SLC16A2 gene mutant and application thereof in AHDS disease risk analysis

Abstract

The invention belongs to the technical field of gene diagnosis, and discloses an SLC16A2 gene mutant and application of the SLC16A2 gene mutant in AHDS disease risk analysis. The specifically disclosed SLC16A2 gene mutant is any one of nucleic acid, a target fragment exists in the nucleic acid, and compared with a wild type SLC16A2 gene with the sequence of SEQ ID NO.1, the target fragment has c.963964delinsAA mutation, and the target fragment has c.963964delinsAA mutation. Compared with a protein coded by a wild type SLC16A2 gene with a sequence of SEQ ID NO.2, the polypeptide has p.Y321 * mutation. Meanwhile, the invention discloses application of a reagent for detecting the SLC16A2 gene mutant in preparation of a product for screening AHDS. According to the invention, the pathogenic gene spectrum of the AHDS is expanded, the cognition of a clinician on the disease is improved, experience is accumulated for clinical screening and diagnosis of the disease, and a basis is provided for prenatal diagnosis.

Description

[0001] The present disclosure is a divisional application of the Chinese patent application No. 2025103622866, entitled SLC16A2, PHEX gene mutant and application thereof, and filed on March 26, 2025. The present disclosure contains the full text of CN119932031A by reference. TECHNICAL FIELD

[0002] The present application belongs to the technical field of genetic diagnosis, and particularly relates to a SLC16A2 gene mutant and application thereof in AHDS risk analysis. BACKGROUND

[0003] Allan-Herndon-Dudley syndrome (AHDS, OMIM 300523) is a rare X-linked recessive genetic disease. It is caused by mutation of the thyroid hormone (TH) transporter SLC16A2 gene (OMIM 300095, formerly known as MCT8 gene), which leads to inactivation of the encoded monocarboxylate transporter 8 (MCT 8) and inability to mediate TH into target cells, resulting in a rare endocrine disease. AHDS can cause damage to the nervous system and thyroid function. Its main clinical manifestations are low muscle tone, feeding difficulties and mild to severe developmental delay / intellectual disability in male children since infancy, followed by pyramidal signs, myelin development delay, extrapyramidal manifestations (dystonia, choreoathetosis, paroxysmal movement disorders), motor function decline, thinness, long face, drug-resistant seizures, easy choking and respiratory tract infections, but growth retardation is not easily observed in early childhood. Other abnormal symptoms may include hypothyroidism and characteristic thyroid test abnormalities, i.e., elevated triiodothyronine (FT3), decreased free tetraiodothyronine (FT4), and normal thyroid-stimulating hormone (TSH). The disease mainly affects males, with a prevalence of 1 / 70000. Most heterozygous females may have no symptoms or only mild thyroid function (thyroid function) abnormalities, but no symptoms of nervous system damage.

