JP2007020426A - Method for judging onset of pulmonary alveolar microlithiasis - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for judging the presence of known alteration in the human SLC34A2 gene of an examinee to cause the onset of pulmonary alveolar microlithiasis, provide a method for judging the onset risk of an examinee before the onset of pulmonary alveolar microlithiasis and establish the cause of the disease for an examinee suspected to be affected by pulmonary alveolar microlithiasis and provide an effective treatment to a patient of pulmonary alveolar microlithiasis. <P>SOLUTION: The invention provides a method for extracting a nucleic acid or a protein from an examinee and detecting a known alteration in the human SLC34A2 gene and human type IIb sodium-phosphate cotransporter protein or an alteration from a wild-type base sequence or an amino acid alteration from a wile-type amino acid sequence, and provides a mutant primer and a clamp primer therefor. The invention further provides a method for the treatment of pulmonary alveolar microlithiasis using a corrected gene based on human SLC34A2 gene. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method for determining the possibility of developing alveolar microlithiasis, a polynucleotide as a probe or primer used in the method, and a polypeptide used as an antigen for preparing an antibody.

  Alveolar microlithiasis is a rare disease whose cause is unknown due to the formation of countless microliths made of layered and annual ring-shaped calcium phosphate in the alveoli (Non-patent Document 1). Although this disease can be seen from children to adults, there is no gender difference in onset, and symptoms vary with age. Usually, childhood to juvenile cases are generally poor in subjective symptoms, despite significant diffuse lung shadows on chest x-rays. For this reason, many cases of this age are often found by chance during health examinations and family examinations. In contrast, cases over 40 years of age complain of subjective symptoms such as dyspnea and cough during exercise. Although the long-term prognosis of this disease varies depending on the age at the time of discovery, it is not necessarily good. In particular, in middle-aged and older people over 40 years old, there are many cases in which respiratory symptoms such as cough and dyspnea occur with the progression of symptoms, resulting in respiratory failure and death. The average mortality of patients with this disease is in their 50s, and half of those who died within 5 years are found to be over 40 years old.

  This disease is considered to be an inherited lung disease due to autosomal recessive inheritance because of the high frequency of siblings and the tendency of horizontal transmission among siblings (Non-patent Document 2). However, the causative gene has not been identified. Although it is a rare disease, its potential incidence is not necessarily low in countries with high siblings, such as island nations consisting of a single ethnic group, or countries with a high relative marriage rate, and can be ignored. is not. Particularly in Japan, it is known that there are very many cases of this disease worldwide (Non-patent Document 3).

From such a background, investigation of the cause of this disease and development of a treatment method are desired, but at present there is no known effective treatment method other than coping therapy such as oxygen therapy or lung transplantation. In addition, this disease tends to worsen as the age of discovery increases or as age increases. However, early detection is difficult if there is no accidental opportunity because of the asymptomatic condition from childhood to early childhood and the very slow progress. In fact, there are many cases where symptoms have already progressed at the time of discovery by subjective symptoms. If you can quickly and easily determine the possibility of developing alveolar microlithiasis when there is no subjective symptom, receive early diagnosis by a doctor and treatment for this disease that is expected to be developed in the near future. Is also possible.
Japanese Patent Application No. 2005-203654 USP 5,424186 Special table flat 10-503841 Japanese Patent Application No. 2005-117698 Mariotta S. et al. , Sarcoidosis Vasc. Diffuse Lung Dis. 21, 173-81 (2004). Castellana G. & Lamorgese V. , Respiration 70, 549-55 (2003). Tachibana T. Hayashi S .; Jokoh T .; UEDA S. & Takahashi H. , Sarcoidosis Vasc. Diffuse Lung Dis. 18 (suppl 1), 58 (2001). Lander E. & Botstein D. , Science 236, 1567-70 (1987). Murer H. Forster I. & Biber J. , Pflugers Arch. -Eur. J. et al. Physiol. 447, 763-67 (2004). Xu H. , Bai L. Collins J .; F. & Ghishan F. K. Genomics 62, 281-4 (1999). NagaiY. , Et al. , Cancer Res (in press). Huquun, Izumi S. , Miyazawa H .; , Ishii K .; Uchiyama B .; , Et al. Submitted.

  The subject of this invention is providing the method of determining the onset possibility of alveolar microlithiasis with respect to the test subject before the subjective symptom of alveolar microlithiasis. Another object of the present invention is to determine the cause of a subject diagnosed as suspected to have alveolar microlithiasis due to clinical symptoms. A further object of the present invention is to use a polynucleotide as a probe or primer used for the onset determination capable of developing alveolar microlithiasis quickly and easily for a subject, and an antigen for producing an antibody. It is to provide a polypeptide.

  In order to solve the above problems, it was first necessary to identify the gene responsible for alveolar microlithiasis. Thus, as a result of intensive studies, the present inventors have discovered that the causative gene of alveolar microlithiasis is a human SLC34A2 gene encoding a human type IIb sodium-phosphate cotransporter protein. The present invention provides the following method for determining the possibility of the onset of alveolar microlithiasis completed based on the discovery of such alveolar microlithiasis-causing gene, and the polynucleotide and polypeptide used therefor. The present invention relates to a polynucleotide used as a probe or primer used in the method and a polypeptide used as an antigen for preparing an antibody. That is, the following invention is provided.

  (1) The present invention relates to a nucleic acid extraction step for extracting a human-derived nucleic acid, and a base sequence of a human SLC34A2 gene that can be contained in the extracted nucleic acid from a wild-type base sequence represented by SEQ ID NO: 1. Provided is a method for determining the onset of alveolar microlithiasis, which comprises determining the possibility of developing alveolar microlithiasis, comprising a genetic alteration detection step for detecting whether or not a known alteration derived from the alteration is present.

  (2) The present invention relates to a nucleic acid extraction step for extracting a human-derived nucleic acid, and the base sequence of the human SLC34A2 gene that can be contained in the extracted nucleic acid is modified from the wild-type base sequence represented by SEQ ID NO: 1. A genetic modification detection step for detecting whether or not a genotype of the human SLC34A2 gene that can be contained in the extracted nucleic acid is a wild type homozygote, a modified homozygote, a wildtype and a modified heterologous, or a modified heterologous The present invention provides a method for determining the onset of alveolar microlithiasis, which comprises a genotype determination step of determining which one of

  (3) The present invention provides a method for determining the onset of alveolar microlithiasis, wherein the nucleic acid extracted in the nucleic acid extraction step is genomic DNA, total RNA, mRNA, or a nucleic acid based thereon.

  (4) The present invention provides a method for determining the onset of alveolar microlithiasis, wherein the total RNA or mRNA is extracted from lung tissue, sputum, or pleural effusion.

  (5) The present invention provides a method for determining the onset of alveolar microlithiasis, wherein the modification to be detected in the genetic modification detection step is a base deletion, substitution, or addition.

  (6) In the present invention, the known modification to be detected in the genetic modification detection step is a base represented by SEQ ID NO: 3 from the 13290th position T to the 13304th position G represented by SEQ ID NO: 1. Provided is a method for determining the onset of alveolar microlithiasis, which is a modification substituted with a sequence.

  (7) The present invention is based on a modification in which the G of the splicing donor site of the eighth intron is replaced with A in the base sequence represented by SEQ ID NO: 1 in the known genetic modification detection step. A method for determining the onset of alveolar microlithiasis is provided.

  (8) In the present invention, the detection method of the modification in the genetic modification detection step is the entire or continuous base sequence of known modifications derived from the modification from the base sequence of the wild-type human SLC34A2 gene represented by SEQ ID NO: 1. A method for determining the onset of alveolar microlithiasis, which is a method of detecting by hybridization using a polynucleotide having a part consisting of at least 14 bases as a probe.

  (9) The present invention provides a region in which a known modification base sequence derived from a modification from the base sequence of the wild-type human SLC34A2 gene represented by SEQ ID NO: 1 is present in the modification detection method in the genetic modification detection step A method for determining the onset of alveolar microlithiasis, which is a method of detecting by determining all or a part of the nucleotide sequence.

  (10) The present invention relates to a polynucleotide comprising a whole of the base sequence of the wild type human SLC34A2 gene represented by SEQ. Provided is a method for determining the onset of alveolar microlithiasis, which is a method of detecting by hybridization using as a probe.

  (11) In the present invention, the modification detection method in the genetic modification detection step determines all or a part of the base sequence of the region where the human SLC34A2 gene may be present in the nucleic acid extracted in the nucleic acid extraction step, Provided is a method for determining the onset of alveolar microlithiasis, which is a method of detecting by comparing the base sequence with the base sequence of the wild-type gene represented by SEQ ID NO: 1.

  (12) The present invention provides a method for determining the onset of alveolar microlithiasis, which is a method for detecting whether a target nucleic acid amplification product is increased by the nucleic acid amplification method in the method for detecting a modification in the genetic modification detection step.

  (13) The present invention provides a method for detecting a modification in the genetic modification detection step by detecting the number of bases of a wild-type nucleic acid amplification product amplified by a nucleic acid amplification method and a modified nucleic acid amplification product. A method for determining the onset of alveolar microlithiasis is provided.

  (14) The present invention provides a method for determining the onset of alveolar microlithiasis, wherein the detection of the modification in the genetic modification detection step is performed based on a cleavage pattern of a wild type and a modified type using a restriction enzyme utilizing RFLP.

  (15) In the present invention, the protein extraction step of extracting a human-derived protein and the amino acid sequence of the human type IIb sodium-phosphate cotransporter protein that can be contained in the extracted protein are represented by SEQ ID NO: 2. And a method for determining the onset of alveolar microlithiasis that determines the possibility of developing alveolar microlithiasis, comprising the step of detecting whether or not there is a known amino acid modification from a wild-type amino acid sequence.

  (16) In the present invention, the protein extraction step of extracting a human-derived protein and the amino acid sequence of the human type IIb sodium-phosphate cotransporter protein that can be contained in the extracted protein are represented by SEQ ID NO: 2. An amino acid modification detecting step for detecting whether the amino acid has been modified from the wild-type amino acid sequence, and a genotype of a human SLC34A2 gene encoding a human type IIb sodium-phosphate cotransporter protein that can be contained in the extracted protein. Provided is a method for determining the onset of alveolar microlithiasis, comprising a second genotype determination step for determining whether a wild-type homozygote, a modified homozygote, a wild-type and a modified heterotype, or a modified heterotype .

  (17) The present invention provides an alveolar microlithiasis onset determination method in which the protein in the protein extraction step is extracted from lung tissue.

  (18) The present invention provides a method for determining the onset of alveolar microlithiasis, wherein the mode of amino acid modification to be detected in the amino acid modification detection step is amino acid deletion, substitution, or addition.

  (19) The present invention provides a method for determining the onset of alveolar microlithiasis, wherein the amino acid modification to be detected in the amino acid modification detection step is an amino acid modification having the amino acid sequence represented by SEQ ID NO: 5.

  (20) The present invention provides a method for determining the onset of alveolar microlithiasis, wherein the amino acid modification to be detected in the amino acid modification detection step is an amino acid modification having the amino acid sequence represented by SEQ ID NO: 6.

  (21) The present invention relates to an amino acid sequence of an amino acid-modified human type IIb sodium-phosphate cotransporter protein whose amino acid modification detection method in the amino acid modification detection step is known, Provided is a method for determining the onset of alveolar microlithiasis, which is a method of detecting by an antigen-antibody reaction method using an antibody whose antigen is a polypeptide having at least 5 consecutive amino acids.

  (22) The present invention provides at least 5 amino acids in the amino acid sequence of the wild-type human type IIb sodium-phosphate cotransporter protein represented by SEQ ID NO: 2 as the method for detecting amino acid modifications in the amino acid modification detection step. The present invention provides a method for determining the onset of alveolar microlithiasis, which is a method of detecting by an antigen-antibody reaction method using an antibody whose antigen is a polynucleotide having a part of the above.

  (23) In the base sequence of a known modified human SLC34A2 gene derived from modification from the wild-type base sequence represented by SEQ ID NO: 1, the present invention provides at least all or a continuous base sequence of the modified region. Provided is a polynucleotide comprising a base sequence having a part of 14 bases or more and used as a probe for use in the method for determining the onset of alveolar microlithiasis according to (8) above.

  (24) The present invention provides a polynucleotide in which the base sequence of the modified region is the base sequence represented by SEQ ID NO: 3.

  (25) The present invention provides a polynucleotide in which the base sequence of the modified region is the base sequence represented by SEQ ID NO: 4.

