the initiator was heterozygous. We selected mutations with an allele frequency of less than 0.01 identified in the dbSNP database, the 1000 Genome Project and gnomAD as rare mutations.

In addition, the mutations found in this study were compared with 1722 normal controls of matched race. For normal control data, KRGDB (Korean Reference Genome Database, http://coda.nih.go.kr/coda/KRGDB/index.jsp) was used.

Sanger sequencing to evaluate variant validity

Novel mutations leading to substitution of all amino acids according to the filtering criteria were evaluated by Sanger sequencing of paternal, maternal and progenitor DNA samples. All exon and exon-intron boundaries of the target gene were amplified by PCR using primers (Table 6). PCR was performed using the gene amplifier model GeneAmp PCR system 9700 (Applied Biosytems, Foster City, CA) under the following conditions: 32 cycles of denaturation at 94°C for 30 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 30 seconds. . The amplicon (5 μl) was treated with 2U shrimp alkaline phosphatase and 10U exonuclease I (USB Corp., Cleveland, OH) at 37°C for 15 minutes, and then incubated at 80°C for 15 minutes to inactivate the enzyme. did it Cycle sequencing was performed using the Big Dye Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems, Foster City, CA, USA) in ABI 3130xl Genetic Analyzer (Applied Biosystems, Foster City, CA, USA).

Mutations were named according to the reference sequence of GenBank. The notation for mutation in the present invention followed the recommendation of the Human Genome Mutation Association (http://www.hgvs.org/mutnomen/) in which the nucleotide corresponding to A of the ATG start codon is denoted by +1.

Example 1: Descriptive statistics for Q.C analysis and exome sequencing

Whole exome sequencing of one ALS progenitor and their healthy parents (n=3) was performed using the Agilent SureSelect all Exon kit 50Mb (Agilent). Total reads of over 50,000,000 bp on average were obtained for each individual. All samples from one progenitor and his parents averaged 106x. For each individual, an average of 99.7% of target bases were processed by at least one independent sequence read, 98.2% were processed by at least 10 independent sequence reads, and 95.2% were processed by at least 20 independent sequence reads became (Table 7).

No mutations were found in previously reported ALS-FTD causes or related genes, HSP for differential diagnosis of ALS, and other disease genes (Table 2-5).

Example 2: Discovery of novel variants of unknown significance (VUS)

After confirming the mutations using Sanger sequencing, the present inventors discovered one novel variants of unknown significance (VUS) from the sALS trio ( FIGS. 2 and 3 ). In this mutation, as the 2790th nucleotide sequence of the LATS1 gene is deleted, leucine, the 930th amino acid among the amino acid sequence of the normal protein LATS1 having the amino acid sequence of SEQ ID NO: 2, is substituted with phenylalanine, and protein synthesis occurs at the 27th (956th) from there. The frameshift mutation that ends. The LATS1 mutant protein expressed by mutation of the LATS1 gene is represented by the amino acid sequence of SEQ ID NO: 3.

This novel VUS was not found in dbSNP, 1000 Genome Project (total and East Asian allele frequencies), gnomAD. Also, it was not found in KRGDB, which is the data of normal control Koreans with matching race. The experimental analysis results suggest that the LATS1 gene is the causative gene for sALS (Table 8).

As a specific part of the present invention has been described in detail above, for those of ordinary skill in the art, it is clear that this specific description is only a preferred embodiment, and the scope of the present invention is not limited thereby. will be. Accordingly, it is intended that the substantial scope of the present invention be defined by the appended claims and their equivalents.

The present invention provides an accurate and efficient marker-based diagnostic method for diagnosing amyotrophic lateral sclerosis that lacks effective molecular diagnostic markers. The marker of the present invention can diagnose amyotrophic lateral sclerosis even when a previously reported marker is not found, and suggests a new diagnostic method for amyotrophic lateral sclerosis. The diagnostic method, marker, and use thereof of the present invention are useful in the diagnosis, prediction, treatment and prevention of amyotrophic lateral sclerosis. Furthermore, the LATS1 gene of the present invention can be used to study the onset mechanism of amyotrophic lateral sclerosis and develop a drug for treatment.

