CN117025747A - Application of reagent for detecting SLC34A3 gene variation in sample in preparation of product for screening low-blood-phosphorus rickets/osteomalacia patients - Google Patents

Application of reagent for detecting SLC34A3 gene variation in sample in preparation of product for screening low-blood-phosphorus rickets/osteomalacia patients Download PDF

Info

Publication number
CN117025747A
CN117025747A CN202310551051.2A CN202310551051A CN117025747A CN 117025747 A CN117025747 A CN 117025747A CN 202310551051 A CN202310551051 A CN 202310551051A CN 117025747 A CN117025747 A CN 117025747A
Authority
CN
China
Prior art keywords
slc34a3
gene
rickets
osteomalacia
reagent
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN202310551051.2A
Other languages
Chinese (zh)
Other versions
CN117025747B (en
Inventor
陈丽
侯新国
蒋玲
孙玉静
刘媛
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Qilu Hospital of Shandong University
Original Assignee
Qilu Hospital of Shandong University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Qilu Hospital of Shandong University filed Critical Qilu Hospital of Shandong University
Priority to CN202310551051.2A priority Critical patent/CN117025747B/en
Priority to CN202410590834.6A priority patent/CN118421783A/en
Publication of CN117025747A publication Critical patent/CN117025747A/en
Application granted granted Critical
Publication of CN117025747B publication Critical patent/CN117025747B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Analytical Chemistry (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Pathology (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The disclosure describes an application of a reagent for detecting SLC34A3 gene variation in a sample in preparing a product for screening patients with hypophosphatemic rickets/osteomalacia, which is characterized in that SLC34A3 C.1336-2A > G gene variation is SLC34A 3. The invention also discloses application of a reagent for detecting SLC34A3 gene variation in a sample in preparing a product for evaluating susceptibility of rickets/osteomalacia hypovolemic, wherein the SLC34A3 gene variation is SLC34A3 c.1336-2A > G. According to the present disclosure, a novel pathogenic gene locus of hypophosphatemic rickets/osteomalacia or a mutation locus of hypophosphatemic rickets/osteomalacia high risk can be provided, which is helpful for diagnosis, treatment and prevention of hypophosphatemic rickets/osteomalacia.