  (26) The present invention consists of the base sequence of the wild-type human SLC34A2 gene represented by SEQ ID NO: 1 all or a base sequence having at least 14 consecutive bases as a part, and is described in (10) above Provided is a polynucleotide used as a probe for use in a method for determining the onset of alveolar microlithiasis.

  (27) In the base sequence of a known modified human SLC34A2 gene derived from modification from the wild-type base sequence represented by SEQ ID NO: 1, the present invention is a case where the base sequence of the modified region is 18 bases or more , Having a part of 18 to 60 bases in the base sequence of the modified region as a part, and used as a primer used in the method for determining the onset of alveolar microlithiasis according to (9) or (12) above Provided polynucleotides.

  (28) The present invention provides a polynucleotide in which the base sequence of the modified region is the base sequence represented by SEQ ID NO: 3.

  (29) The present invention provides a polynucleotide in which the base sequence of the modified region is the base sequence represented by SEQ ID NO: 4.

  (30) The present invention includes a part of a continuous 18 to 60 bases in the base sequence of the wild-type human SLC34A2 gene represented by SEQ ID NO: 1, and the lung according to (11) or (13) Provided is a polynucleotide used as a primer for use in a method for determining the development of cyst microlithiasis.

  (31) The present invention provides an alveolar microlithiasis onset determination kit for determining the possibility of onset of alveolar microlithiasis using any one or more of the polynucleotides (23) to (30). .

  (32) The present invention relates to an amino acid sequence of a region in which amino acid modification is performed in the amino acid sequence of a known amino acid-modified human type IIb sodium-phosphate cotransporter protein from the wild-type amino acid sequence represented by SEQ ID NO: 2 As an antigen for preparing an antibody that comprises an amino acid sequence having at least 5 consecutive amino acids as a part thereof and that detects an amino acid modification in the method for determining the onset of alveolar microlithiasis according to (15) above A polypeptide is provided.

  (33) The present invention provides a polypeptide, wherein the amino acid sequence of the amino acid-modified region is the amino acid sequence represented by SEQ ID NO: 5.

  (34) The present invention provides a polypeptide, wherein the amino acid sequence of the amino acid-modified region is the amino acid sequence represented by SEQ ID NO: 6.

  (35) The present invention consists of an amino acid sequence having, as a part, at least 5 or more consecutive amino acids of the entire amino acid sequence of the wild-type human type IIb sodium-phosphate cotransporter protein represented by SEQ ID NO: 2; Provided is a polypeptide as an antigen for preparing an antibody for detecting a modification of the method for determining the onset of alveolar microlithiasis according to (16) above.

  (36) The present invention relates to a nucleic acid extraction step for extracting a human-derived nucleic acid, and a wild-type nucleic acid represented by SEQ ID NO: 7 in which the base sequence of the expression control region of the human SLC34A2 gene that can be contained in the extracted nucleic acid An expression control region modification detection step for detecting whether the base sequence has been modified, and an alveolar microlithiasis onset determination method for determining the possibility of onset of alveolar microlithiasis.

  According to the “onset determination method” of the present invention, it is possible to quickly and easily detect whether the human SLC34A2 gene has a known modification that causes onset of alveolar microlithiasis. If the subject is a subject before the onset of subjective symptoms of alveolar microlithiasis, the possibility of developing alveolar microlithiasis as a subjective symptom in the future can be determined by the detection. If the determination result is “possible to develop”, it is possible to confirm the genotype of the human SLC34A2 gene of the subject and receive early diagnosis and early treatment by a doctor. Moreover, if it is a test subject who has already been diagnosed with alveolar microlithiasis from clinical symptoms, it can be determined by this detection whether the human SLC34A2 gene of the test subject has a known modification. If the subject has a known modification, it can be determined that the subject may have alveolar microlithiasis even at the genetic level.

  According to the “onset determination method” of the present invention, the genotype related to the human SLC34A2 gene of a subject can be determined quickly and easily before the subjective symptoms of alveolar microlithiasis. The possibility of future onset of the disease can be determined from the determination result. In addition, this method can be performed even if the subject is a fetus. Furthermore, even when it is determined by the method that the subject is highly likely to develop the disease, it is possible to receive early diagnosis and early treatment by a doctor.

  According to the “onset determination method” of the present invention, the present invention is carried out on a subject who has developed a disease suspected of having alveolar microlithiasis from clinical symptoms, thereby detecting the modification of the human SLC34A2 gene. It can be determined that the subject has alveolar microlithiasis. Moreover, if it turns out that it is due to the modification of the gene, it becomes possible to perform appropriate treatment accordingly.

  According to the “onset determination method” of the present invention, by revealing the genotype of a couple's human SLC34A2 gene, the possibility of developing alveolar microlithiasis in a child born between the couple can be predicted. Furthermore, early diagnosis and early treatment of alveolar microlithiasis in the fetus can be performed by determining the possibility of fetal development at an early stage after the pregnancy is determined according to the result.

  According to the “kit containing a polypeptide used in the onset determination method” of the present invention, the possibility of the onset of alveolar microlithiasis due to the modification of the human SLC34A2 gene can be determined quickly and easily.

  According to the determination method or the kit of the present invention, alveolar microlithiasis caused by alteration of the human SLC34A2 gene without requiring specialized knowledge or skill as a doctor if a person who has acquired a certain operation technique is acquired. The possibility of onset can be determined. In addition, some of the methods do not require special dedicated equipment, and can be carried out with equipment that is always available in most research facilities. In addition, regarding the method that requires a special dedicated device, analysis can be easily entrusted to a specialist.

  The best mode for carrying out each invention will be described below. The present invention is not limited to these embodiments, and can be implemented in various ways without departing from the scope of the invention.

  In the present invention, nucleic acid, base, base sequence, gene, genotype, wild type, homo, hetero, genomic DNA, cDNA, total RNA, mRNA, intron, splicing donor site, hybridization, probe, primer, protein, amino acid The definitions of the terms of amino acid sequences and codes are the same as the meanings widely used by those skilled in the art in the fields of molecular biology, genetics, genetic engineering, etc. Explanation of the definition of is omitted.

The first embodiment relates to claims 1, 3 to 9, 12, 23 to 30, and the like.
The second embodiment relates to claims 2 to 14, 23 to 30, and the like. The third embodiment relates to claims 15, 17 to 21, 32 to 35, and the like. The fourth embodiment relates to claims 16 to 22, 32 to 35, and the like. The fifth embodiment relates to claim 31 and the like. The sixth embodiment relates to claim 36 and the like.

  Prior to the description of the present embodiment, the discovery of a causative gene for alveolar microlithiasis will be described. Past research results suggest that this disease is a simple recessive genetic disease (Non-patent Document 3). As a method for identifying a recessive disease gene, a homozygosity mapping method is known as a conventional technique (Non-patent Document 4). However, it has been difficult to identify the causative genes of rare genetic diseases such as alveolar microlithiasis by this method, which is based on pedigree analysis. Therefore, the present inventors have expanded the concept of the homozygote mapping method and newly developed a homozygous fingerprint method. Regarding the principle of the homozygous fingerprint method, etc., a patent application invented by the present inventors “Homozygous region determination method, homoeologous region determination device, and gene screening method by homozygous fingerprint method (Patent Document 1)” Please refer to.

  As a result of screening the causative gene using samples obtained from 3 alveolar microlithiasis patients by the homozygous fingerprint method, the causative gene of this disease was 11.5 Mb (Mb = 1 million bases) It was found to exist in the candidate area. The candidate region contains 35 genes, 25 of which were known genes or genes whose functions were predicted. The only gene directly involved in the metabolism of calcium and phosphate considered to be highly related to this disease was the human SLC34A2 gene in the candidate region. The human SLC34A2 (Unigene Hs. 479372) gene is one of the members of the Solute Carrier Family 34 (SLC34) and encodes a human type IIb sodium-phosphate cotransporter protein. This protein is a membrane protein that functions to transport phosphate ions mainly on the cell membrane by a sodium-dependent method (Non-patent Document 5). Therefore, focusing on the gene as a candidate gene for the cause of alveolar microlithiasis, the genomic base sequences of the gene of the 3 patients with alveolar microlithiasis and 10 healthy individuals were determined. As a result, all of the healthy individuals had the same wild-type nucleotide sequence, but all three patients with alveolar microlithiasis had homozygous modifications to the gene. The types of alterations detected from alveolar microlithiasis patients were the two types shown in FIG. 1 and FIG. In addition, it was found that neither of the amino acid-modified proteins based on the modification of the two genes has activity as a human type IIb sodium-phosphate cotransporter. From the above results, it is clear that the human SLC34A2 gene is a causative gene for alveolar microlithiasis, and that the inactivation of the function of the human type IIb sodium-phosphate cotransporter is associated with the onset of alveolar microlithiasis. (Non-Patent Document 8). Hereinafter, two types of modifications discovered by the present inventors will be described in detail.

  The first modification was a modification by substitution as shown in FIG. Specifically, in the wild-type base sequence (0101), a sequence corresponding to 15 bases from T at position 13290 to G at position 13304 in SEQ ID NO: 1 is represented by SEQ ID NO: 3 in the modified type (0102). The 19-base sequence (0103) was replaced. Since this modification causes an amino acid frameshift, a stop codon appears in the middle of wild-type CDS. As a result, the amino acid after isoleucine at position 286 of SEQ ID NO: 2 was substituted with 28 amino acids represented by SEQ ID NO: 5, and as shown in FIG. Transporter protein (0105) is produced. The modified protein lacks five transmembrane domains (TM) present on the C-terminal side among eight transmembrane domains (TM) predicted from the amino acid sequence of the wild-type protein (0104). In the present specification, hereinafter, the modification of the base sequence is referred to as modification A, and the amino acid sequence generated by the modification A is referred to as amino acid modification A.

  The second modification was also a modification by substitution as shown in FIG. Specifically, in the wild type base sequence (0201), GT represented as the splicing donor site (double underlined portion) of the 8th intron had a point mutation that replaced AT in the modified type (0202). As a result of this modification, the 8th intron is not removed by mRNA splicing after transcription of the gene. As shown in FIG. 2B, the mature mRNA has a nucleotide sequence (0203) in which the modified 8th intron is left. ) That is, in the wild-type CDS (Cording sequence: amino acid coding sequence) of the gene described in SEQ ID NO: 1, the nucleotide sequence represented by SEQ ID NO: 4 is present between T at position 14304 and G at position 15670. The arrangement is similar to the inserted state. Since this modification causes an amino acid frame shift, a stop codon appears in the base sequence of SEQ ID NO: 4. As a result, the cysteine after position 350 of SEQ ID NO: 2 was substituted with 10 amino acids represented by SEQ ID NO: 6, and an amino acid-modified human type IIb sodium-phosphate codon consisting of 359 amino acids as shown in FIG. Transporter proteins are produced. The modified protein (0205) lacks 5 of the 8 transmembrane domains predicted from the amino acid sequence of the wild-type protein (0204), present on the C-terminal side. In this specification, the modification of the base sequence is referred to as modification B, and the amino acid sequence generated by modification B is referred to as amino acid modification B.

  Here, SEQ ID NO: 1 represents the entire nucleotide sequence (including 5 ′ untranslated region and 3 ′ untranslated region) on the genome of the wild type human SLC34A2 gene and the amino acid sequence corresponding to the coding region. . Position information such as exon and intron numbers is described in the sequence listing. SEQ ID NO: 2 represents the entire amino acid sequence of wild-type human type IIb sodium-phosphate cotransporter protein. SEQ ID NO: 3 represents a sequence consisting of 19 bases that replaces the modification A. SEQ ID NO: 4 represents the base sequence of the eighth intron having a point mutation at the splicing donor site in the modified B. SEQ ID NO: 5 represents an amino acid sequence obtained by amino acid modification with the amino acid modification A. SEQ ID NO: 6 represents an amino acid sequence obtained by amino acid modification with the amino acid modification B.

  The following embodiments have been invented based on the above findings.

  << Embodiment 1 >>

  <Embodiment 1: Overview> Embodiment 1 extracts a human-derived nucleic acid and detects whether the to SLC34A2 gene, which is a causative gene for alveolar microlithiasis, that can be contained in the extracted nucleic acid has a known modification This is based on a method for determining the possibility of developing alveolar microlithiasis.

  <Embodiment 1: Process Flow> FIG. 3 illustrates a process flow of the first embodiment. First, in the “nucleic acid extraction step” (S0301), human-derived nucleic acid is extracted from a sample by an appropriate method. Next, the base sequence of the human SLC34A2 gene that can be contained in the human-derived nucleic acid obtained in the nucleic acid extraction step in the “gene modification detection step” (S0302) is determined based on the wild-type base sequence represented by SEQ ID NO: 1. It is detected whether it has a known modification derived from the modification. The onset possibility of alveolar microlithiasis is determined from the results obtained by the above steps. Hereinafter, each step will be described in detail.