[0004] The SLC16A2 gene is located in the q13.2 region of the X chromosome, and the total length of the genome is about 112.46 kilobases. It is composed of 6 exons and 5 introns, and the total length of the exons is about 4161 base pairs, which is responsible for encoding the MCT8 protein containing 539 amino acids. The MCT8 protein is composed of 12 transmembrane domains, and it is a specific transporter for thyroid hormone T3 into cells, and it has the function of bidirectional transport of T3 and T4. The protein is mainly distributed on the membranes of endothelial cells, brain neurons and oligodendrocytes that constitute the human blood-brain barrier. The MCT8 protein on the endothelial cells is responsible for transporting thyroid hormones in the circulatory system to the central nervous system, and then the MCT8 protein on the membranes of brain neurons and oligodendrocytes transports these hormones into the corresponding cells. The active thyroid hormone (T3) or the active thyroid hormone converted from T4 enters the cell and binds to the nuclear receptor, and the nuclear receptor binds to the thyroid hormone response element in the promoter region of the thyroid hormone target gene, thereby guiding the transcription and translation process of the cell, and affecting cell proliferation, neurogenesis, cell migration, cell differentiation, myelination, synapse formation and apoptosis. It is generally believed that the occurrence of Allan-Herndon-Dudley (AHDS) is mainly due to the variation of SLC16A2 gene leading to the partial or complete deletion of MCT8 protein, causing the obstacle of MCT8 protein binding to cell membrane and thyroid hormone, leading to the reduction or complete disappearance of thyroid hormone in brain neurons and oligodendrocytes, affecting the transcription and translation process of brain neurons and oligodendrocytes, and then causing the formation, migration, differentiation and maturation of neural cells and oligodendrocytes to be disturbed, triggering a series of nervous system symptoms, such as mental retardation, motor and language development delay, convulsions, microcephaly, hearing abnormalities, muscle tension disorders, ataxia, etc. In addition, the defect of MCT8 protein can also lead to the increased expression of deiodinase D1 and D2 in thyroid tissue, causing the decrease of T4 level and the increase of T3 level in peripheral blood, leading to the hypermetabolism of peripheral tissues, the enhancement of gluconeogenesis and glycolysis, the acceleration of fat synthesis and degradation, and the acceleration of protein decomposition in peripheral tissues and skeletal muscle. Patients may have facial features similar to myopathy, such as long face, emaciation, etc. Friesema EC et al. (DOI:10.1016 / S0140-6736(04)17226-7) have previously confirmed that AHDS is caused by variation of the gene SLC16A2 encoding MCT8 protein.

[0005] At present, the relationship between the above diseases and specific genes needs to be further studied, and the screening method for the above diseases needs to be improved to realize the early screening of potential susceptible diseases and provide new ideas for subsequent treatment and early intervention. SUMMARY

[0006] In order to solve the above problems, the application provides a SLC16A2 gene mutant and application thereof in AHDS risk analysis, mainly to provide a new diagnosis method for Allan-Herndon-Dudley syndrome, and further deepen the understanding of the SLC16A2 gene in the field.

[0007] In order to solve the above problems, the application adopts the following technical scheme:

[0008] The SLC16A2 gene mutant and application thereof provide a new pathogenic mutant, and propose a scheme for the application of the mutant in clinic.

[0009] As to the SLC16A2 gene mutant, the SLC16A2 gene mutant is any of the following: a nucleic acid, the nucleic acid has a target fragment, and the target fragment has a c.963_964delinsAA mutation, i.e. deletion of bases at positions 963 and 964 and insertion of two bases A, compared with a wild-type SLC16A2 gene with a sequence of SEQ ID NO. 1; a polypeptide, the polypeptide has a p.Y321* mutation, compared with a protein encoded by a wild-type SLC16A2 gene with a sequence of SEQ ID NO. 2.

[0010] As to the influence of the SLC16A2 gene mutant on cells, tissues or organs of the body, the protein encoded by the SLC16A2 gene refers to MCT8 protein. If the MCT8 protein is combined with the cell membrane and thyroid hormone, the distribution of thyroid hormone in brain neurons and oligodendrocytes will be reduced or completely disappeared. This will affect the transcription and translation processes of brain neurons and oligodendrocytes, and further interfere with the formation, migration, differentiation and maturation of neural cells and oligodendrocytes, and finally trigger a series of nervous system symptoms, including mental retardation, motor and language development delay, convulsions, microcephaly, hearing abnormalities, muscle tone disorders, ataxia, etc. In addition, the functional defect of MCT8 protein can also cause the expression of deiodinase D1 and D2 in thyroid tissue to increase, resulting in a decrease in T4 level and an increase in T3 level in peripheral blood. The increase in T3 level will make the peripheral tissues in a hypermetabolic state, the processes of gluconeogenesis and glycolysis are enhanced, fat synthesis and decomposition are accelerated, and protein decomposition in peripheral tissues and skeletal muscle is accelerated, which can cause patients to have facial features similar to myopathy, such as long face, emaciation, etc.