Description

Application of reagent for detecting SLC34A3 gene variation in sample in preparation of product for screening low-blood-phosphorus rickets/osteomalacia patients
Technical Field
The invention relates to the technical field of biological medicine, in particular to application of a reagent for detecting SLC34A3 protein variation or gene variation in a sample in preparation of a product for screening patients with hypophosphatemic rickets/osteomalacia.
Background
Rickets/osteomalacia hypovolemic (hypophosphoric-micckets/osteomalacia) is a group of bone mineralization disorder diseases which are caused by hereditary or acquired reasons and are mainly characterized by rickets/osteomalacia hypovolemic, and has higher disability and teratogenesis rate. The rickets in childhood are mainly manifested by chicken breast, rib beading, limb deformity (lower limb gonal inversion or gonal valgus), and growth retardation. The adult onset is called osteomalacia, and is manifested by debilitation, bone changes, short stature, multiple fracture, limited movement, and even disability. Clinically, hypophosphatemic rickets/osteomalacia is not uncommon and is frequently seen in hereditary diseases, wherein hereditary rickets/osteomalacia with hypophosphorous disease include X-linked inheritance (X-linked hypophosphatemia, XLH, OMIM #307800, OMIM # 300554), autosomal dominant inheritance (autosomal dominant hypophosphatemic ricket, ADHR, OMIM # 193100), autosomal recessive inheritance (autosomal recessive hypophosphatemic ricket, ARHR, OMIM #241520, OMIM #613312, OMIM # 259775), rickets with hypercalcuria (hereditary hypophosphatemic rickets with hypercalciuria, HHRH, OMIM # 241530). Wherein XLH rickets are most common, account for more than 80% of familial hypophosphatemic rickets, 50% -70% of XLH patients can detect PHEX gene mutation due to PHEX (regulating gene with homolog to endopeptidases on the X chromosome) gene abnormality.
Wherein, the low-phosphorus rickets with high calcium urea disease (HHRH) is caused by mutation of a gene SCL34A3 encoding a renal tubular sodium phosphate cotransporter NaPi2c, the genetic mode is autosomal recessive inheritance, and the homozygous or compound heterozygous loss of function mutation in the SLC34A3 can lead to waste of urine phosphate, so that clinical manifestations are generated, but the clinical manifestations of bones are highly variable, some patients have no obvious bone abnormality, and some patients have mild to severe osteomalacia. Due to variability in clinical presentation, it is important to conduct genetic diagnosis of the disease. Furthermore, unlike other types of hypophosphatemic rickets/osteomalacia, HHRH is the only disease in which vitamin D is elevated in 1, 25 dihydroxys in patients, and administration of active vitamin D to HHRH patients can lead to the appearance or exacerbation of the original kidney stones in patients, and thus active vitamin D is a contraindication for treatment of such patients. In the past, due to the lack of gene diagnosis technology, HHRH patients are misdiagnosed as XLH and are treated by active vitamin D, so that the phenomena of serious side effects such as kidney stones and the like are more common. Therefore, the genotyping of the rickets/osteomalacia caused by hypovolemia is clearly diagnosed, and the method has a key effect on selecting a proper treatment scheme.
At present, a plurality of unknown pathogenic gene loci related to familial hypophosphatemic rickets/osteomalacia still exist, the pathogenic mechanism of the hypophosphatemic rickets/osteomalacia is further researched, new pathogenic gene variation of familial hypophosphatemic rickets/osteomalacia is separated, and the method has important significance for diagnosing, treating and preventing the hypophosphatemic rickets/osteomalacia.
Disclosure of Invention
The present disclosure has been made in view of the above-mentioned prior art, and an object thereof is to provide a disease-causing mutation site of rickets/osteomalacia with low blood phosphorus or a mutation site of rickets/osteomalacia with high risk, which is helpful for diagnosis, treatment and prevention of rickets/osteomalacia with low blood phosphorus.
Therefore, the first aspect of the present disclosure provides an application of a reagent for detecting SLC34A3 gene variation in a sample in preparing a product for screening patients with hypophosphatemic rickets/osteomalacia, wherein the SLC34A3 gene variation is SLC34A3 c.1336-2A > G. In the disclosure, SLC34A3 c.1336-2A > G mutation (the 1336 th base of a DNA sequence coding region of SLC34A3 gene is replaced by G base at the forward 2 nd base of a non-coding region) is identified through family research, and the SLC34A3 c.1336-2A > G mutation can influence SLC34A3 activity through family research, so that a novel disease-causing gene locus of rickets with low blood phosphorus or a mutation locus of rickets with high risk of rickets with low blood phosphorus/osteomalacia is provided, and whether a sample carries SLC34A3 c.1336-2A > G mutation or not can be used for assisting in screening rickets with low blood phosphorus/osteomalacia patients.
In the application of the first aspect to which the present disclosure relates, optionally, the rickets/osteomalacia patient with hypophosphorous acid is a rickets patient with hypercalcemia. Thus, patients with low-phosphorus rickets and hypercalcemia can be screened.
In an application of the first aspect of the present disclosure, the SLC34A3 gene variation is optionally detected using at least one of pyrosequencing technology, sanger sequencing, NGS sequencing, polymerase chain reaction-single strand conformational polymorphism analysis, taqMan probe method. Thus, the SLC34A3 gene can be detected using these methods to aid in screening patients with low phosphorus rickets with hypercalcemia.
In an application of the first aspect to which the present disclosure relates, optionally, the reagent comprises a primer pair for amplifying the SLC34A3 gene and/or a probe for detecting a variation of the SLC34A3 gene. Thus, the SLC34A3 gene variation c.1336-2A > G can be captured and/or detected by primer pairs and/or probes.
In the application of the first aspect of the present disclosure, optionally, the primer pair is designed according to a nucleotide sequence upstream and downstream of a base of an intron region located in the 1336 th position and forward 2 nd position of the coding region of the SLC34A3 gene in the human genome, and the probe is designed according to a nucleotide sequence upstream and downstream of a base of an intron region located in the 1336 th position and forward 2 nd position of the coding region of the SLC34A3 gene in the human genome. Thus, the primer can bind to the sequence of the upstream and downstream region of the SLC34A3 gene c.1336-2, and the probe can bind to the sequence of the SLC34A3 gene c.1336-2 and the upstream and downstream region thereof, to detect the region.
In an application of the first aspect to which the present disclosure relates, optionally, the reagents further comprise dNTPs, a DNA polymerase and a PCR reaction buffer. Thus, a reaction substrate, catalytic enzyme and buffer can be provided to facilitate detection of SLC34A3 c.1336-2A > G.
In an application of the first aspect to which the present disclosure relates, optionally, the product further comprises a nucleic acid extraction reagent. Thus, the sample can be subjected to nucleic acid extraction to facilitate subsequent further detection of SLC34A3 gene variation.
In an application of the first aspect of the disclosure, optionally, the sample is at least one of a peripheral blood, saliva, and tissue sample from a subject, and the SLC34A3 gene variation is a germline variation of the SLC34A3 gene. Thus, by detecting a subject's peripheral blood, saliva and/or tissue sample, a subject's germline mutation of the SLC34A3 gene (germline mutation refers to a variation that has been carried during the development of a human embryo, carried by each cell in the body) can be detected.
The second aspect of the present disclosure provides an application of a reagent for detecting SLC34A3 gene variation in a sample in preparing a product for evaluating susceptibility of rickets/osteomalacia hypovolemia, wherein SLC34A3 gene variation is SLC34A3 c.1336-2A > G. In the disclosure, SLC34A3 c.1336-2A > G mutation (the 1336 th base of a DNA sequence coding region of SLC34A3 gene is substituted by G base) is identified through family research, and the SLC34A3 c.1336-2A > G mutation can influence SLC34A3 activity through family research, so that a novel disease-causing gene locus of low-blood phosphorus rickets/osteomalacia or a mutation locus of high risk of low-blood phosphorus rickets/osteomalacia is provided, and the susceptibility of the low-blood phosphorus rickets/osteomalacia can be evaluated in an auxiliary manner by detecting whether the sample carries the SLC34A3 c.1336-2A > G mutation.