  The “nucleic acid extraction step” (S0301) is a step of extracting human-derived nucleic acid from a sample. The sample is usually obtained from the human body, but may be obtained from a transformant containing a human-derived nucleic acid and a human-derived nucleic acid. Use a sample derived from a single individual. However, this is not the case when the presence or absence of alteration of the human SLC34A2 gene is examined using two or more units using the method of the present invention. In the case where the sample is obtained from the human body, the type and state of the sample are not limited as long as the nucleic acid of the collected individual can be extracted. For example, a part of the tissue that constitutes the human body such as blood, mucous membrane, skin, hair, and nails, or that is secreted or excreted from the human body such as saliva, semen, pus, stool, etc. In this case, villus or amniotic fluid is applicable. The sample may be cultured for a certain period of time immediately after collection (limited to living cells), frozen after collection, or stored as a paraffin-embedded specimen after formalin fixation. You may use things. The type of nucleic acid extracted in the nucleic acid extraction step is preferably genomic DNA, total RNA, mRNA, or a nucleic acid based on them that can contain the nucleotide sequence information of the target human SLC34A2 gene.

  “Nucleic acid based on them” means a nucleic acid processed based on human-derived genomic DNA, total RNA, or mRNA. For example, cDNA obtained by reverse transcription reaction of mRNA, genomic DNA, cDNA, or cloned DNA obtained by incorporating all or part of EST into a vector such as a plasmid or virus.

  “Nucleic acid based on them” means a nucleic acid processed based on human-derived genomic DNA, total RNA, or mRNA. For example, cDNA obtained by reverse transcription reaction of mRNA, clone DNA in which all or part of genomic DNA, cDNA, or EST is incorporated into a vector such as a plasmid or virus is applicable.

  When total RNA or mRNA is extracted in the nucleic acid extraction step, the sample is preferably lung tissue, sputum or pleural effusion of the subject. This is based on the fact that the human SLC34A2 gene, which is a causative gene for alveolar microlithiasis, is most frequently expressed in the lung (Non-patent Document 6).

  The method for extracting nucleic acid from cells is not particularly limited as long as the target nucleic acid can be extracted from a sample. However, usually, as shown in FIG. 4, (A) a step of disrupting cells (S0401), (B) a step of removing proteins (S0402), and (C) a step of removing non-target nucleic acids (S0403). The method is common. Hereinafter, each step of this method will be described as an example of a method for extracting nucleic acid from cells.

  (A) The step of disrupting cells (S0401) is a step of destroying the membrane structure of the collected cells and causing cell contents such as cytoplasm to flow out. The destruction of the cell membrane structure can be achieved, for example, by chemical destruction of the cell membrane lipid structure by a surfactant, or by enzymatic chemical destruction by the combined use of a protease and a surfactant such as proteinase K.

  (B) The step of removing protein (S0402) is a step of denaturing and removing the protein contained in the cell lysate in which the cell contents are dissolved. The protein denaturation treatment method can be achieved by, for example, phenol treatment.

  (C) The step of removing non-target nucleic acid (S0403) is a step of removing the non-target nucleic acid from the nucleic acid contained in the sample obtained in the step of removing the protein and purifying the target nucleic acid. . The nucleic acid to be removed here is either DNA or RNA. The method of removing nucleic acids other than the target can be achieved by an enzyme treatment using a nucleolytic enzyme that specifically degrades only one of the nucleic acids, such as an RNA degrading enzyme or a DNA degrading enzyme.

  When extracting nucleic acid from a sample, it is convenient to use a commercially available kit in addition to the above method. When using a kit, use a kit of the type appropriate for the type of sample. The procedure for using the kit may be in accordance with the protocol attached to the kit.

  By the way, the amount of nucleic acid extracted in the nucleic acid extraction step is often insufficient for the amount required in the subsequent steps. In such a case, it is preferable to amplify the nucleic acid as necessary before performing the subsequent steps. The amplification method is not particularly limited as long as the target nucleic acid region can be amplified. For example, a gene cloning method may be used, but a nucleic acid amplification method is convenient because it is quick and simple. The nucleic acid amplification method is a method of amplifying a specific nucleic acid region by an enzymatic reaction using an amplification primer. For example, the PCR method, ICAN method, LAMP method, or NASBA method is applicable. Among these methods, the PCR method is the most widely used method in the fields of medicine and molecular biology all over the world, and is convenient because various application techniques are known.

  The “gene modification detection step” (S0302) is a wild type wherein the nucleotide sequence of the human SLC34A2 gene (hereinafter referred to as a modification detection gene) that can be contained in the nucleic acid extracted in the nucleic acid extraction step is represented by SEQ ID NO: 1. It is the process of detecting whether it has a known modification derived from the modification from the base sequence of.

  In the present embodiment, “modified” means a state in which the base sequence of the modified detection gene does not match the base sequence of the wild-type human SLC34A2 gene. The base sequence of the wild type human SLC34A2 gene is based on the base sequence on the genome represented by SEQ ID NO: 1. “Based on the base sequence on the genome” means that the base sequence on the genome represented by SEQ ID NO: 1 is transcribed and spliced into the wild-type base sequence of mature mRNA, and human IIb type sodium-phosphate co-transport It is also meant to include wild-type CDS that encodes body amino acids. In addition, the modification of the present embodiment means a substantial modification that causes alveolar microlithiasis particularly by homozygosity. That is, when only a protein having no function as a wild-type human type IIb sodium-phosphate cotransporter is produced by homozygote, or only a wild-type protein necessary for functioning as the cotransporter This is the case when the amount is not produced.

  Examples of the modification to be detected in the present embodiment include base deletion, substitution, or addition. “Deletion” refers to a modification in which one or more bases are lost from the base sequence of the wild-type gene when the base sequence of the modified detection gene is compared with the base sequence of the wild-type human SLC34A2 gene. In the present embodiment, “substitution” means that when the base sequence of the modified detection gene is compared with the base sequence of the wild-type human SLC34A2 gene, one or more consecutive bases other than all of the base sequence of the wild-type gene A modification that is replaced with a different base. For example, duplications, insertions, or translocations with point mutations, inversions, or deletions. Furthermore, in this embodiment, “addition” means that one or more consecutive positions are not found in the base sequence of the wild-type human SLC34A2 gene when the base sequence of the modified detection gene is compared with the base sequence of the wild-type gene. A modification in which a base is newly added. For example, an insertion, duplication, or translocation without deletion is applicable.

  In this embodiment, the “known modification” is a modification that occurs on the base sequence of the human SLC34A2 gene obtained from a patient with alveolar microlithiasis and is described in a publication distributed in Japan or abroad. Or an alteration that can cause alveolar microlithiasis that has become publicly known through a telecommunication line. The above-mentioned modification A or B corresponds to a specific example of the known modification.

  In the present embodiment, the “known modification to be detected” includes a known modification derived from a modification from the wild-type base sequence represented by SEQ ID NO: 1. The term “derived from modification” refers not only to the modification site on the genome base sequence, but also to the entire region of modification that occurs due to the modification. For example, when the nucleic acid to be detected is mRNA, the known modification to be detected by modification B is left not only in the base of the splicing donor site of the eighth intron shown in FIG. 2B but also in the mature mRNA. This corresponds to the entire region of the 8th intron having the modified base.

  The method for detecting a known modification in this step may be, for example, (1) a detection method by hybridization using a probe, or (2) a detection method by determination of a base sequence, (3) A detection method based on comparison of amplification products using a nucleic acid amplification method may be used, or (4) a method using a polymorphic marker may be used. These four detection methods will be described below.

  (1) A detection method by hybridization using a probe will be described. This method is a method for detecting the presence or absence of a known modification by hybridization using a polynucleotide having a base sequence of a known modification as a probe. For example, a method using a gel blotting method or a method using a substrate on which a nucleic acid is immobilized may be used. Specific examples of the method using the gel blotting method include Southern blot hybridization method and Northern blot hybridization method. Specific examples of the method using a substrate on which a nucleic acid is immobilized include an analysis method using a microarray or a DNA chip.

  The “polynucleotide” used in the present invention includes not only a normal definition but also an oligonucleotide composed of several bases to 20 bases. Moreover, the structure of the said polynucleotide may be comprised from DNA, RNA, LNA etc. and those combinations.

  The base sequence of the polynucleotide used as the probe is the entire base sequence derived from modification from the base sequence of the wild-type human SLC34A2 gene represented by SEQ ID NO: 1, or a sequence having at least 14 consecutive bases as a part It is. For example, in the case of modification A, it is sufficient if it has the entire 19 base sequence represented by SEQ ID NO: 3 or a sequence of 14 bases or more and 18 bases or less of the 19 bases. Further, in the case of modification B, it is sufficient if it has the entire base sequence represented by SEQ ID NO: 4 or a sequence of 1464 or more and 1364 bases or less in the base sequence. The base sequence of the polynucleotide used as the probe may be the sense strand side or the antisense strand side when the nucleic acid to be detected is DNA, but when the nucleic acid to be detected is RNA, the nucleotide sequence is The sequence on the sense strand side is used.

  The polynucleotide used as the probe may be modified. For example, labeling with a radionuclide, a chromogenic substance, or a fluorescent substance, addition of a quenching substance, a chemical group, or a sugar chain is applicable. One probe may be subjected to a plurality of modifications of the same type or different types. The modification part does not matter. For example, it may be the 5 ′ end portion or any other portion. Further, the end or the inside of the polynucleotide may contain a restriction enzyme site or a modified sequence such as a tag sequence.

  The polynucleotide used as a probe may be fixed to a support. The support is not particularly limited in shape and size as long as it is a material to which nucleic acid can be directly bound or a material coated with a material to which nucleic acid is bound. For example, it may be a fine particle composed of silica, a substrate composed of glass, or a fiber composed of an organic polymer chain. As a method for immobilizing a polynucleotide, for example, a method of directly synthesizing a polynucleotide on the material (Patent Document 2), a method of supporting a previously prepared polynucleotide on the material later (Patent Document 3), or the like can be used.

  When the hybridization result is positive by this method, the base sequence of the human SLC34A2 gene contained in the sample is determined to have a base sequence based on a known modification.

  (2) A detection method based on determination of a base sequence will be described. In this method, the human SLC34A2 gene that can be contained in the nucleic acid extracted in the nucleic acid extraction step determines the presence or absence of the modification by determining the whole or part of the region where the base sequence derived from a known modification can exist. It is a method of detecting. The method for determining the base sequence is not particularly limited. A method combining the method based on the nucleic acid amplification method currently most widely used in the world and the dideoxy method is convenient. Dedicated reagents and automatic base sequence readers (sequencers) based on this principle are commercially available from various companies, which is very convenient in terms of speed and simplicity.

  The detection method requires a nucleic acid as a template, a polynucleotide as a primer, and a nucleic acid such as dNTP (dATP, dGTP, dTTP, dCTP) as a raw material for a synthetic DNA strand.

  It is preferable to use the genomic DNA or cDNA obtained in the nucleic acid extraction step as the template nucleic acid. When the nucleic acid used as the template is RNA, one that has been converted into cDNA by reverse transcription is used. The template nucleic acid may be amplified as necessary. In the case of a sequencing method using the dideoxy method, ddNTP (ddATP, ddGTP, ddTTP, ddCTP) is added together with dNTP. The ddNTP may be modified with a radionuclide, a fluorescent substance, or the like.

  The nucleotide sequence of the polynucleotide as a primer can determine all or part of the nucleotide sequence derived from the modification from the nucleotide sequence of the wild-type human SLC34A2 gene represented by SEQ ID NO: 1, and Any base sequence may be used as long as the base sequence of the corresponding wild type gene cannot be detected. For example, in the case of modification A, the base sequence of the polynucleotide used as a primer can be the entire 19 base sequence represented by SEQ ID NO: 3.

  The orientation of the base sequence of the polynucleotide as a primer may be the sense strand side, the antisense strand side, or both strands of the nucleic acid serving as the template. In view of the accuracy of the results, it is preferable to design both strands and determine the base sequences of both strands.

  The length of the base of the polynucleotide as a primer may be in the range of 18 to 100 bases, but 18 to 80 bases thereof are preferably all or part of the base sequence of the human SLC34A2 gene. .

  The polynucleotide used as a primer may be modified. For example, labeling with a radionuclide, a chromogenic substance, or a fluorescent substance, addition of a quenching substance, a chemical group, or a sugar chain is applicable. One primer may be subjected to a plurality of modifications of the same type or different types. The modification part does not ask | require. For example, it may be the 5 ′ end portion or any other portion.