[0011] In terms of the use of the reagent for detecting the aforementioned SLC16A2 gene mutant in the preparation of a product for screening Allan-Herndon-Dudley syndrome. By analyzing whether a specific SLC16A2 gene mutant exists in a sample (such as peripheral blood), it can be determined whether the sample is derived from an Allan-Herndon-Dudley syndrome patient or a potential risk group (here, the fetus is regarded as a patient or a potential risk group regardless of its developmental stage), because individuals carrying the specific SLC16A2 gene mutant must have Allan-Herndon-Dudley syndrome or belong to the risk group. Specifically, the reagent for detecting the SLC16A2 gene mutant should at least include at least one of a probe or a primer specific to the SLC16A2 gene mutant, and techniques such as Sanger sequencing, NGS sequencing, etc. (all within the scope of the present application) can also be used. Further, the primer should at least include a pair of primers: F-GTGGTGTCTGCTGGGAGTAG and R-GCAGGTTGAGGGTAGCTTCT; and the probe should at least be AACGCACTTACCGCATCTGGG. In order to more clearly explain the application and principle of SLC16A2 c.963_964delinsAA mutation in the diagnosis of Allan-Herndon-Dudley syndrome, it is necessary to explain the role of SLC16A2 gene mutant in the screening of Allan-Herndon-Dudley syndrome patients or potential risk groups: since the SLC16A2 gene is located on the X chromosome, when the SLC16A2 gene on the X chromosome is mutated and there is no normal allele to cover up, it will cause disease symptoms; for men, since they only carry one X chromosome, the SLC16A2 c.963_964delinsAA mutation is a hemizygous mutation, if the only copy has a loss-of-function mutation (such as a nonsense mutation, a frameshift mutation, including deletion or insertion of bases), due to the combined effects of gene dosage deficiency and loss of function, the total activity of the gene will decrease below the critical threshold, leading to cell or organ dysfunction, and thus inducing disease, and the loss-of-function mutation of the only copy directly leads to the occurrence of disease; for women, since they carry two X chromosomes, if it is an X-linked recessive genetic disease (such as Allan-Herndon-Dudley syndrome), when one chromosome is normal, they will become an asymptomatic carrier, but if they give birth to a son, the son has a high risk of disease (at least a 50% probability of disease). Further, Allan-Herndon-Dudley syndrome is an X-linked recessive genetic disease.Since males have only one X chromosome, when they carry the mutation, due to the lack of compensation by the normal allele, the encoded MCT8 protein is dysfunctional, which in turn triggers the symptoms of Allan-Herndon-Dudley syndrome in males. Since females carry two X chromosomes, when they carry the aforementioned mutation, if the mutation only exists on one X chromosome, due to the compensation function of the normal allele on the other X chromosome, the SLC16A2 gene can normally encode the MCT8 protein, maintain the function of the MCT8 protein, and therefore will not have symptoms of the disease; if both X chromosomes of the female carry the aforementioned mutation, the MCT8 protein cannot be normally encoded, and the symptoms of the disease will appear. In addition, by detecting whether the SLC16A2 gene in the two X chromosomes of the female carries the aforementioned mutation through Sanger sequencing, NGS sequencing and other technologies, it can be determined whether it is a carrier or a patient.

[0012] In terms of application for screening Allan-Herndon-Dudley syndrome, the screening of Allan-Herndon-Dudley syndrome not only involves the identification of patients and high-risk populations, but also includes in-depth examination of specific target groups as a basis for auxiliary diagnosis. In addition, in clinical practice, when used for prenatal diagnosis, combined with genetic counseling, genetic testing and imaging examination, high-risk fetuses can be diagnosed, aiming to reduce the incidence of birth defects and improve the level of eugenics and population quality. If a male carries the SLC16A2 gene c.963_964delinsAA mutation, he can be determined to be a patient; if a female carries this mutation, she belongs to the risk population and may only be a carrier or a potential patient. However, this situation is still of great significance to clinical practice, specifically, if a female is a carrier and gives birth to a male baby, the male baby has at least a 50% chance of being ill; if a female is a patient of Allan-Herndon-Dudley syndrome, her male baby will inevitably suffer from this syndrome.