In a second aspect of the present disclosure, optionally, the rickets/osteomalacia patient with hypophosphatemia is a rickets patient with hypercalcemia. Thus, the susceptibility of low-phosphorus rickets with hypercalcemia can be evaluated in an assisted manner.
According to the present disclosure, a pathogenic mutation site of hypophosphatemic rickets/osteomalacia or a mutation site of hypophosphatemic rickets/osteomalacia high risk can be provided, which is helpful for diagnosis, treatment and prevention of hypophosphatemic rickets/osteomalacia.
Drawings
FIG. 1 is a diagram of sequencing peaks of the precursor and his parent SLC34A3 c.1336-2A > G according to an embodiment of the present invention.
Fig. 2 is a diagram of sequencing peaks of the proband and its parent SLC34A3 c.575c > T according to an embodiment of the present invention.
Fig. 3 is a family diagram of a prover according to an embodiment of the present invention.
FIG. 4 is a protein structure of SLC34A3 according to an embodiment of the present invention.
FIG. 5 is a diagram of amino acid sequences of SLC34A3 protein according to an embodiment of the present invention between different species.
FIG. 6 is a protein topology prediction graph of SLC34A 3.
FIG. 7 is a diagram showing the results of minigene splicing experiments on the pcMINI-C vector.
FIG. 8 is a diagram showing the results of minigene splicing experiments on pcDNA3.1 vector.
Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to like or similar elements throughout or elements having like or similar functionality. The embodiments described below by way of the drawings are exemplary only and should not be construed as limiting the invention.
It will be understood by those skilled in the art that, unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the prior art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.
As used herein, the singular forms "a", "an", "the" and "the" are intended to include the plural forms as well, unless expressly stated otherwise, as understood by those skilled in the art. It will be further understood that the terms "comprises" and/or "comprising," when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, and/or groups thereof.
In order that the invention may be readily understood, a further description of the invention will be rendered by reference to specific embodiments that are illustrated in the appended drawings and are not to be construed as limiting embodiments of the invention. It will be appreciated by those skilled in the art that the drawings are merely schematic representations of examples and that the elements of the drawings are not necessarily required to practice the invention.
In this embodiment, any one of the following applications is referred to:
application of reagent for detecting SLC34A3 gene variation in sample in preparing product for screening low blood phosphorus rickets/osteomalacia patient;
application of reagent for detecting SLC34A3 gene variation in sample in preparing product for screening patients with low-phosphorus rickets and hypercalcemia;
application of reagent for detecting SLC34A3 gene variation in sample in evaluating susceptibility of rickets/osteomalacia with low blood phosphorus;
application of reagent for detecting SLC34A3 amino acid mutation in sample in evaluating susceptibility of low-phosphorus rickets with hypercalcemia.
The novel mutation site provided by the invention supplements the hereditary mutation spectrum of familial low-phosphorus rickets with hypercalcuria, can be beneficial to diagnosing patients with low-phosphorus rickets with hypercalcuria so as to facilitate treatment, and carrying out genetic diagnosis on carriers in families so as to facilitate health management, and can guide fertility according to genotypes of both parents so as to avoid inheritance of pathogenic genes and guide prepotency.
The reagent according to the present embodiment may include a reagent for detecting the SLC34A3 gene mutation. In some examples, a variation of the SLC34A3 gene to SLC34A3 c.1336-2A>G,SLC34A3 c.1336-2A > G refers to a mutation of the base from A (adenine) to C (cytosine) at position 1336 to 2 forward of the coding region of the wild-type SLC34A3 gene. In other words, the SLC34A3 c.1336-2A > G mutation may be detected, or alternatively, whether the subject carries the SLC34A3 c.1336-2A > G mutation may be detected.
In the embodiment, the SLC34A3 c.1336-2A > G mutation is identified through family research, and functional research proves that the SLC34A3 c.1336-2A > G mutation can influence the activity of SLC34A3, so that the pathogenicity evidence of the mutation is perfected, and the mutation is proved to be a pathogenic mutation. Therefore, a novel pathogenic gene locus of low-phosphorus rickets with hypercalcuria or a mutation locus of low-phosphorus rickets with hypercalcuria high risk is provided, and whether SLC34A3 c.1336-2A > G mutation is carried in a sample is detected, so that screening of patients with low-phosphorus rickets with hypercalcuria can be assisted.
In some examples, the SLC34A3 c.1336-2A > G variation can be detected using at least one of a pyrosequencing technique, sanger sequencing, NGS sequencing, polymerase chain reaction-single strand conformation polymorphism analysis, taqMan probe method. Thus, whether SLC34A3 c.1336-2A > G mutation is carried or not can be detected to assist in screening patients with low phosphorus rickets with hypercalcemia.
In some examples, the above-described reagents according to the present embodiment may include a primer pair for amplifying the SLC34A3 gene and/or a probe for detecting SLC34A3 gene variation. Thus, the SLC34A3 gene variation can be captured and/or detected by primer pairs and/or probes. In some examples, the reagents may also include dNTPs, DNA polymerase, and PCR reaction buffers. This facilitates detection of the SLC34A3 gene variation.
In some examples, the primer pair may be designed based on the nucleotide sequence upstream and downstream of the nucleotide sequence of the SLC34A3 gene c.1336-2 in the human genome, and the probe may be designed based on the nucleotide sequence of the nucleotide sequence upstream and downstream of the nucleotide sequence of the SLC34A3 gene coding region c.1336-2 in the human genome. Thus, SLC34A3 c.1336-2A > G can be detected.
In some examples, the reagent according to the present embodiment may include a reagent for detecting a gene related to rickets with low blood phosphorus/osteomalacia other than the SLC34A3 gene. In some examples, the gene associated with hypophosphatemic rickets/osteomalacia may include at least one gene of PHEX, FGF23, DMP-1, ENPP1, FAM20C, GNAS, OGD, NRAS, KRAS, and HRAS. In other words, the reagent according to the present embodiment can detect at least one of SLC34A3, PHEX, FGF23, DMP-1, ENPP1, FAM20C, GNAS, OGD, NRAS, KRAS and HRAS genes. Thus, genotyping of patients with hypophosphatemic rickets/osteomalacia can be facilitated.
In some examples, the reagent according to the present embodiment may include a reagent for detecting a SLC34A3 mutation other than SLC34A3 c.1336-2A > G. In some examples, the other SLC34A3 mutations may include all pathogenic mutations and suspected pathogenic mutations of the SLC34A3 gene currently known for rickets with hypercalcemia of low phosphorus. Therefore, the relevant sites of the low-phosphorus rickets and the hypercalcuria can be detected, and the one-time and more comprehensive screening of the low-phosphorus rickets and the hypercalcuria is facilitated.
In some examples, the reagent according to the present embodiment may include a reagent for detecting a gene or protein associated with another disease. For example, reagents for detecting genes or proteins associated with diseases for which differential diagnosis is desired (e.g., vitamin D dependent cartilage disease) may also be included. This makes it possible to perform differential diagnosis of the subject at the same time. For example, reagents for detecting genes or proteins associated with a related genetic bone disorder (e.g., cartilage dysplasia) may also be included. Thus, the test subject can be screened for a plurality of diseases at the same time.
In some examples, the above-described product according to the present embodiment may also include a nucleic acid extraction reagent. Thus, the subsequent detection of SLC34A3 gene variation can be facilitated.