  When a modification is detected in the base sequence determined by the method, it is determined that the base sequence of the human SLC34A2 gene contained in the sample has a base sequence based on a known modification.

  (3) A detection method using an amplification product using a nucleic acid amplification method will be described. This method is a method for detecting the presence or absence of amplification of a nucleic acid amplification product having the base sequence of the modified human SLC34A2 gene. In this method, a base sequence is selected such that only the target modified human SLC34A2 gene is amplified when designing primers for amplification. For example, if one of the amplification primers is designed so that all of the base sequences of one of the amplification primers have a modified gene-specific base sequence, the pair of amplification primers has the modified type. An amplification product is obtained only if present. Further, if the PNA-LNA-PCR clamp method developed by the present inventors (Patent Document 4, Non-Patent Document 7) is used, it is convenient because the modified site of the modified gene can be specifically detected.

  When a nucleic acid amplification product having the base sequence of human SLC34A2 gene is amplified by this method, it is determined that the base sequence of human SLC34A2 gene contained in the sample has a base sequence based on a known modification. The

  (4) Finally, a method using a polymorphic marker will be described. This method is a method for detecting a modification from the presence or absence of the polymorphic marker using a polymorphic marker of DNA that can specifically exist on the base sequence of the modified human SLC34A2 gene.

  “Polymorphism” refers to a difference in bases on DNA, and it is defined that a change of a certain base is present at a frequency of 1% or more in the population. In the present invention, a wild type and a modified type are defined. All polymorphisms that differ in nucleotide sequence between and For example, SNP (Single Nucleide Polymorphism), microsatellite, VNTR (Variable Nucleotide Tandem Repeat), RFLP (Restriction Fragment Length Polymorphism), and the like are applicable.

  The polymorphic marker is a marker using a specific polymorphism as a marker (label). In the method using a polymorphic marker, it is necessary to select a polymorphic marker specific for the modified human SLC34A2 gene used for detection of the modification in advance. When the selected polymorphic marker is SNP, microsatellite, or VNTR, whether the target to be detected is a base sequence derived from modification or a polymorphic marker specific to the modified gene, and the detection method is The same method as the detection method by hybridization of (1) or the detection method by determination of the base sequence of (2) may be used. Therefore, the detection method using the polymorphic marker may be used in combination as a method for confirming the modification of the modified detection gene detected by either of the two methods.

  The detection method using RFLP is a method that can be used when a restriction enzyme site specific to the modified human SLC34A2 gene is present as a polymorphic marker. The base region containing the restriction enzyme site is amplified by a method according to the nucleic acid amplification method (3). The obtained amplification product is cleaved with the restriction enzyme, and the presence or absence of modification can be detected by the presence or absence of cleavage.

  When a polymorphic marker specific to the modified gene is detected by this method, the base sequence of the human SLC34A2 gene contained in the sample is determined to have a base sequence based on a known modification. .

  The genetic modification detection step may be performed by a contractor. In particular, the analysis method using the microarray or the DNA chip should use a specialized contractor that provides a contract analysis service for the microarray or the DNA chip in consideration of the technology, labor, and cost for producing the substrate for the present invention. Convenient. In addition, there are many companies that offer nucleotide sequence trust services, so you can also use them.

  In the present embodiment, it is desirable to verify a plurality of known modified types. Most preferably, all known variants are detected. For example, instead of detecting only the modification A, the modification A and the modification B are detected.

  In the present invention, “onset of alveolar microlithiasis” means that symptoms such as coughing, respiratory symptoms such as dyspnea, or respiratory failure appear as subjective symptoms of alveolar microlithiasis. Therefore, the present invention does not consider symptoms that progress unconsciously even if microlith formation or accumulation in the alveoli occurs, as the onset of alveolar microlithiasis.

  <Effect of Embodiment 1> According to the present embodiment, it is possible to easily determine whether there is a known modification that can cause alveolar microlithiasis in the human SLC34A2 gene of the subject. If a known modification is detected in the gene, the subject can be determined as “has a possibility of developing with a probability of at least 50% in the future” even if alveolar microlithiasis has not yet occurred. Moreover, when it is diagnosed as alveolar microlithiasis from the clinical symptom of the subject, it can be confirmed at the gene level that the disease is concerned by confirming a known modification of the human SLC34A2 gene.

  << Embodiment 2 >>

  <Embodiment 2: Overview> In Embodiment 2, a human-derived nucleic acid is extracted, and the base sequence of the alveolar microlithiasis causal gene SLC34A2 that can be contained in the extracted nucleic acid is a wild-type represented by SEQ ID NO: 1. By detecting whether the nucleotide sequence has been modified, the genotype of the human SLC34A2 gene that can be contained in the extracted nucleic acid is wild-type homo, modified homo, wild-type and modified hetero, or modified hetero It is based on the method of determining which one is.

  <Second Embodiment: Process Flow> FIG. 5 illustrates a process flow of the second embodiment. First, in the “nucleic acid extraction step” (S0501), human-derived nucleic acid is extracted from a sample by an appropriate method. Next, in the “gene modification detection step” (S0502), it is detected whether the nucleotide sequence of the modification detection gene obtained in the nucleic acid extraction step has been modified from the wild-type nucleotide sequence represented by SEQ ID NO: 1. . Finally, in the “genotype determination step” (S0503), the genotype of the human SLC34A2 gene of the subject who provided the sample is determined from the result obtained in the genetic modification detection step.

  In the present embodiment, the “nucleic acid extraction step” (S0501) is the same as the “nucleic acid extraction step” (S0301) of the first embodiment, and thus the description thereof is omitted. Since the “gene modification detection step” (S0502) is also based on the “gene modification detection step” (S0501) of the first embodiment, only the characteristic points of this embodiment will be described here. Furthermore, since the “genotype determination step” (S0503) is a step characteristic of the present embodiment, it will be described in detail below.

  The characteristic point of the “gene modification detection step” (S0502) of the present embodiment is that the first embodiment detects whether the base sequence of the modification detection gene has a known modification, whereas this The embodiment is to detect whether the base sequence of the modified detection gene is modified from the wild-type base sequence represented by SEQ ID NO: 1. That is, not only is it detected whether there is a known modification, but it is also detected that there is no modification.

  The definitions of “modification”, modification mode, “known modification”, “derived from modification”, “polynucleotide” and the like in the present embodiment are the same as those in the first embodiment.

  The method for detecting a known modification in this step is also based on the genetic modification detection step of the first embodiment. That is, (1) a detection method by hybridization using a probe may be used, (2) a detection method by determination of a base sequence may be used, or (3) an amplification product using a nucleic acid amplification method may be used. It may be a detection method by comparison, or (4) a method using a polymorphic marker. Hereinafter, the characteristic points of this embodiment in each method will be described. Since the other points are the same as those of the first embodiment, the description thereof is omitted.

  (1) In the detection method by hybridization using a probe, a characteristic point of this embodiment is that two types of a detection polynucleotide and a wild-type detection polynucleotide are used as probes. As in the first embodiment, the “modified polynucleotide for detection” refers to all of the base sequence derived from the modification from the base sequence of the wild-type human SLC34A2 gene represented by SEQ ID NO: 1, or at least 14 consecutive bases. Part of the base sequence. “Wild-type detection polynucleotide” has the entire base sequence of the wild-type human SLC34A2 gene represented by SEQ ID NO: 1, or a base sequence comprising at least 14 consecutive bases, and modified detection It is a polynucleotide capable of detecting a wild-type base sequence corresponding to the position of the polynucleotide for use. For example, in the case of modification A, the base sequence of the “modified detection polynucleotide” has the entire 19 base sequence represented by SEQ ID NO: 3 or the continuous 14 bases to 18 bases of the 19 bases. It only has to be. On the other hand, the base sequence of the “wild type detection polynucleotide” is 15 bases (from 13290th position T to 13304th position of SEQ ID NO: 1) before being replaced with the 19 base sequence represented by SEQ ID NO: 3. (Equivalent to G)) or 14 bases. In the case of modification B, the base sequence of the “modified detection polynucleotide” is the entire base sequence represented by SEQ ID NO: 4, or a sequence of 14 bases or more and 1364 bases or less continuous among the base sequences. As long as it has. On the other hand, the base sequence of the “wild type detection polynucleotide” may be the sequence after splicing ligation in the 8th and 9th exons, including the ligation site and having 14 or more consecutive bases.

  As long as one modified detection polynucleotide and one wild type detection polynucleotide are selected, two or more kinds may be used in one detection. For example, one or more wild-type detection polynucleotides may be used for one modified detection polynucleotide.

  Even when there is no modification by the detection method, it can be detected. Therefore, even in the case of the heterozygous wild type and modified type that could only be detected as having the modified type in the first embodiment, the genotype can be accurately detected for the modified type.

  (2) The detection method based on the determination of the base sequence is characterized by this embodiment in that the human SLC34A2 gene that can be included in the nucleic acid extracted in the nucleic acid extraction step is desirably the entire base sequence, at least derived from known modifications By determining all or a part of the base sequence of the region where the base sequence to be present can be determined, whether the human SLC34A2 gene has only the wild type, only the modified type, or both the wild type and the modified type In the point.

  When determining the entire nucleotide sequence of the human SLC34A2 gene, the nucleotide sequence of the polynucleotide as the primer is SEQ ID NO: 1 if the template is derived from genomic DNA, and SEQ ID NO: 1 if the template is derived from mRNA (eg, cDNA). Any region of the CDS of the type human SLC34A2 gene may be selected. The number of types of primers used in one detection may be one or more as long as it is designed so that the entire base sequence of the human SLC34A2 gene can be determined.

  When determining the base sequence of a region where a base sequence derived from a known modification can be present, the base sequence of the polynucleotide as a primer has only a wild type, a modified type, or a wild type. The base sequence of any region may be used as long as it is possible to detect whether both types and modified types are present. For example, in the case of modification A, the base sequence of the polynucleotide used as a primer is upstream (5 ′) from position 856, which is the modification position of SEQ ID NO: 1, so that all 19 sequences represented by SEQ ID NO: 3 can be detected. Side) What is necessary is just to design the base sequence of the position of about 100 bases as a primer. Thus, if the region where a base sequence derived from a known modification can exist is only wild-type, only the band of the wild-type base sequence, and if only the modification A, only the band of the base sequence of modification A only Is detected. Further, when the modified A is heterozygous, two types of bands of wild type and modified A are detected after the 856th position.

  In this embodiment, it is desirable to detect the presence / absence of all known modified types in the modified detection gene, and most desirably, the presence / absence of all known modified types is detected, and the entire base sequence is determined to determine other modified types. It is to confirm that there is no.

  (3) Nucleic acid amplification products using the nucleic acid amplification method are characteristic in this embodiment. (3-1) When detecting the presence or absence of nucleic acid amplification products, each of wild type and modified type is detected. And (3-2) the point of detecting the wild type and the modified type as the difference in the number of bases of the nucleic acid amplification product.

  (3-1) A method for detecting the presence or absence of amplification of a nucleic acid amplification product is a method for detecting only one modification of a human SLC34A2 gene, wherein only a nucleic acid amplification product having a modification is amplified or a wild-type base sequence corresponding to the modification It is detected whether only the nucleic acid amplification product having the above is amplified or both are amplified. The base sequence of the amplification primer is selected so that only one of the region containing the modification or the wild-type region corresponding thereto is amplified. At least one set of amplification primers is selected for each of the modified type detection and the wild type detection. Desirably, at least one set each for wild-type detection and modified-type detection is selected for all known modifications. Others are in accordance with the method of the first embodiment. When only a nucleic acid amplification product having a modification with respect to one modification is amplified by this method, only a nucleic acid amplification product having a modified nucleotide sequence corresponding to the modification is amplified. In some cases, it can be detected as a wild-type homozygote for the modification, and when both are amplified, the modification can be detected as a wild-type and modified heterozygote.

  (3-2) The method of detecting the difference in the number of bases of the nucleic acid amplification product is a method that can be used only when a difference in the number of bases occurs between the wild type and the modified type with respect to one modification. The case where the difference in the number of bases occurs corresponds to, for example, deletion, addition, or substitution with a difference in the number of bases. In such a case, the gene amplification method is performed by designing at least one set of amplification primers so as to sandwich a region on the base sequence where the difference in the number of bases occurs. For detection, the difference in the number of bases of the nucleic acid amplification product may be confirmed by gel electrophoresis or the like, or the nucleic acid amplification product may be stained with a fluorescent substance and the fluorescence intensity measured by real-time PCR or the like. The distance between the pair of amplification primers is preferably such that the expected number of bases of the nucleic acid amplification product is at least 50 bases. When only the nucleic acid amplification product corresponding to the number of bases in the case of having one modification is amplified by the method, the modified homozygote with respect to the modification, and having a wild-type base sequence corresponding to the modification When only a nucleic acid amplification product corresponding to the number of bases is amplified, it can be detected as a wild-type homozygote for the modification, and when both are amplified, it can be detected as a heterozygous of the wild-type and the modified type. .