[0013] In this disclosure, the pathogenic gene spectrum of Allan-Herndon-Dudley syndrome is expanded, which not only deepens the understanding and recognition of clinicians for this disease, but also provides valuable experience for the screening and diagnosis of such diseases in clinical practice. In addition, this research also provides a scientific basis for prenatal diagnosis, which is helpful for early identification and intervention, thereby improving the prognosis of patients. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 Family tree of Family 1;

[0015] Figure 2Imaging results of proband 1 of family 1; wherein (A) myelination is obviously delayed, (B) perivascular space is obvious, (C) corpus callosum is slightly thin, (D) normal infant MR corpus callosum;

[0016] Figure 3 Second-generation sequencing graph of proband 1 of family 1;

[0017] Figure 4 First-generation sequencing graph of members of family 1;

[0018] Figure 5 Pedigree of family 2;

[0019] Figure 6 Imaging results of proband 2 of family 2; wherein (A) (B) distal metaphyseal expansion of femur and tibia, curved diaphysis, (C) left distal ulnar and radial metaphyseal expansion, edge brush-like change, (D) left proximal humeral metaphyseal expansion, reduced bone density;

[0020] Figure 7 Second-generation sequencing graph of proband 2 of family 2;

[0021] Figure 8 First-generation sequencing graph of members of family 2. DETAILED DESCRIPTION

[0022] The above content of the present application will be further described in detail below by combining specific research examples. However, this should not be understood as the scope of the above subject matter of the present application being limited to the following examples.

[0023] I. Research on pathogenic genes and mutation sites

[0024] 1. Sample collection object: The proband and his family members of the present research all signed the informed consent form.

[0025] Family 1: Collect the Allan-Herndon-Dudley syndrome family, □ represents a normal male, ○ represents a normal female, ■ represents a male patient, ● represents a female patient, and ☉ represents an XR trait female carrier (such as Figure 1 ). Proland 1: growth and development retardation, unstable muscle tension in limbs; head MRI shows that the bilateral cerebral hemispheres are basically symmetrical, the gray and white matter contrast is blurred, the central pre-posterior gyrus T2WI low signal is blurred, DWI does not show abnormal signal; the right basal ganglia perivascular space is obvious, the bilateral lateral ventricles are slightly full, the size and shape of the remaining ventricles and cisterns are normal, and no obvious widening is found; the midline structure is centered, the cerebellum and brainstem are normal, and the corpus callosum is slightly thin (such as Figure 2) and the mother of proband 1 had no related abnormality, considering that the variant was a heterozygous carrier. The elder brother of proband 1 was also diagnosed as Allan-Herndon-Dudley syndrome. In family 1: through family analysis, it was considered that the X chromosome of the second brother of proband 1 was inherited from the healthy X chromosome of his mother, while the X chromosomes of proband 1 and his elder brother were inherited from the abnormal X chromosome of their mother.

[0026] Family 2: X-linked hypophosphatemic rickets family was collected, □ represents normal male, o represents normal female, ■ represents ill male, and ● represents ill female (as shown in Figure 5 ) Proband 2: 84.3 cm (P10) in height and 11.99 kg (P25) in weight at 2 years and 4 months old; O-shaped legs, stumbling when walking, easy to fall down, and shaking left and right when walking; low blood phosphorus (0.81 mmol / L), high urine phosphorus (55.18 mmol / L), and abnormally high alkaline phosphatase (489 U / L); bone DR showed that the shape, size and bone density of each bone of the pelvis were normal, and the bone structure was complete; there was no abnormality in the position of the bilateral femoral head epiphysis, the Shenton line was continuous, and the acetabular shape was acceptable; the distal femur and the proximal and distal tibia metaphysis of both sides were enlarged, the bone shaft was curved, and the bilateral knee joint space was acceptable. Two wrist bones were visible in the left wrist, the distal metaphysis of the left ulna and radius was blurred, the metaphysis of the ulna and radius was enlarged, and the edge was changed to a brush-like change; the proximal metaphysis of the left humerus was slightly enlarged, and the bone density of the humerus was reduced Figure 6 ) The elder brother of proband 2: congenital bone dysplasia, low bone density, multiple joint inflexibility, O-shaped legs (orthopedic surgery has been performed), 145 cm in height (adult), low blood phosphorus (0.76 mmol / L). The mother of proband 2 was 145 cm in height, and had the same clinical phenotype as her son. The maternal grandmother of proband 2 had similar symptoms and was dead.