The product according to the present embodiment may be in the form of a reagent, a kit of reagents, or a kit of reagents. In some examples, the product may also include a system of instruments. In some examples, the product according to the present embodiment may also include a system composed of an apparatus for detecting SLC34A3 gene mutation. For example, the product may be a system consisting of PCR reagents and DNA sequencing reagents and DNA sequencers, or a system consisting of TaqMan probes, PCR primer pairs, quantitative PCR instruments and other reagents required for genotyping and TaqMan probe technology, or a system consisting of probes, PCR primer pairs and other reagents and instruments required for the Ligase Detection Reaction (LDR), or a system consisting of PCR primer pairs, single base extension primers, chips, PCR instruments, modules for genotyping and/or other reagents and instruments required for Sequenom MassArray technology. Thus, detection of the SLC34A3 gene can be facilitated.
In some examples, in the above-described application according to the present embodiment, the SLC34A3 gene may be detected by detecting at least one of peripheral blood, saliva, and tissue samples of the subject. In other words, the sample to be tested may be derived from at least one of peripheral blood, saliva, and tissue samples of the subject.
In some examples, the subject may be a general population, a population suspected of having rickets/osteomalacia with low blood phosphorus, or a high risk population of rickets/osteomalacia with low blood phosphorus. In some examples, an individual suspected of having hypophosphatemic rickets/osteomalacia may be an individual with symptoms of hypophosphatemia, lower limb deformity, false fracture, slow growth, and the like. In some examples, the high risk population of rickets/osteomalacia high risk population may be a population having a family history of rickets/osteomalacia high risk population of low blood phosphorus, e.g., at least one lineal family member diagnosed as an individual of rickets/osteomalacia low blood phosphorus.
In some examples, the germline mutation of the SLC34A3 gene in the sample may be detected. Germ line mutations, also called germ cell mutations, are mutations carried by germ cells, such as sperm or ovum. In some examples, the germline mutation results of the SLC34A3 gene may be obtained by extracting gDNA (genomic DNA) of the subject and detecting the genomic DNA.
In the embodiment, through the SLC34A3 c.1336-2A > G gene variation as a marker, the patients with low-blood phosphorus rickets/osteomalacia/low-phosphorus rickets and hypercalcuria can be screened, and further the application of the reagent for detecting SLC34A3 gene variation in a sample in preparing a product for screening the patients with low-phosphorus rickets and hypercalcuria is provided. Similarly, the application of the reagent for detecting SLC34A3 gene variation in the sample in preparing a product for screening susceptibility of rickets with low blood phosphorus/osteomalacia/rickets with low phosphorus and hypercalcuria can be provided.
The above applications of the present invention will be further explained in detail with reference to examples, but they should not be construed as limiting the scope of the present invention.
Examples (example)
Clinical cases:
(1) Case information
The first-stage syndrome is a 30-year-old female with low phosphorus bone diseases, double lower limb deformity, blood examination results show that blood phosphorus is low, double lower limb deformity starts at 3 years old, the height is 142mm, and the weight is 60kg.
(2) Sample detection:
a) Collecting peripheral blood of a prover, a father of the prover and a mother of the prover, extracting genomic DNA (gDNA) and performing total exon (WES) gene sequencing, wherein the prover adopts high-throughput sequencing and Sanger verification, and the father of the prover and the mother of the prover adopt Sanger verification. All exon sequencing, sanger validation and sequence analysis were performed by the university of Shandong, qilu Hospital Cooperation, jinan Ainew Zuoer medical detection Co.
In this embodiment, all data are acquired and applied legally based on compliance with legal regulations and user consent. The forensics and their families agree and sign on the informed consent. In addition, the reagents and apparatus used in this example are commercially available unless otherwise indicated.
(3) Detection result:
the results of the proband test are shown in Table 1 below, which shows that the proband SLC34A3 gene has two variants, c.1336-2A > G (splicing) and c.575C > T (p.S192L), from the proband parents, respectively, to form a composite heterozygous variant.
TABLE 1 detection results of first-evidence patients
SLC34A3 c.1336-2A > G (exon 13, NM-080877), the mutation site is located in the splicing region of the exon, and is expected to result in amino acid changes in splicing, which is a splice mutation. According to ACMG genetic variation classification criteria and guidelines, the variation is initially judged to be suspected pathogenic (Likely pathogenic), and evidence includes pvs1_modifying+pm2_supporting+pm3 (Trans) +pp4, in particular. Wherein pvs1_modelate: this variation is a null mutation (splice mutation) that may result in loss of gene function; PM2_supporting: changes to be less frequent in the normal population database as 0.0006418; PM3 (Trans): recessive genetic disease, trans-existing with another pathogenic suspected pathogenic variation (composite heterozygous with another pathogenic/suspected pathogenic mutation); PP4: the phenotype of the patient is highly specific to low phosphorus rickets with hypercalcemia. In addition, the literature database has no relevance report of the site, and the ClinVar database has no pathogenicity analysis result of the site.
SLC34A3 c.575c > T (exon 7, nm_080877), resulting in an amino acid change p.s192l, a missense mutation. According to ACMG genetic variation classification criteria and guidelines, the variation is initially determined as a Pathogenic variation (pathgenic), and evidence includes, in particular, pm1+pm2_supporting+pm3_strong+pm5+pp3+pp4. Wherein PM1: the variation is located in a mutation hot spot region; PM2_supporting: the frequency in the normal population database is lower, 0.0009; PM3_Strong: the literature database has a case report of recessive inheritance of the locus (Hereditary hypophosphataemic rickets with hypercalciuria), a mutation label is DM (Pathogenic mutation), and the pathogenicity analysis of the locus by the ClinVar database is Pathogenic, autosomal recessive hypophosphatemic bone disease |non-provided; PM5: mutations at the same position have been reported in literature databases/Clinvar but with different amino acid changes; PP3: the biological informatics protein function comprehensive prediction software REVEL predicts that the result is possibly harmful, SIFT, polyPhen _2 and MutationTaster, GERP + predicts that the result is harmful, benign and harmful respectively; PP4: the phenotype of the patient is highly specific to low phosphorus rickets with hypercalcemia.
Fig. 1 is a sequencing peak diagram of the prover and the parent SLC34A3 c.1336-2a > g according to the embodiment of the present invention, fig. 2 is a sequencing peak diagram of the prover and the parent SLC34A3 c.575c > T according to the embodiment of the present invention, and fig. 3 is a family diagram of the prover according to the embodiment of the present invention. According to FIGS. 1, 2 and 3, it is shown that the prover carries a composite heterozygous variation of SLC34A3 c.1336-2A > G and SLC34A3 c.575C > T from the prover mother and SLC34A3 c.575C > T from the prover father through family verification analysis. And (3) injection: since Sanger's verification uses forward or reverse sequencing, the peak pattern shows bases that are likely to be reverse complements of the detected bases, such as: 163G > A, the peak pattern may be shown as G > A or its reverse complement C > T.
In this example, the heterozygous mutation (SLC 34A3 c.1336-2A > G) was found to be carried in a manner consistent with the clinical phenotype of the carrier, and thus this mutation may impair the function of ALC34A3 and is a pathogenic mutation (deleterious mutation) for hyperphosphatemia. In order to further investigate the pathogenicity of SLC34A3 c.1336-2A > G variation, functional studies were subsequently performed.
SLC34A3 c.1336-2a > g functional study:
(1) Prediction of letter generation
The analysis of the letter indicates that the SLC34A3 (c.1336-2A > G) mutation might disrupt the conserved splice acceptor of exon12, located at position 2 of intron12, belonging to the "class I mutant region" affecting splicing. Computational predictions using SpliceAI, HSF and MatEntScan show that this mutation affects the cut.