  (4) Finally, the characteristic feature of this embodiment in the method using a polymorphic marker is that the polymorphic marker of the polymorphic marker is different from the wild type and the modified human SLC34A2 gene using a DNA polymorphic marker. The point is to detect the wild type and the modified type from the presence or absence.

  In the method using the polymorphic marker, the polymorphic marker in the human SLC34A2 gene needs to be specified in advance before detection. The polymorphic marker specified at this time needs to show a different distribution pattern or a different base sequence between the wild type and the modified type. Among the polymorphic markers, SNP, microsatellite, and VNTR are detected only by different detection targets. The detection method by the hybridization (1) of the present embodiment or the detection by determination of the base sequence (2) It can be achieved in a manner similar to the method. Therefore, the detection method using the polymorphic marker may be used in combination as a method for confirming the result of the modified detection gene detected by either of the two methods.

  The detection method using RFLP is a method that can be used when there are restriction enzyme sites different between the wild type and the modified type in the human SLC34A2 gene, or when restriction enzyme sites are present only in one of them. The base region containing the restriction enzyme site is amplified by a method according to the nucleic acid amplification method (3). The obtained nucleic acid amplification product is cleaved with the restriction enzyme, and the presence or absence of modification can be detected by the presence or absence of cleavage.

  When only a polymorphic marker specific to the modified gene was detected for one modification by the method, only the polymorphic marker specific to the wild type gene was detected as a modified homozygote for the modification. In some cases, it can be detected as a wild type homozygote for the modification, and when both polymorphic markers are detected, it can be detected as a heterozygous wild type and modified type for the modification.

  The genetic modification detection step in this embodiment may also be performed by a contractor.

  The “genotype determination step” (S0503) is a step of determining whether the genotype of the modified detection gene is a wild type homozygote, a modified homozygote, a wild type or a modified heterogene, or a modified heterogeneous. It is. The determination step is performed based on the detection result of each known alteration confirmed in the genetic alteration detection step.

  FIG. 6 is a diagram showing the concept of genotype determination in the determination step. a, b, and c represent known modifications of the human SLC34A2 gene, and the numbers represent combinations (cases) of detection results of the respective known modifications.

  The case where “wild type homology” is determined is when the entire nucleotide sequence of the modified detection gene is determined and no modification is detected at all, or when all the modifications in the confirmed range are detected only in the wild type. Cases 1 to 3 in FIG. Judgment is made only from the result of the confirmed modification, and unconfirmed modification is not added to the determination. For example, in 2 it is unconfirmed with respect to modified c, but if c actually has modification, even if it is a wild type and modified heterozygous or modified homozygous, as long as it is unconfirmed I can't enter.

  “Hetero of wild type and modified type” is determined when both wild type and modified type are detected in one of the modifications confirmed in the modified detection gene, and other modifications are unconfirmed. This is the case when all wild types are detected. Cases 4 to 6 in FIG. This determination is also determined only from the confirmed modification result, and unconfirmed modification is not added to the determination.

  “Modified heterogeneity” is determined when both wild type and modified type are detected in two or more of the modifications confirmed in the modified detection gene, and other modifications are unconfirmed or wild type This is the case when it is detected. Cases 7 to 9 in FIG. This determination is also determined only from the confirmed modification result, and unconfirmed modification is not added to the determination.

  “Modified homo” is determined when only the modified type is detected by at least one of the modifications confirmed in the modified detection gene. That is, if only one of the other modifications is “wild type only”, “both wild type and modified type”, or “both wild type and modified type with two or more modifications”, only “modified type” is If any one is detected, it is always determined as “modified homo”. Cases 10 to 19 in FIG.

  The genotype determined in this step as described above is comprehensively determined for the alterations confirmed in the genetic alteration detection step. The accuracy of the genotype is applied only to the alteration confirmed in the genetic alteration detection step. Therefore, the genotype is not necessarily determined for the entire modified detection gene, except when one or more of the detected modifications are “modified homozygous” and when the entire nucleotide sequence of the modified detection gene is determined. Not.

  For example, if only the modification a is confirmed in the genetic modification detection step as shown in case 1 of FIG. 6 and only the wild type is detected, it is determined to be a wild type homology in the genotype determination step. However, this is because only the modified a was determined that the modified detection gene was a wild type homology, and the entire modified detection gene was not determined to be a wild type homology. Nonetheless, even if genotyping only for modification a, the determination of the occurrence of alveolar microlithiasis, which has never been heretofore, is wild-type for at least one known modification by the process The significance of being able to be judged is great. Similarly, as in case 5 of FIG. 6, two known modifications, modification a and b, were confirmed, and both modification a and wild type were detected, and modification b was detected only in the wild type. If it is a result, it will be judged as the heterozygous of a wild type and a modified type. This determination is also comprehensively determined from the results for modification a and modification b, and the entire gene is not determined to be wild-type homozygote. However, as the number of known modifications to be confirmed becomes more than one, the accuracy of determination increases and the risk of erroneous determination decreases, so the significance of determination increases.

  The possibility of developing alveolar microlithiasis in the subject from the genotype of the human SLC34A2 gene determined in this embodiment will be described below.

  If the genotype is a modified homozygote, it can be determined that the subject is very likely to develop even if alveolar microlithiasis has not yet occurred.

  If the genotype is wild-type homozygous, the subject can determine that there is no possibility of developing alveolar microlithiasis for the confirmed modification. However, this does not apply to modifications not confirmed. In order to completely eliminate the possibility of developing alveolar microlithiasis with respect to the modified detection gene, the entire region of the gene is confirmed by a method such as determining the entire base sequence of the modified detection gene, and alveolar microlithiasis It is necessary to confirm that there is no substantial modification that causes However, this method is time consuming and costly and is inefficient. Since alveolar microlithiasis is a rare disease, it can be expected that the number of substantial alterations in the human SLC34A2 gene that causes the occurrence of alveolar microlithiasis currently present worldwide is very limited. Therefore, the genotype determination by this method is not limited to the entire human SLC34A2 gene, even if it is a partial modification, since only a substantially known modification is detected pinpointed, the determination accuracy is low. Absent. Furthermore, compared with the case of determining the entire base sequence, it is excellent in the efficiency, speed and cost of modification detection.

  When the genotype is heterozygous between the wild type and the modified type, the subject can determine that there is no possibility of developing alveolar microlithiasis for the confirmed modification. However, this does not apply to modifications not confirmed.

  When the genotype is a modified heterogeneity, the possibility of developing alveolar microlithiasis differs depending on the combination of the modified types. For example, it can be determined that the possibility of alveolar microlithiasis is extremely high if the heterogeneity of modification A and modification B is present, but it is assumed that modification A and modification c (modification lacking the three TM domains on the N-terminal side). ), The modified A and modified c gene products may compensate each other and have the same function as the wild-type human type IIb sodium-phosphate cotransporter protein. In this case, the subject can determine that there is no possibility of developing alveolar microlithiasis for the confirmed modification. Therefore, in the case of modified heterogeneity, the possibility of onset can be determined only from the results obtained from clinical data or experimental data.

  <Effect of Embodiment 2> According to this embodiment, the possibility that a subject develops alveolar microlithiasis can be determined by genotype. Further, if the subject has already developed alveolar microlithiasis, if the genotype of the human SLC34A2 gene of the subject is a modified homozygote or a modified heterogeneous by substantial modification, at least the modification of the gene It can be determined that it is the cause of the disease.

  << Embodiment 3 >>

  <Embodiment 3: Overview> Embodiment 3 extracts human-derived protein and detects whether the human type IIb sodium-phosphate cotransporter protein that can be contained in the extracted protein has an amino acid modification. The method is based on a method for determining the possibility of developing microcystiasis.

  <Third Embodiment: Process Flow> FIG. 7 illustrates a process flow of the third embodiment. First, in the “protein extraction step” (S0701), a human-derived protein is extracted from a sample by an appropriate method. Next, the amino acid sequence of the human type IIb sodium-phosphate cotransporter protein that can be contained in the human-derived protein obtained in the protein extraction step in the “amino acid alteration detection step” (S0702) is a known amino acid alteration. Is detected. The onset possibility of alveolar microlithiasis is determined from the results obtained by the above steps. Hereinafter, each step will be described in detail.

  The “protein extraction step” (S0701) is a step of extracting a human-derived protein from a sample. The sample corresponds to a sample obtained from the human body, but may be a transformant that is a biological species other than human and that produces a human-derived protein. Use a sample from one individual. However, this is not the case when the presence or absence of amino acid modification of the human IIb type sodium-phosphate cotransporter protein is examined using two or more units using the method of the present invention. In the case where the sample is obtained from the human body, the type of the sample is not limited as long as the protein of the collected individual can be extracted, but the extracted protein must contain human type IIb sodium-phosphate cotransporter protein . Therefore, the sample type is preferably the lung tissue of the subject. This is based on experimental data that the protein is localized in the lung and small intestine (Non-patent Document 8). Lung tissue may be collected by bronchoscopic lung biopsy or the like.

  Protein extraction is generally performed by (A) a cell disruption step, (B) a soluble fraction collection step, and (C) a target protein purification step. Each step will be described below, but the specific method is not limited as long as the target protein can be extracted from the sample.

  (A) The cell disruption step is a step of disrupting cells contained in the sample and causing the cytoplasm to flow out. Cell disruption can be achieved by, for example, a mechanical disruption method using a homogenizer, a sonicator (ultrasonic disruption device), or the like, or a chemical disruption method of a cell membrane lipid structure using a surfactant. Both methods must be extracted from cells with minimal protein degradation, such as at low temperatures. A solution containing the crushed cells obtained in this step is used as a cell lysate.

  (B) The soluble fraction collection step is a step of separating the cell lysate obtained in the cell disruption step into a soluble fraction and an insoluble fraction, and collecting the soluble fraction. Collection of the soluble fraction can be achieved by centrifugation. The cell lysate is centrifuged at an appropriate gravitational acceleration at a low temperature, and the supernatant is used as a soluble fraction. It should be noted that the human type IIb sodium-phosphate cotransporter protein that detects amino acid modifications is a membrane protein. Membrane proteins are basically not solubilized because they have hydrophobic transmembrane domains. Therefore, when a method other than the chemical disruption method using a surfactant is used in the step (A), an appropriate surfactant is added to the cell disruption solution before the step (B) is executed. Thereby, the cell membrane lipid structure is destroyed, and the membrane protein can be solubilized.

  (C) The target protein purification step is a step of obtaining the target protein by affinity purification, HPLC purification or the like. In the protein extraction in the present embodiment, up to the (B) soluble fraction collection step. Is sufficient, and this step is not always necessary.

  In the “amino acid modification detection step” (S0702), the base sequence of human type IIb sodium-phosphate cotransporter protein (hereinafter referred to as amino acid modification detection protein) that can be contained in the protein extracted in the protein extraction step is This is a step of detecting whether or not there is a known amino acid modification from the wild type amino acid sequence represented by SEQ ID NO: 2.

  In the present embodiment, “amino acid modification” means a state where the amino acid sequence of the amino acid modification detection protein does not match the amino acid sequence of the wild-type human type IIb sodium-phosphate cotransporter protein represented by SEQ ID NO: 2. The amino acid modification of this embodiment means a substantial amino acid modification that loses its function as a wild-type human type IIb sodium-phosphate cotransporter.

  Examples of the amino acid modification to be detected in the present embodiment include amino acid deletion, substitution, or addition. “Deletion” means that one or more amino acids are lost from the amino acid sequence of the wild-type protein when the amino acid sequence of the amino acid modification detection protein is compared with the amino acid sequence of the wild-type human type IIb sodium-phosphate cotransporter protein. Amino acid modification. Further, in this embodiment, “substitution” is not all from the amino acid sequence of the wild-type protein when the amino acid sequence of the amino acid modification detection protein is compared with the amino acid sequence of the wild-type human type IIb sodium-phosphate cotransporter protein. An amino acid modification in which one or more consecutive amino acids are replaced with other different amino acids. Furthermore, in this embodiment, “addition” means that the amino acid sequence of the wild-type human type IIb sodium-phosphate cotransporter protein is compared with the amino acid sequence of the amino acid modification detection protein compared to the amino acid sequence of the wild-type protein. An amino acid modification in which one or more consecutive amino acids are newly added at positions that are not seen.