[0027] 2. Sample collection: 5 ml of venous peripheral blood was taken for examination, and 2 ml of EDTA anticoagulant was added. 2 ml of genomic DNA was extracted by DNABlood Mini Kit (QIAGEN) kit, and the concentration was determined by Qubit( dsDNA HS Assay Kit, Invitrogen), and was stored at -20℃ for standby.

[0028] 3. Whole exome sequencing and result analysis

[0029] 3.1 Whole exome sequencing: First, the genomic DNA was fragmented using a Covaris ultrasonic disruptor, and the broken products were end-repaired, A-tailed, adapter-ligated, and amplified using a library construction kit to complete the pre-library construction. Then, the pre-library was treated with a human whole exome capture kit, and the target region was enriched by probe hybridization capture method to obtain the final library. The library was analyzed for concentration and fragment distribution by Qubit and QIAGEN QIAxcel Advanced automated nucleic acid analysis system, and the qualified library was quantified using a quantitative kit. Finally, the sequencing reaction was completed on a Huada MGISEQ-T7 gene sequencer.

[0030] The capture probe sequence used:

[0031] SLC16A2 c.963_964delinsAA: AACGCACTTACCGCATCTGGG.

[0032] PHEX c.112_113insA: ACGATCCTCTTTCTAGTGAGT.

[0033] 3.2 Data processing: After sequencing, the sequences passing quality control were aligned to the human genome reference sequence using BWA software; the mutation sites in the target sequence were identified using GATK software, and the mutation sites were annotated to the public mutation database using Annovar annotation software. According to the frequency of the mutation site in the normal population, the sequence conservation, the amino acid change caused by the mutation, and the position in the protein structure, the degree of influence of the mutation on protein function was predicted; then, combined with the sample clinical phenotype, the mutation was interpreted for pathogenicity according to the ACMG (The American College of Medical Genetics and Genomics) variation classification standard and guidelines.

[0034] 3.3 Proband whole exome sequencing results:

[0035] Proband 1: The sample analysis found that there was a semi-variant in exon 3 of SLC16A2 gene: c.963_964delinsAA (NM_006517.5), i.e. deletion of cytosine nucleotides 963 and 964 and insertion of two adenines, which ultimately resulted in the change of amino acid 321 from tyrosine to stop codon (p.Y321*) (PVS1); the variation was not reported in the normal population gene database (allele frequency (%): gnomeAD:.) (PM2_PP); the clinical symptoms of the case were consistent with Allan-Herndon-Dudley syndrome (PP4).

[0036] Proband 2: The sample analysis found a heterozygous variation in exon 1 of the PHEX gene: c.112_113insA (NM_000444.6), which is an insertion of an adenine between thymine nucleotides 112 and 113, resulting in a mutation of the 38th amino acid from phenylalanine to tyrosine and causing an early termination codon (p.F38Yfs*13) (PVS1). This variation has not been reported in the normal population gene database (allele frequency (%): gnomeAD: 0.0000000000) (PM2_PP). The clinical manifestations of this case are consistent with X-linked hypophosphatemic rickets (PP4).

[0037] 3.4 Bioinformatics prediction analysis:

[0038] (1) Harmfulness and pathogenicity analysis

[0039] Proband 1: SLC16A2 c.963_964delinsAA (p.Y321*), according to the ACMG (The American College of Medical Genetics and Genomics) variation classification guidelines, this variation is a pathogenic variation (ACMG: PVS+2PP).

[0040] Proband 2: PHEX c.112_113insA (p.F38Yfs*13), according to the ACMG (The American College of Medical Genetics and Genomics) variation classification guidelines, this variation is a pathogenic variation (ACMG: PVS+2PP).