To further investigate the functional impact of the SLC34A3 mutation on protein function, we predicted the impact of the SLC34A3 mutation on protein structure. Fig. 4 is a protein structure diagram of SLC34A3 according to an embodiment of the present invention, fig. 5 is an amino acid sequence diagram of SLC34A3 protein according to an embodiment of the present invention between different species, and fig. 6 is a protein topology prediction diagram of SLC34 A3. From FIG. 4, it can be seen that the conserved domain of human SLC34A3 consists of a Na+/pi-cotransporter region, and that SLC34A3, a member of the transporter family, participates in positive transport of phosphate into cells via Na+ cotransporter in the renal brush border membrane. As can be seen from FIG. 5, the SLC34A3 c.1336-2A > G position is highly conserved, amino acid 466 being valine (Val) in all four species, according to a conservation analysis. Referring to FIG. 6, localization and topology prediction of SLC34A3/NaPi2 c.1336-2A > G (p.v166) was performed, assuming that the structure consisted of 6 transmembrane domains based on sequence similarity to NaPi2b, where Val446 (indicated by the arrow) is part of the 6 th transmembrane helix, which is close to the substrate binding site, and this mutation was expected to affect SLC34A3 protein function.
(2) In vitro functional study
To further demonstrate that the SLC34A3 c.1336-2A > G variant affects cleavage, we performed minigene splicing experiments using two different vectors, pcMINI-C and pcDNA3.1, including: the vector pcMINI-C-wt/mut and the vector pcDNA3.1-wt/mut were constructed. The minigene construction strategy of pcMINI-C-SLC34A3-wt/mut was to insert intron12 (539 bp) -Exon13 (603 bp) into a pcMINI-C vector containing the universal sequence Exona-intronA-MCS, and to observe whether the cleavage pattern of Exona-Exon13 is abnormal after transfection of cells. The minigene construction strategy of pcDNA3.1-SLC34A3-wt/mut was to insert Exon12 (125 bp) -intron12 (1220 bp) -Exon13 (603 bp) into pcDNA3.1 vector and observe whether the cleavage pattern of Exon12-Exon13 was abnormal after cell transfection. Specifically, the experimental procedure is:
(1) primers were designed and the primer information is shown in Table 2 below:
TABLE 2 primer information
(2) Recombinant vector construction:
the nested first round of amplification is carried out by taking gDNA as a template 2965-SLC34A3-F and 6202-SLC34A3-R as primers to obtain nested first round products (3238 bp), and the nested first round products as templates 3329-SLC34A3-F and 5964-SLC34A3-R as primers are carried out for the nested second round of amplification to obtain nested second round products (2636 bp).
PcMINI-C fragment amplification: the products of the second round of nested PCR were used as templates, pcMINI-C-SLC34A3-BamHI-F and pcMINI-C-SLC34A3-EcoRI-R, and the wild type fragment (1142 bp) of pcMINI-C was obtained by amplification. Mutant fragment 1 (563 bp) was amplified using pcMINI-C-SLC34A3-BamHI-F and SLC34A3-mut-R as primers, and mutant fragment 2 (630 bp) was amplified using SLC34A3-mut-F and pcMINI-C-SLC34A3-EcoRI-R as primers. Fragment 1 and fragment 2 were subjected to 1:1, and using the primers pcMINI-C-SLC34A3-BamHI-F and pcMINI-C-SLC34A3-EcoRI-R as primers to amplify to obtain pcMINI-C mutant fragment (1162 bp)
pcDNA3.1 fragment amplification: the products of the second round of nested PCR were used as templates and amplified using pcDNA3.1-SLC34A3-BamHI-F and pcDNA3.1-SLC34A3-EcoRI-R as primers to give wild type fragments (1972 bp), and pcDNA3.1-SLC34A3-BamHI-F and SLC34A3-mut-R as primers to give wild type and mutant fragment 1 (1358 bp). Wild-type and mutant fragment 2 (615 bp) were amplified using SLC34A3-mut-F and pcDNA3.1-SLC34A3-EcoRI-R as primers. Fragment 1 and fragment 2 were subjected to 1:1, and using pcDNA3.1-SLC34A3-BamHI-F and pcDNA3.1-SLC34A3-EcoRI-R as primers to make amplification so as to obtain pcDNA3.1 mutant fragment (1972 bp).
The vector pcMINI-C and the fragment are subjected to enzyme digestion, recovery, connection, transformation, colony PCR identification and sequencing.
The vector pcDNA3.1 and the fragment are subjected to enzyme digestion, recovery, connection, transformation, colony PCR identification and sequencing.
(3) The PCR amplification system was as follows:
1.1×Mix22ul；
Primer-F 1 ul；
Primer-R1 ul；
gDNA(wt/mut)1 ul (0.5ug)；
Total25ul；
the annealing temperature of the primer was 57 ℃.
(4) The enzyme digestion system is as follows:
10×NEB buffer3 ul；
Enzyme 10.6 ul；
Enzyme 20.6 ul；
Vector/DNA Fragment500ng / 25ul；
ddH2O complements to 30 ul;
after enzyme digestion reaction for 2 hours at 37 ℃, electrophoresis detection and gel recovery are carried out.
(5) The connection system is as follows:
10×ligase buffer1ul；
7ul of digested DNA Fragment (wt/mut);
vector 1ul after cleavage;
Ligase1ul；
after overnight ligation at 4 ℃, DH5a competence was transformed.
(6) Cell transfection: the recombinant vector is respectively and transiently transfected into two cell lines of Hela and 293T, the transfection method is carried out according to the instruction of liposome, and the sample is collected after 48 hours.
(7) minigene transcription analysis: total RNA in the cell samples is extracted, and the extraction method is carried out according to the instruction of the kit. After concentration measurement, cDNA synthesis was performed with equal amounts of RNA. PCR amplification was performed using primers pcDNA3.1-F and SLC34A3-RT-R on both sides of minigene, and the size of the gene transcribed band was detected by agarose gel and sequenced.
48hr after plasmid transfection of cells, RNA sample extraction and cDNA preparation were performed, and the RNA sample concentrations and purities are shown in Table 3 below:
TABLE 3 concentration and purity results for RNA samples
As shown in Table 3, the concentration and purity of the RNA samples were determined to be acceptable.
(8) Detection result:
FIG. 7 is a diagram showing the result of minigene splicing experiments on pcMINI-C vector, and FIG. 8 is a diagram showing the result of minigene splicing experiments on pcDNA3.1 vector.
Referring to FIG. 7, RT-PCR detection results show that: wild type has a band of expected size (555 bp) in 293T and Hela cells, designated band a, mutation has a band larger than wild type, designated band b, and wild type and mutant bands are sequenced separately; sequencing results showed: wild-type band a is a normal sheared band, sheared in the following manner: exonA-Exon13 (272 bp); mutant band b was the complete retention of the inserted introns intron A and intron12 in the manner of Exona-intron A-intron12 (539 bp) -Exon13 (272 p).
Referring to FIG. 8, RT-PCR detection results show that: wild type has a band of expected size (507 bp) in 293T and Hela cells, designated band a; the mutant has a larger band than the wild type, designated as band b, and the wild type and the mutant bands are respectively sequenced; sequencing results showed: wild type band a is a normal cut band, and the cut mode is Exon12 (125 bp) -Exon13 (272 bp); the mutant band b is all retention of the intron12, and the shearing mode is Exon12 (125 bp) - [ V ] intron12 (1220 bp) -Exon13 (272 bp).
In conclusion, the mutation SLC34A3 c.1336-2A > G affects the normal cleavage of the gene mRNA as detected by minigene in vitro experiments. Both sets of vector detection show that mutation can cause 1220bp total retention of intron12 introns, namely, mutation damages the shearing of the original acceptors site to cause the total retention of the introns where the mutation is located; the Intron12 1220bp retention resulted in a premature stop codon in the middle of Intron12, which was expressed at the cDNA and protein level in c.1335_1336ins1220bp p.Gln445fs*71.
Taken together, the above studies and results of the studies according to the examples show that the SLC34A3 c.1336-2A > G mutation impairs the function of the SLC34A3 protein. The pathogenicity of SLC34A3 c.1336-2A > G is confirmed to be pathogenicity variation (pathogenicity) by the pedigree analysis and functional study of the invention. The SLC34A3 c.1336-2A > G mutation found in the example is a new pathogenic gene mutation of familial low-phosphorus rickets with hypercalcemia in low-phosphorus rickets/osteomalacia, and is inherited recessively in autosomal fashion.
While the disclosure has been described in detail in connection with the drawings and embodiments, it should be understood that the foregoing description is not intended to limit the disclosure in any way. Modifications and variations of the present disclosure may be made as desired by those skilled in the art without departing from the true spirit and scope of the disclosure, and such modifications and variations fall within the scope of the disclosure.