  In this embodiment, the “known amino acid modification” refers to an amino acid modification that occurs on the amino acid sequence of the wild-type human type IIb sodium-phosphate cotransporter protein represented by SEQ ID NO: 2, and thereby Losing protein activity means amino acid modification described in a publication distributed in Japan or abroad, or amino acid modification that has become publicly known through a telecommunication line. The above-mentioned amino acid modification A or B corresponds as a specific example of the known amino acid modification.

  As a method for detecting a known amino acid modification in this step, an antigen-antibody reaction method or the like may be used. In the antigen-antibody reaction method in the present embodiment, an antigen-antibody reaction is performed on an amino acid-modified detection protein using an antibody prepared by using a polypeptide having all or part of a known amino acid-modified amino acid sequence as an antigen. This is a method for detecting the presence or absence of a known amino acid modification. For example, an immunoblotting method such as a Northern blotting method or an ELISA method may be used.

  In the present invention, “polypeptide” also includes the meaning of an oligopeptide composed of several amino acids to 10 amino acids.

  The amino acid sequence of the polypeptide used as the antigen is the entire amino acid sequence derived from the amino acid modification from the amino acid sequence of the wild-type human type IIB sodium-phosphate cotransporter protein represented by SEQ ID NO: 2, or at least continuous. It is a sequence having 5 amino acids or more as a part. For example, in the case of amino acid modification A, it is sufficient to have the entire 28 amino acid sequence represented by SEQ ID NO: 5 or a sequence of 5 to 27 amino acids that are consecutive among the 28 amino acids. In the case of amino acid modification B, it is sufficient that the amino acid sequence has the entire amino acid sequence represented by SEQ ID NO: 6 or a sequence of 5 amino acids or more and 9 amino acids or less in the amino acid sequence.

  The antibody may be a polyclonal antibody or a monoclonal antibody. In any case, the antibody is preferably an antibody having immunoreactivity specific to the antigen. In the case of polyclonal antibodies, antibodies may be prepared by inoculating and immunizing laboratory livestock such as rabbits with the antigen. In the case of a monoclonal antibody, a generally performed method may be used. For example, a mouse or the like is inoculated with the antigen and immunized, and then cell fusion between B lymphocytes and myeloma cells is performed, and a hybridoma cell producing the desired monoclonal antibody can be obtained by screening. Since the production of these antibodies requires time and labor, it is somewhat costly, but it is convenient to outsource them to companies that offer antibody production contract services.

  The antibody may be labeled. For example, a labeling substance such as a chromogenic substance, a fluorescent substance, an enzyme or biotin is applicable.

  The antibody may be immobilized on a support. The support may be of any shape and size as long as it is a material that can be directly bound by an antibody or a material that is coated with a material that can be directly bound by an antibody. For example, it may be fine particles made of glass or a plate made of plastic. The material of the support is preferably one in which the antibody is not denatured by fixation.

  When the result of the antigen-antibody reaction is positive by the method, the amino acid sequence of the human type IIB sodium-phosphate cotransporter protein contained in the sample has an amino acid sequence based on a known amino acid modification. It is determined that

  <Embodiment 3: Effect of treatment> According to this embodiment, it is possible to easily determine whether a known amino acid modification is present in the human IIB sodium-phosphate cotransporter protein of the subject. If a known substantial amino acid modification is detected in the protein, the subject is determined to have “at least a 50% probability of developing” even if alveolar microlithiasis has not yet occurred. it can. In addition, when there is a suspicion of alveolar microlithiasis from the subject's symptoms, the disease can be confirmed by confirming a known amino acid modification of human type IIB sodium-phosphate cotransporter protein.

  << Embodiment 4 >>

  <Embodiment 4: Overview> In Embodiment 4, a human-derived protein is extracted, and the amino acid sequence of a human type IIB sodium-phosphate cotransporter protein that can be contained in the extracted protein is represented by SEQ ID NO: 2. By detecting whether the amino acid sequence has been modified from the type amino acid sequence, the genotype of the human SLC34A2 gene encoding the human type IIB sodium-phosphate cotransporter protein that can be contained in the extracted protein is a wild type homozygous, modified This method is based on a method for determining whether a heterozygous type, a wild type and a modified type of heterogeneity, or a modified type of heterogeneity.

  <Flow of Embodiment 4> FIG. 8 illustrates a flow of processing of Embodiment 4. First, in the “protein extraction step” (S0801), a human-derived protein is extracted from a sample by an appropriate method. Next, regarding the amino acid modification detection protein obtained in the protein extraction step in the “amino acid modification detection step” (S0802), whether the amino acid sequence has been amino acid modified from the wild-type amino acid sequence represented by SEQ ID NO: 2 To detect. Finally, in the “second genotype determination step” (S0803), human SLC34A2 encoding the human type IIB sodium-phosphate cotransporter protein of the subject who provided the sample from the result obtained in the amino acid modification detection step Determine the genotype of a gene.

  In the present embodiment, the “protein extraction step” (S0801) is the same as the “protein extraction step” (S0701) of the third embodiment, and thus the description thereof is omitted. Further, since the “amino acid modification detection step” (S0802) is also based on the “amino acid modification detection step” (S0801) of the third embodiment, only the characteristic points of this embodiment will be described here. Furthermore, the “second genotype determination step” (S0803) is a step characteristic of the present embodiment, and will be described in detail below.

  The characteristic point of the “amino acid modification detection step” (S0802) of the present embodiment is that the third embodiment detects whether the amino acid sequence of the amino acid modification detection protein has a known amino acid modification. This embodiment is to detect whether the amino acid sequence of the amino acid modification detection protein has been amino acid modified from the wild-type amino acid sequence represented by SEQ ID NO: 2. That is, not only is it detected whether or not it has a known amino acid modification, but it is also detected that it has no amino acid modification.

  The definitions of “amino acid modification”, amino acid modification mode, “known amino acid modification”, “derived from amino acid modification”, “polypeptide” and the like in the present embodiment are the same as in the third embodiment.

  The method for detecting an amino acid modification in this step is also based on the amino acid modification detection step of the third embodiment. That is, a method may be used in which an antigen-antibody reaction is performed on an amino acid modification detection protein to detect the presence or absence of amino acid modification. Hereinafter, a characteristic point of the present embodiment will be described in the detection method of amino acid modification using an antigen-antibody reaction method. Since the other points are the same as those of the third embodiment, the description thereof is omitted.

  A characteristic feature of this embodiment in the detection method using an antigen-antibody reaction using an antibody is that two types of antibodies for detection, that is, a modified detection antibody and a wild type detection antibody are used as detection antibodies.

  The “modified antibody for detection” refers to the entire amino acid sequence derived from amino acid modification from the amino acid sequence of the wild human type IIB sodium-phosphate cotransporter protein represented by SEQ ID NO: 2 as in Embodiment 3, or It is an antibody prepared by using a polypeptide having an amino acid sequence including at least 5 consecutive amino acids as an antigen as an antigen.

  For example, in the case of amino acid modification A, the amino acid sequence of the polypeptide used as an antigen in the preparation of the “modified antibody for detection” may be the entire 28 amino acid sequence represented by SEQ ID NO: 5 or the 28 amino acid sequence. Of these, it is only necessary to have 5 or more consecutive amino acids. In the case of amino acid modification B, the amino acid sequence may be the entire amino acid sequence represented by SEQ ID NO: 6, or the amino acid sequence may have at least 5 consecutive amino acids.

  The “wild-type detection antibody” is an amino acid sequence comprising all of the amino acid sequence of the wild-type human type IIB sodium-phosphate cotransporter protein represented by SEQ ID NO: 2 or a part of at least 5 consecutive amino acids. It is an antibody prepared by using a polypeptide having an antigen as an antigen, and an antibody capable of detecting a wild-type amino acid sequence corresponding to the position of the amino acid-modified detection antibody.

  For example, in the case of amino acid modification A, the amino acid sequence of the polypeptide used as an antigen in the preparation of the “wild-type detection antibody” is 405 amino acids before being substituted with the 28 amino acid sequence represented by SEQ ID NO: 5. It should suffice to have all of the sequences (after isoleucine at position 286 represented by SEQ ID NO: 2) or 5 amino acids or more. Further, in the case of amino acid modification B, it is sufficient that all or a continuous 5 amino acids or more in the 350th or lower amino acid sequence at position 350 represented by SEQ ID NO: 2 is included.

  As long as at least one modified detection antibody and one wild type detection antibody are selected, two or more types may be used in one detection. For example, one or more wild-type detection antibodies may be used for one modified detection antibody.

  In the “second genotype determination step” (S0803), based on the result of amino acid modification in the amino acid modification detection protein detected in the amino acid modification detection process, the human IIB type sodium-phosphate cotransporter protein of the sample is determined. This is a step of determining whether the genotype of the encoded human SLC34A2 gene is wild-type homozygote, modified homozygote, wild-type and modified heterogeneity, or modified heterogeneity.

  The method for determining the genotype of the human SLC34A2 gene in the sample in this step is in accordance with the “genotype determination step” (S0703) of the second embodiment. That is, “wild type homozygous” is determined when only the wild type is detected for all the amino acid modifications verified in the amino acid modification detection protein. Judgment is made only from the result of verified amino acid modification, and unverified amino acid modification is not included in the determination. Moreover, it is judged that both wild type and modified type are detected by one amino acid modification among the amino acid modifications verified in the amino acid modification detection protein. These amino acid modifications are unverified or all wild type is detected. This determination is also determined only from the verified amino acid modification result, and unverified amino acid modification is not added to the determination. Furthermore, “modified heterozygous” is determined when both wild type and modified type are detected by two or more amino acid modifications among the amino acid modifications verified in the amino acid modification detection protein. Is when unverified or wild type is detected. This determination is also determined only from the verified amino acid modification result, and unverified amino acid modification is not added to the determination. Finally, “modified homolog” is determined when only the modified type is detected by at least one amino acid modification among the amino acid modifications verified in the amino acid modification detection protein. That is, even if the other amino acid modification is either “wild type only”, “both wild type and modified type”, or “both wild type and modified type with two or more amino acid modifications”, “modified type only” If even one is detected, it is always determined as “modified homo”. Others are the same as the “genotype determination step” (S0703) of the second embodiment, and thus the description thereof is omitted.

  The possibility that the subject will develop alveolar microlithiasis from the genotype determined in the present embodiment is the same as that in the “genotype determination step” (S0703) of the second embodiment, and a description thereof will be omitted.

  <Effects of Embodiment 4> According to the present embodiment, the detection target can be a protein, and substantially the same effects as those of Embodiment 2 can be obtained.

  << Embodiment 5 >>

  <Embodiment 5: Overview> Embodiment 5 is a lung using a polynucleotide as a probe for determining the possibility of developing alveolar microlithiasis used in the method of Embodiment 1 or 2, or a primer. This is a kit for determining the onset of microlithiasis.

  <Embodiment 5: Configuration>

  The configuration of the fifth embodiment will be described. The kit for determining the onset of alveolar microlithiasis according to this embodiment is (1) a kit that uses a probe or a polynucleotide as a primer. Hereinafter, each of (1) and (2) will be described in detail.

  The kit for determining the onset of alveolar microlithiasis according to the present embodiment is a polymorph as an antigen or primer required for determining the possibility of the onset of alveolar microlithiasis by the method of the first or second embodiment. Includes nucleotides. The polynucleotide is pre-prepared to be readily available for detecting substantial alterations in the human SLC34A2 gene. For example, a polynucleotide having an appropriate base sequence or base length for detection of alteration is synthesized or cloned in advance, and the polynucleotide is modified in advance with a fluorescent substance necessary for detection. And / or pre-fixed to a substrate or the like. The kit may include one or both of the polynucleotides for wild-type detection and each modified type detection. Preferably both are included. More preferably, all of the polynucleotides for detecting each modified type are included so that all known modified types can be detected. The polynucleotide included in the kit may be either one used as an antigen, one used as a primer, or both. The kit may include not only the polynucleotide but also reagents such as buffers and enzymes necessary for the detection method using the polynucleotide. A protocol may be attached. The polynucleotides and reagents included in the kit are preferably those that can be stably stored for a certain period under predetermined conditions. The predetermined conditions correspond to low temperature, light shielding, drying, and the like. The fixed period varies depending on the nature of the reagent and the like, but is preferably one month or longer.

  <Embodiment 5: Effects> According to this embodiment, it is possible to quickly and easily detect substantial alteration of the human SLC34A2 gene in order to determine the onset of alveolar microlithiasis and the like. Furthermore, by following the attached protocol, those who have undergone a certain amount of training can determine the onset of alveolar microlithiasis without the need for special knowledge or skills.