An electronic file is attached.

Claims

In the isolated biological sample, LATS1 (Large tumor suppressor kinase 1) mutant gene or LATS1 encoded by the LATS1 mutant gene A method for providing information for diagnosis of amyotrophic lateral sclerosis (ALS), comprising the step of detecting a mutated protein.
The method of claim 1, wherein the LATS1 mutant gene is a single nucleotide variant (Single Nucleotide variant: SNV), insertion or deletion (Indel), and any one or more selected from the group consisting of copy-number variation (CNV) An information providing method for the diagnosis of amyotrophic lateral sclerosis (ALS), characterized in that the mutation has occurred.
According to claim 1, wherein the LATS1 (Large tumor suppressor kinase 1) mutant gene,

In the nucleotide sequence of SEQ ID NO: 1, a gene in which the 2790th nucleotide is deleted; or

Information providing method, characterized in that the gene encoding the protein represented by the amino acid sequence of SEQ ID NO: 3.
According to claim 1, wherein detecting the LATS1 mutant gene amplifies the gene,

An information providing method, characterized in that the gene mutation is analyzed using sequencing data of the amplified product.
The method of claim 1, wherein a primer capable of specifically amplifying the mutation site of the LATS1 mutant gene or

An information providing method, characterized in that the detection is performed using a probe that is complementary to a region including the mutation site of the LATS1 mutant gene.
According to claim 1, LATS1 encoded by the LATS1 mutant gene The mutant protein is

In the nucleotide sequence of SEQ ID NO: 1, a protein encoded by a gene in which the 2790th nucleotide is deleted; or

Information providing method, characterized in that the protein represented by the amino acid sequence of SEQ ID NO: 3.
According to claim 1, LATS1 encoded by the LATS1 mutant gene The step of detecting the mutant protein is an information providing method, characterized in that the detection using an antibody or aptamer specific for the mutant protein.
As a diagnostic marker of amyotrophic lateral sclerosis, LATS1 (Large tumor) containing at least one of single nucleotide variant (SNV), insertion or deletion (Indel), and copy-number variation (CNV) suppressor kinase 1) mutant gene.
The mutant gene according to claim 8, wherein the LATS1 mutant gene has the 2790th nucleotide deleted from the nucleotide sequence of SEQ ID NO: 1.
As a diagnostic marker for amyotrophic lateral sclerosis, LATS1 (Large tumor) containing at least one of single nucleotide variant (SNV), insertion or deletion (Indel), and copy-number variation (CNV) suppressor kinase 1) LATS1 mutant protein encoded by mutant gene.
11. The method of claim 10, wherein the LATS1 mutant protein,

In the nucleotide sequence of SEQ ID NO: 1, a protein encoded by a gene in which the 2790th nucleotide is deleted; Or a protein characterized in that the protein represented by the amino acid sequence of SEQ ID NO: 3.
A composition for diagnosis of amyotrophic lateral sclerosis comprising an agent capable of detecting a large tumor suppressor kinase 1 ( LATS1) mutant gene or a LATS1 mutant protein encoded by the LATS1 mutant gene.
The method of claim 12, wherein the formulation is characterized in that in the region containing the mutation occurs region of LATS1 variation Mutagenic capable of amplifying the region with specific primers or LATS1 mutant gene of a gene comprising a probe that binds complementarily composition to do.
The composition according to claim 13, wherein the primer is represented by the nucleotide sequence of SEQ ID NO: 4 or 5.
The composition of claim 12, wherein the agent comprises an antibody or aptamer specific for the LATS1 mutant protein.
A kit for diagnosis of amyotrophic lateral sclerosis, comprising an agent capable of detecting a large tumor suppressor kinase 1 ( LATS1) mutant gene or a LATS1 mutant protein encoded by the LATS1 mutant gene.