Claims (10)

1. The application of a reagent for detecting SLC34A3 gene variation in a sample in preparing a product for screening patients with hypophosphatemic rickets/osteomalacia is characterized in that SLC34A3 C.1336-2A > G.
2. The use according to claim 1, wherein the hypophosphatemic rickets/osteomalacia patient is a hypophosphatemic rickets with hypercalcemia patient.
3. The use of claim 1 or 2, wherein the SLC34A3 gene variation is detected using at least one of pyrosequencing technology, sanger sequencing, NGS sequencing, polymerase chain reaction-single strand conformation polymorphism analysis, taqMan probe method.
4. The use according to claim 3, characterized in that said reagent comprises a primer pair for amplifying the SLC34A3 gene and/or a probe for detecting the SLC34A3 gene variation.
5. The use of claim 4, wherein the primer pair is designed based on the nucleotide sequence upstream and downstream of the base of the intronic region located 1336 forward of the coding region of the SLC34A3 gene in the human genome, and the probe is designed based on the nucleotide sequence upstream and downstream of the base of the intronic region located 1336 forward of the coding region of the SLC34A3 gene in the human genome.
6. The use of claim 4, wherein the reagents further comprise dNTPs, DNA polymerase and PCR reaction buffer.
7. The use of claim 1, wherein the product further comprises a nucleic acid extraction reagent.
8. The use of claim 1, wherein the sample is derived from at least one of a peripheral blood, saliva, and tissue sample of the subject, and the SLC34A3 gene variation is a germline variation of the SLC34A3 gene.
9. The application of a reagent for detecting SLC34A3 gene variation in a sample in preparing a product for evaluating susceptibility of rickets/osteomalacia with hypophosphorus is characterized in that SLC34A3 c.1336-2A > G.
10. The use according to claim 9, wherein the hypophosphatemic rickets/osteomalacia patient is a hypophosphatemic rickets with hypercalcemia patient.
CN202310551051.2A 2023-05-16 2023-05-16 Application of reagent for detecting SLC34A3 gene variation in sample in preparation of product for screening low-blood-phosphorus rickets/osteomalacia patients Active CN117025747B (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CN202310551051.2A CN117025747B (en) 2023-05-16 2023-05-16 Application of reagent for detecting SLC34A3 gene variation in sample in preparation of product for screening low-blood-phosphorus rickets/osteomalacia patients
CN202410590834.6A CN118421783A (en) 2023-05-16 2023-05-16 Product for screening patients with low-phosphorus rickets and hypercalcemia