  << Embodiment 6 >>

  <Embodiment 6: Overview> Embodiment 6 extracts human-derived nucleic acids and detects whether the expression control region of the alveolar microlithiasis gene SLC34A2 that may be contained in the extracted nucleic acids has a modification. This is based on a method for determining the possibility of developing alveolar microlithiasis.

  <Sixth Embodiment: Process Flow> In the process flow of the sixth embodiment, first, a nucleic acid derived from human is extracted from a sample by an appropriate method in the “nucleic acid extraction step”. Next, the base sequence of the expression control region of the human SLC34A2 gene that can be contained in the human-derived nucleic acid obtained in the nucleic acid extraction step in the “expression control region modification detection step” is a wild type represented by SEQ ID NO: 7 It is detected from the base sequence of. The onset possibility of alveolar microlithiasis is determined from the results obtained by the above steps. The “nucleic acid extraction step” is almost the same as the “nucleic acid extraction step” (S0301) of the first embodiment, and the “expression control region alteration detection step” is mostly the same as the “gene alteration detection step” (S0302) of the first embodiment. It is. Therefore, only the features characteristic of this embodiment in each step will be described below.

  A characteristic point of the “nucleic acid extraction step” of the present embodiment is that the type of nucleic acid used is genomic DNA or nucleic acid derived from genomic DNA. A nucleic acid derived from genomic DNA refers to a nucleic acid obtained by incorporating a part of a polypeptide chain of genomic DNA into a vector such as cosmid or an artificial chromosome such as YAC. The nucleic acid extraction method may be in accordance with the genomic DNA extraction method if it is genomic DNA. Moreover, if it is a nucleic acid derived from genomic DNA, what is necessary is just to follow the extraction method according to the kind. For example, if it is a cosmid, it may extract according to the cosmid extraction method.

  In the “expression control region modification detection step”, the nucleotide sequence of the expression control region of human SLC34A2 gene (hereinafter referred to as detection target gene expression control region) that can be contained in the nucleic acid extracted in the nucleic acid extraction step is SEQ ID NO: 7. This is a step of detecting whether or not the wild-type nucleotide sequence is modified.

  The “expression control region” is a region as a base sequence on the genome sequence that controls or regulates the expression of a specific gene. For example, an enhancer region or a promoter region is applicable. The expression control region often has a difference in position and base sequence depending on the gene, and it is difficult to specify them. However, at least in the case of a promoter, 3 Kb upstream from the transcription start point of the gene to be normally controlled ( It is often included in the range of Kb = 1000 bases). Therefore, in the present invention, the “expression control region” is set to 2.7 Kb upstream from the predicted transcription start point (1st position of SEQ ID NO: 1) of the human SLC34A2 gene as shown by SEQ ID NO: 7.

The modification mode of the expression control region, the detection method of the modification, and the like may be the same as in the first embodiment. Modifications detected in the present embodiment include (1) a case where expression is suppressed and (2) a case where expression is enhanced with respect to the human SLC34A2 gene under the control of an expression control region having the modification, And (3) Although there is no particular influence, it is preferable to detect the modification of (1) or (2).
<Embodiment 6: Effect> According to the present embodiment, the possibility of onset of alveolar microlithiasis can be determined from abnormal expression control of the human SLC34A2 gene in the subject. If a modification that suppresses the expression in the expression control region of the human SLC34A2 gene is detected, the subject may have “at least a 50% probability of developing alveolar microlithiasis even if the alveolar microlithiasis has not yet occurred. Can be determined.

  The present invention will be described in more detail with reference to the following examples, which are merely illustrative and the present invention is not limited thereto.

  In addition, since alveolar microlithiasis is a rare disease, even if the onset determination method of the present invention is performed on a normal subject, in most cases, it becomes negative for a known modification, and the human SLC34A2 gene The genotype is also a wild-type homozygote. In other words, since it is rare that the subject contains an alveolar microlithiasis carrier before the onset of this disease, positive results such as modified homozygos or wild type and modified heterozygous in the onset determination method It is difficult to illustrate. Therefore, in this example, the present onset determination method was performed on patients diagnosed with alveolar microlithiasis as clinical symptoms, and a positive example showing that this onset determination method works effectively was used.

  << Example 1 >>

  <Verification of existence of known alterations in human SLC34A2 gene between alveolar microlithiasis patients and healthy volunteers>

  In this example, whether specimens (patients) diagnosed with alveolar microlithiasis from clinical symptoms and 188 healthy volunteer specimens have modification A or modification B that is a known modification of the human SLC34A2 gene. In order to verify this, the alveolar microlithiasis onset determination method of the present invention based on Embodiment 1 was performed. Hereinafter, the present embodiment will be specifically described.

(Nucleic acid extraction step) Extraction of genomic DNA First, peripheral blood was obtained from samples diagnosed as alveolar microlithiasis and 188 healthy volunteer samples. Next, lysis buffer (final concentration: 100 μg / ml proteinase K, 50 mM Tris-HCl pH 7.5, 10 mM CaCl ₂ , 1% SDS) is added to 5 ml of peripheral blood of each specimen, and incubated at 50 ° C. for 30 minutes. Cells were lysed. Subsequently, an equal amount of phenol saturated with TE buffer was added to the cell lysate, and the container was inverted and mixed several times. Next, centrifugation was performed at 3000 × g for 10 minutes at room temperature to separate the aqueous layer and the phenol layer. Subsequently, only the upper water layer was collected and transferred to a new container. Next, an equal amount of a phenol / chloroform mixed solution (1: 1 mixing ratio) was added to the aqueous layer, and the container was inverted again and mixed several times. Centrifugation was again performed at 3000 xg for 10 minutes at room temperature to separate into three layers, an aqueous layer, an intermediate layer (denatured protein layer), and a phenol / chloroform layer. After collecting only the aqueous layer so as not to mix the denatured protein constituting the intermediate layer, the treatment with the phenol / chloroform mixed solution was repeated several times until the intermediate layer could not be confirmed. Next, RNase A was added to the finally obtained aqueous layer sample to a final concentration of 50 μg / ml, and the mixture was incubated at 50 ° C. for about 1 hour to degrade RNA. Subsequently, the above lysis buffer was added and the proteinase K was again treated to deactivate RNase A in the aqueous layer sample. Next, an equal amount of the above-mentioned phenol / chloroform mixed solution was added, and the phenol / chloroform treatment was performed again. 1/10 volume of 3M sodium acetate and an equivalent amount of iso-propanol were added to the volume of the aqueous layer after the treatment, and the mixture was gently stirred. Finally, the precipitated genomic DNA was entangled with a glass rod or centrifuged at 3000 × g for 10 minutes at room temperature to obtain the desired genomic DNA from each specimen.

(Gene alteration detection process)
Known alterations were detected using the genomic DNA of the specimen obtained in the nucleic acid extraction step. For detection of modification A, a 5 ′ → 3 ′ nuclease assay real-time PCR method using an LNA probe was used, and for detection of modification B, a PNA-LNA-PCR clamp method was used. 5 ′ → 3 ′ nuclease assay / real-time PCR method using LNA probe, 5 ′ → 3 ′ nuclease activity of Taq DNA polymerase using a probe containing LNA modified at both ends with a fluorescent substance and a quencher The principle is the same as that of Takara Bio's TaqMan probe method. The PNA-LNA-PCR clamp method was approached by Non-Patent Document 7.

  A. Preparation of real-time PCR reaction solution

(1) Preparation of PCR basic reaction solution The "PCR basic reaction solution" was prepared by mixing the following reagents. 25 mM TAPS (pH 9.3), 50 mM KCl, 2 mM MgCl ₂ , 1 mM 2-mercaptoethanol, 200 μM each dNTP (= dATP, dTTP, dGTP, dCTP), 1.25 U TAKARA Ex Taq HS (Takara Bio).

  (2) Preparation of modified A detection reaction solution 200 mM each of forward (F) primer and reverse (R) primer were added to the “PCR basic reaction solution” as modified A amplification primers. The sequence of the F primer of the modified A amplification primer is represented by SEQ ID NO: 8. The sequence of the R primer of the modified A amplification primer is represented by SEQ ID NO: 9. Furthermore, 100 nM of a modified A detection LNA probe was added. The base sequence of the modified A detection LNA probe is represented by SEQ ID NO: 12. Of the base sequence of the modified A detection LNA probe, the 4th T, 7th C, 11th T, 15th C and 18th C are composed of LNA. Further, the 5 ′ end of the LNA probe is modified with a fluorescent dye 6-FAM, and the 3 ′ end is modified with a fluorescence inhibitor BHQ1.

  (3) Preparation of reaction solution for detection of modified B 200 mM each of F primer and R primer as amplification primers for modified B were added to the “PCR basic reaction solution”. The sequence of the F primer of the modified B amplification primer is represented by SEQ ID NO: 10. The sequence of the R primer of the modified B amplification primer is represented by SEQ ID NO: 11. Further, 100 nM of the modified B detection LNA probe and 5 μM of the PNA clamp primer were added. The base sequence of the modified B detection LNA probe is represented by SEQ ID NO: 13. In the base sequence of the modified B detection LNA probe, the 10th T is constituted by LNA. Further, the 5 ′ end of the LNA probe is modified with a fluorescent dye 6-FAM, and the 3 ′ end is modified with a fluorescence inhibitor BHQ1. The base sequence of the PNA clamp primer is represented by SEQ ID NO: 14. The PNA clamp primer is composed of PNA at all bases.

  The LNA probes were outsourced to IDT, and the PNA clamp primer was outsourced to Greiner Japan.

  25 μg of genomic DNA of the sample obtained in the nucleic acid extraction step was added to each modified detection reaction solution of (2) and (3) to adjust the final total amount to 25 μl. Subsequently, the following real-time PCR was performed for each reaction solution.

B. Real-Time PCR Reaction Real-time PCR using the 5 ′ → 3 ′ nuclease activity of Ex Taq was monitored using Smart Cycler II (Cepheid Sunnyvale). The PCR reaction cycle was maintained at 95 ° C. for 30 seconds, and then a step of 95 ° C. for 3 seconds and 56 ° C. for 30 seconds was performed in 45 cycles, and the fluorescence intensity in each cycle was measured. A real-time PCR reaction was performed for each specimen, and known alterations in the human SLC34A2 gene were detected.

(result)
FIG. 9 shows the result. The vertical axis of each graph represents the fluorescence intensity, and the horizontal axis represents the number of PCR cycles. First, since all the 188 healthy volunteer samples showed the same fluorescence pattern, the result of one of them is shown in A here as a representative diagram. In the modification detection reaction solution, neither fluorescence modification A nor modification B showed a significant change in fluorescence intensity even when the number of PCR cycles increased. This result means that neither the modified A nor the B exists in the human SLC34A2 gene of the 188 healthy volunteer samples (in the 376 genome).

  On the other hand, the specimen diagnosed as alveolar microlithiasis from the clinical symptoms showed the fluorescence pattern shown in B. In the modified A detection reaction solution, no significant change in fluorescence intensity was observed even when the number of PCR cycles was increased. This result means that the modified A does not exist in the human SLC34A2 gene of the sample. In contrast, in the modified B detection reaction solution, a rapid increase in fluorescence intensity was observed when the PCR cycle number was 25 cycles or more. This result means that the modified B exists in the human SLC34A2 gene of the sample.

(Discussion)
From the above results, it was found that neither modification A nor modification B, which are known modifications, were present in the human SLC34A2 gene of 188 healthy volunteer samples (in the 376 genome). On the other hand, it has been clarified that the modified B is heterozygous or homozygous in the human SLC34A2 gene of the specimen diagnosed as alveolar microlithiasis based on clinical symptoms. Therefore, it was suggested at the gene level that inactivation of the function of human IIB type sodium-phosphate cotransporter protein by the modified B is highly likely to cause alveolar microlithiasis.

  << Example 2 >>

  <Confirmation of genotype in human SLC34A2 gene in patients with alveolar microlithiasis>

  It was found that the specimen diagnosed as alveolar microlithiasis from the clinical symptoms of Example 1 had modification B, which is a known modification. Therefore, the nucleotide sequence of the human SLC34A2 gene was determined for the specimen using the primer represented by SEQ ID NO: 10 capable of determining the modified B and the wild-type nucleotide sequence corresponding to the modified B. The same procedure was performed for one healthy volunteer sample that was found to contain neither modified A nor modified B as a control.

(Confirmation of sequence of PCR amplification product)
The PCR amplification product of the modified B detection reaction solution obtained by the real-time PCR reaction of Example 1-B was purified using Wizard PCR Prep DNA purification Kit (Promega), and then used for cloning. The reaction was carried out using BigDye Terminator v1.1 cycle sequencing Kit (ABI) according to the attached protocol using the F primer represented by The base sequence of this reaction product was directly read with an automatic DNA sequencer (ABI PRISM 310: ABI).