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202310551051.2A CN117025747B (en) 2023-05-16 2023-05-16 Application of reagent for detecting SLC34A3 gene variation in sample in preparation of product for screening low-blood-phosphorus rickets/osteomalacia patients

Related Child Applications (1)

Application Number Title Priority Date Filing Date
CN202410590834.6A Division CN118421783A (en) 2023-05-16 2023-05-16 Product for screening patients with low-phosphorus rickets and hypercalcemia

Publications (2)

Publication Number Publication Date
CN117025747A true CN117025747A (en) 2023-11-10
CN117025747B CN117025747B (en) 2024-04-09

Family

ID=88630550

Family Applications (2)

Application Number Title Priority Date Filing Date
CN202410590834.6A Pending CN118421783A (en) 2023-05-16 2023-05-16 Product for screening patients with low-phosphorus rickets and hypercalcemia
CN202310551051.2A Active CN117025747B (en) 2023-05-16 2023-05-16 Application of reagent for detecting SLC34A3 gene variation in sample in preparation of product for screening low-blood-phosphorus rickets/osteomalacia patients

Family Applications Before (1)

Application Number Title Priority Date Filing Date
CN202410590834.6A Pending CN118421783A (en) 2023-05-16 2023-05-16 Product for screening patients with low-phosphorus rickets and hypercalcemia

Country Status (1)

Country Link
CN (2) CN118421783A (en)

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050249718A1 (en) * 2004-05-09 2005-11-10 Technion Research & Development Foundation Ltd. Compositions and methods for treating disorders associated with abnormal phosphate metabolism
US20100221707A1 (en) * 2006-08-17 2010-09-02 The Curators Of The University Of Missouri Diagnostic methods for detecting congenital bone defects
CN112813156A (en) * 2021-02-03 2021-05-18 山东第一医科大学附属省立医院(山东省立医院) DNA library for detecting and diagnosing bone development disorder pathogenic gene and application thereof
CN113308527A (en) * 2021-03-09 2021-08-27 上海市第六人民医院 Gene composition, chip and kit for screening refractory hereditary bone diseases
CN113549637A (en) * 2021-07-21 2021-10-26 山东第一医科大学附属省立医院(山东省立医院) Mouse PHEX gene SNP locus and application thereof
CN113549638A (en) * 2021-07-21 2021-10-26 山东第一医科大学附属省立医院(山东省立医院) Method for constructing mouse model of X-linked rickets with low blood phosphorus

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050249718A1 (en) * 2004-05-09 2005-11-10 Technion Research & Development Foundation Ltd. Compositions and methods for treating disorders associated with abnormal phosphate metabolism
US20100221707A1 (en) * 2006-08-17 2010-09-02 The Curators Of The University Of Missouri Diagnostic methods for detecting congenital bone defects
CN112813156A (en) * 2021-02-03 2021-05-18 山东第一医科大学附属省立医院(山东省立医院) DNA library for detecting and diagnosing bone development disorder pathogenic gene and application thereof
CN113308527A (en) * 2021-03-09 2021-08-27 上海市第六人民医院 Gene composition, chip and kit for screening refractory hereditary bone diseases
CN113549637A (en) * 2021-07-21 2021-10-26 山东第一医科大学附属省立医院(山东省立医院) Mouse PHEX gene SNP locus and application thereof
CN113549638A (en) * 2021-07-21 2021-10-26 山东第一医科大学附属省立医院(山东省立医院) Method for constructing mouse model of X-linked rickets with low blood phosphorus

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
PRINCE JACOB ET AL.: "Exome Sequencing in Monogenic Forms of Rickets", INDIAN JOURNAL OF PEDIATRICS, vol. 90, no. 12, 24 January 2023 (2023-01-24), pages 1182 - 1190 *
刘阳洋 等: "低血磷性佝偻病的诊治进展", 中国儿童保健杂志, vol. 23, no. 5, 10 May 2015 (2015-05-10), pages 486 - 488 *

Also Published As

Publication number Publication date
CN117025747B (en) 2024-04-09
CN118421783A (en) 2024-08-02

Similar Documents

Publication Publication Date Title
EP2257644B1 (en) Genetic alterations associated with autism and the autistic phenotype and methods of use thereof for the diagnosis of autism
Stephens et al. A keratin 14 mutational hot spot for epidermolysis bullosa simplex, Dowling-Meara: implications for diagnosis
US20210024999A1 (en) Method of identifying risk for autism
US20140127685A1 (en) Compositions and methods for diagnosing autism
Chouery et al. A novel locus for autosomal recessive primary torsion dystonia (DYT17) maps to 20p11. 22–q13. 12
Wong et al. Association between schizophrenia and the syntaxin 1A gene
US7655403B2 (en) CHD7 gene polymorphisms are associated with susceptibility to idiopathic scoliosis
Steele et al. De novo mutations causing Shwachman-Diamond syndrome and a founder mutation in SBDS in the French Canadian population
CN117025747B (en) Application of reagent for detecting SLC34A3 gene variation in sample in preparation of product for screening low-blood-phosphorus rickets/osteomalacia patients
EP1749891A1 (en) Method and device for the in vitro detection of polycystic ovarian syndrome (pcos) and pathologies involving cardiovascular risk
Vuillaumier-Barrot et al. Intragenic rearrangements in LARGE and POMGNT1 genes in severe dystroglycanopathies
TW201311908A (en) Method and kit for diagnosis of canine glaucoma
Dalal et al. Genetics of bleeding disorders
KR20210042614A (en) A SNP marker composition and a method for diagnosis or prediction of osteochondrodyplasia in cats
CN114622013B (en) Juvenile idiopathic scoliosis detection product
US20230279493A1 (en) Testing assay for screening and diagnosis of usher, pendred, jervell, and lange-nielsen syndromes
Abolghasemi et al. MMP9 (RS20544) and ADCY2 (RS58502974) as susceptibility factors for schizophrenia in Iranian population
Yoo et al. Prenatal molecular evaluation of six fetuses in four unrelated Korean families with ornithine transcarbamylase deficiency
Ahmed Molecular Diagnosis of Genetic Haemoglobin Disorders
CN116287191A (en) Primer, method and kit for detecting HENMT1 gene mutation
CN113308534A (en) VDAC1 mutant gene, primer, kit and method for detecting same and application thereof
CN116769902A (en) Nucleic acid product for detecting DNMT3A gene mutation and application thereof
CN117004708A (en) Screening method, detection composition and detection kit for hereditary Imerslund-Grasbeck syndrome
CN113265409A (en) TIMM21 mutant gene, primer, kit and method for detecting same and application thereof
WO2008093343A2 (en) Schizophrenia associated genes and markers

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant
CB03 Change of inventor or designer information

Inventor after: Sun Yujing

Inventor after: Liu Yuan

Inventor after: Jiang Ling

Inventor after: Zhang Xiaoli

Inventor before: Chen Li

Inventor before: Hou Xinguo

Inventor before: Jiang Ling

Inventor before: Sun Yujing

Inventor before: Liu Yuan

CB03 Change of inventor or designer information