(result)
FIG. 10 shows the result. First, as shown by A, the base sequence of the amplification product of the specimen diagnosed as alveolar microlithiasis from clinical symptoms is obtained only as a waveform representing the modified B sequence, and mixed with the waveform representing the wild type sequence. The waveform could not be observed. This result indicates that the specimen diagnosed as alveolar microlithiasis from clinical symptoms has the modified B homozygous and is not a heterozygous of the wild type and the modified type. On the other hand, as indicated by B, only the waveform representing the wild type sequence was obtained as the base sequence of the amplification product of the healthy volunteer sample. As a result, it was determined that the genotype of the human SLC34A2 gene in the sample is a modified homozygote and that at least the modified B is homozygous is the cause of the alveolar microlithiasis.

The conceptual diagram for demonstrating the aspect of the modification A in human SLC34A2 gene, and the structure of the amino acid modification A which is the said gene product. The conceptual diagram for demonstrating the aspect of the modification B in human SLC34A2 gene, and the structure of the amino acid modification B which is the said gene product. 3 is a flowchart for explaining the first embodiment. Flow diagram of a general method for extracting nucleic acids from cells. 9 is a flowchart for explaining the second embodiment. The figure which showed the concept of the genotype determination in the genotype determination process of Embodiment 2. FIG. 10 is a flowchart for explaining the third embodiment. 10 is a flowchart for explaining the fourth embodiment. Results of Example 1. Results of Example 2.

Claims (36)

A nucleic acid extraction step of extracting human-derived nucleic acid;
A genetic modification detection step for detecting whether the base sequence of the human SLC34A2 gene that can be contained in the extracted nucleic acid has a known modification derived from the modification from the wild-type base sequence represented by SEQ ID NO: 1.
A method for determining the onset of alveolar microlithiasis, which comprises determining the possibility of the onset of alveolar microlithiasis. A nucleic acid extraction step of extracting human-derived nucleic acid;
A genetic modification detection step for detecting whether the base sequence of the human SLC34A2 gene that can be contained in the extracted nucleic acid is modified from the wild-type base sequence represented by SEQ ID NO: 1;
Genotyping to determine whether the genotype of the human SLC34A2 gene that can be contained in the extracted nucleic acid is wild-type homo, modified homo, wild-type and modified hetero, or modified hetero Process,
A method for determining the onset of alveolar microlithiasis.   The method for determining the onset of alveolar microlithiasis according to claim 1 or 2, wherein the nucleic acid extracted in the nucleic acid extraction step is genomic DNA, total RNA, mRNA, or nucleic acid based thereon.   The method for determining the onset of alveolar microlithiasis according to claim 3, wherein the total RNA or mRNA is extracted from lung tissue, sputum, or pleural effusion. The mode of modification to be detected in the genetic modification detection step is:
The method for determining the onset of alveolar microlithiasis according to any one of claims 1 to 4, wherein the deletion, substitution, or addition is a base. Known modifications to be detected in the genetic modification detection step are:
The base sequence from T at position 13290 to G at position 13304 represented by SEQ ID NO: 1 is a modification in which the base sequence represented by SEQ ID NO: 3 is substituted. Method for determining the onset of alveolar microlithiasis. Known modifications to be detected in the genetic modification detection step are:
The determination of the onset of alveolar microlithiasis according to any one of claims 1 to 4, which is based on a modification in which G in the splicing donor site of the eighth intron is substituted with A in the base sequence represented by SEQ ID NO: 1. Method. The method for detecting a modification in the genetic modification detection step is:
A probe comprising a polynucleotide having the entire base sequence of a known modification derived from the base sequence modification of the wild-type human SLC34A2 gene represented by SEQ ID NO: 1 or a part consisting of at least 14 consecutive bases. The method for determining the onset of alveolar microlithiasis according to any one of claims 1 to 7, which is a method of detecting by hybridization. The method for detecting a modification in the genetic modification detection step is:
A method for detection by determining the base sequence of all or part of a region where a known modified base sequence can be present derived from a modification from the base sequence of the wild-type human SLC34A2 gene represented by SEQ ID NO: 1. The method for determining the onset of alveolar microlithiasis according to any one of 1 to 7. The method for detecting a modification in the genetic modification detection step is:
The method according to claim 2, wherein the detection is carried out by hybridization using as a probe a polynucleotide having the entire nucleotide sequence of the wild-type human SLC34A2 gene represented by SEQ ID NO: 1 or a portion comprising at least 14 consecutive nucleotides. The lung according to any one of the method for determining the onset of alveolar microlithiasis or the method for determining the onset of alveolar microlithiasis dependent on claim 2 among the methods for determining the onset of alveolar microlithiasis according to claim 3 or 4. Cystolithiasis onset determination method. The method for detecting a modification in the genetic modification detection step is:
In the nucleic acid extracted in the nucleic acid extraction step, the whole or part of the region where the human SLC34A2 gene can exist is determined, and the base sequence and the base sequence of the wild-type gene represented by SEQ ID NO: 1 are determined. The alveolar microlithiasis onset determination method according to claim 2, or the alveolar microlithiasis onset determination method according to claim 3 or 4, which is a detection method by comparison. The method for determining the onset of alveolar microlithiasis according to any one of the methods for determining the onset of stone disease. The method for detecting a modification in the genetic modification detection step is:
The method for determining the onset of alveolar microlithiasis according to any one of claims 1 to 7, which is a method of detecting whether the target nucleic acid amplification product has increased by the nucleic acid amplification method. The method for detecting a modification in the genetic modification detection step is:
The method for determining the onset of alveolar microlithiasis according to claim 2, which is a method of detecting by comparing the number of bases of a wild-type nucleic acid amplification product amplified by a nucleic acid amplification method and a modified nucleic acid amplification product. 5. The method for determining the onset of alveolar microlithiasis according to any one of the methods for determining the onset of alveolar microlithiasis according to claim 2 among the methods for determining the onset of alveolar microlithiasis according to item 3 or 4. The detection of the modification in the genetic modification detection step,
5. The method for determining the onset of alveolar microlithiasis according to claim 2, or the alveolar microscopic disease according to claim 3 or 4, wherein the detection method is based on a cleavage pattern of wild type and modified type using a restriction enzyme utilizing RFLP. The method for determining the onset of alveolar microlithiasis according to any one of the methods for determining the onset of alveolar microlithiasis according to claim 2 among the methods for determining the onset of stone disease. A protein extraction step of extracting human-derived protein;
An amino acid for detecting whether the amino acid sequence of the human type IIb sodium-phosphate cotransporter protein that can be contained in the extracted protein has a known amino acid modification from the wild-type amino acid sequence represented by SEQ ID NO: 2 A modification detection step;
A method for determining the onset of alveolar microlithiasis, which comprises determining the possibility of the onset of alveolar microlithiasis. A protein extraction step of extracting human-derived protein;
An amino acid modification detection step for detecting whether the amino acid sequence of the human type IIb sodium-phosphate cotransporter protein that can be contained in the extracted protein has been modified from the wild-type amino acid sequence represented by SEQ ID NO: 2; ,
The genotype of the human SLC34A2 gene encoding the human type IIb sodium-phosphate cotransporter protein that can be contained in the extracted protein is a wild type homozygote, a modified homozygote, a wildtype and a modified heterotype, or a modified type A second genotyping process to determine which is a heterogeneity,
A method for determining the onset of alveolar microlithiasis. The protein in the protein extraction step is
The method for determining the onset of alveolar microlithiasis according to claim 15 or 16, wherein the method is extracted from lung tissue. The mode of amino acid modification to be detected in the amino acid modification detection step is:
The method for determining the onset of alveolar microlithiasis according to any one of claims 15 to 17, which is deletion, substitution, or addition of an amino acid. The amino acid modification to be detected in the amino acid modification detection step is:
The method for determining the onset of alveolar microlithiasis according to any one of claims 15 to 17, which is an amino acid modification having the amino acid sequence represented by SEQ ID NO: 5. The amino acid modification to be detected in the amino acid modification detection step is:
The method for determining the onset of alveolar microlithiasis according to any one of claims 15 to 17, which is an amino acid modification having the amino acid sequence represented by SEQ ID NO: 6. The method for detecting amino acid modification in the amino acid modification detection step,
An antibody comprising, as an antigen, a polypeptide having the amino acid sequence of a known amino acid-modified human type IIb sodium-phosphate symporter protein having all or a part of at least 5 consecutive amino acids modified in the amino acid sequence 21. The method for determining the possibility of developing alveolar microlithiasis according to any one of claims 15 to 20, which is a method of detection by an antigen-antibody reaction method used. The method for detecting an amino acid modification in the amino acid modification detection step is a method of detecting a polymorphism having the whole amino acid sequence of the wild-type human type IIb sodium-phosphate symporter protein represented by SEQ ID NO: 2 or a part of at least 5 consecutive amino acids. The method for determining the onset of alveolar microlithiasis according to claim 16, or the method for determining the onset of alveolar microlithiasis according to claim 17 or 18, which is a method of detecting by an antigen-antibody reaction method using an antibody having nucleotides as antigens. The method for determining the onset of alveolar microlithiasis according to any one of the preceding claims.   In the base sequence of the known modified human SLC34A2 gene derived from the modification from the wild-type base sequence represented by SEQ ID NO: 1, the base sequence of the modified region is all or part of at least 14 consecutive bases A polynucleotide comprising a nucleotide sequence having a nucleotide sequence and used as a probe for use in the method for determining the onset of alveolar microlithiasis according to claim 8.   The polynucleotide according to claim 23, wherein the base sequence of the modified region is the base sequence represented by SEQ ID NO: 3.   The polynucleotide according to claim 23, wherein the base sequence of the modified region is the base sequence represented by SEQ ID NO: 4.   The method for determining the onset of alveolar microlithiasis according to claim 10, wherein the method comprises the base sequence of the wild-type human SLC34A2 gene represented by SEQ ID NO: 1 all or a base sequence having at least 14 consecutive bases as a part. A polynucleotide used as a probe used in the above.   In the base sequence of the known modified human SLC34A2 gene derived from the modification from the wild-type base sequence represented by SEQ ID NO: 1, when the base sequence of the modified region is 18 bases or more, the modified region A polynucleotide having a part of 18 to 60 bases in a continuous base sequence and used as a primer used in the method for determining the onset of alveolar microlithiasis according to claim 9 or 12.   The polynucleotide according to claim 27, wherein the base sequence of the modified region is the base sequence represented by SEQ ID NO: 3.   The polynucleotide according to claim 27, wherein the base sequence of the modified region is the base sequence represented by SEQ ID NO: 4.   It has 18 to 60 bases as a part in the base sequence of the wild type human SLC34A2 gene represented by SEQ ID NO: 1, and is used in the method for determining the onset of alveolar microlithiasis according to claim 11 or 13 A polynucleotide used as a primer.   A kit for determining the onset of alveolar microlithiasis, wherein the possibility of the onset of alveolar microlithiasis is determined using the polynucleotide of any one of claims 23 to 30.   In the amino acid sequence of the known amino acid-modified human type IIb sodium-phosphate symporter protein from the wild-type amino acid sequence represented by SEQ ID NO: 2, all or a continuous amino acid sequence of the amino acid-modified region is at least 5 A polypeptide as an antigen which comprises an amino acid sequence having a part of amino acid or more as a part thereof and which produces an antibody for detecting an amino acid modification in the method for determining the onset of alveolar microlithiasis according to claim 15. The polypeptide according to claim 32, wherein the amino acid sequence of the amino acid-modified region is the amino acid sequence represented by SEQ ID NO: 5. The polypeptide according to claim 32, wherein the amino acid sequence of the amino acid-modified region is the amino acid sequence represented by SEQ ID NO: 6.   The lung according to claim 16, comprising the amino acid sequence of the wild-type human type IIb sodium-phosphate cotransporter protein represented by SEQ ID NO: 2 as a part of the whole or at least 5 consecutive amino acids. A polypeptide as an antigen for preparing an antibody for detecting a modification of the method for determining the onset of cyst microlithiasis. A nucleic acid extraction step of extracting human-derived nucleic acid;
An expression control region modification detection step for detecting whether the base sequence of the expression control region of the human SLC34A2 gene that can be contained in the extracted nucleic acid is modified from the wild-type base sequence represented by SEQ ID NO: 7;
A method for determining the onset of alveolar microlithiasis, which comprises determining the possibility of the onset of alveolar microlithiasis.