[0041] (2) Protein function and conservation analysis

[0042] SLC16A2 c.963_964delinsAA (p.Y321*): This mutation is a deletion of two cytosine nucleotides at positions 963 and 964 and an insertion of two adenines, resulting in a change of the 321st amino acid of the encoded protein from tyrosine to a termination codon, affecting the normal translation of the protein, and thus causing the SLC16A2 gene-encoded MCT8 protein to be abnormal and leading to the production of Allan-Herndon-Dudley syndrome. The MCT8 protein encoded by the SLC16A2 gene has high conservation, and abnormal expression of the MCT8 protein encoded by the SLC16A2 gene will lead to a deficiency of the MCT8 protein, inducing Allan-Herndon-Dudley syndrome.

[0043] PHEX c.112_113insA (p.F38Yfs*13): This mutation inserts an adenine between the two thymine nucleotides at positions 112 and 113, resulting in a mutation of the 38th amino acid of the encoded protein from phenylalanine to tyrosine, affecting the normal translation of the protein, and thus making the PHEX protein encoded by the PHEX gene abnormal and leading to the occurrence of X-linked hypophosphatemia resistant to vitamin D rickets; the PHEX protein encoded by the PHEX gene has high conservation, and if the PHEX gene encodes an abnormal PHEX protein, it will lead to a deficiency of the PHEX protein, and thus the PHEX protein cannot effectively protect MEPE from being hydrolyzed by hydrolytic enzymes, inducing X-linked hypophosphatemia resistant to vitamin D rickets.

[0044] 4. Sanger sequencing verification: Sanger sequencing was used to verify the site of the mutation found. 20 ng of DNA (such as peripheral blood genomic DNA) from family members was taken, primers were used, and PCR reactions were performed according to the TaKaRa LA PCR TM Kit Ver.2.1 (TaKaRa) operation procedure;

[0045] Primer sequence 1: F-GTGGTGTCTGCTGGGAGTAG, R-GCAGGTTGAGGGTAGCTTCT.

[0046] Primer sequence 2: F-CAGCCACCAAACCACGAAA, R-ATGAACGCAGGCAAACAGC.

[0047] Sequencing analysis was performed on the ABI3730XL (Applied Biosystems) platform, and the presence of each mutation in the gene was finally confirmed (only the family members involved in the verification and detection were analyzed): (1) In family 1, the mother and older brother of proband 1 also had the SLC16A2 c.963_964delinsAA mutation, but the mother of proband 1 was a heterozygous mutation with only one X chromosome having the SLC16A2 c.963_964delinsAA mutation, which was consistent with the previous suspected carrier, and the father and second brother did not have the mutation. (2) In family 2, the brother and mother of proband 2 also had the PHEX c.112_113insA mutation, the father did not have the mutation, and the mutation of the proband was heterozygous (consistent with the father not having the mutation), the mother was also heterozygous, and the proband's grandmother was deceased and could not be detected.

[0048] 5. Comprehensive conclusion

[0049] (1) Based on the conclusion of the analysis of the mutation of the proband 1: the hemizygous mutation of c.963_964delinsAA in the SLC16A2 gene detected by whole exome sequencing technology is the pathogenic cause of this Allan-Herndon-Dudley syndrome family; the research results expand the gene spectrum of Allan-Herndon-Dudley syndrome, provide experience for the screening and diagnosis of clinical Allan-Herndon-Dudley syndrome, and also provide a basis for prenatal diagnosis; therefore, targeted genetic research on Allan-Herndon-Dudley syndrome provides research direction and new theoretical basis for early diagnosis and effective treatment of Allan-Herndon-Dudley syndrome, and also provides new molecular targets for the development of specific drugs for the treatment of Allan-Herndon-Dudley syndrome in practice.

[0050] (2) Based on the conclusion of the analysis of the mutation of the proband 2: the hemizygous / heterozygous mutation of c.112_113insA in the PHEX gene detected by whole exome sequencing technology is the pathogenic cause of this X-linked hypophosphatemic rickets family; the research results expand the gene spectrum of X-linked hypophosphatemic rickets, provide experience for the screening and diagnosis of clinical X-linked hypophosphatemic rickets, and also provide a basis for prenatal diagnosis; therefore, targeted genetic research on X-linked hypophosphatemic rickets provides research direction and new theoretical basis for early diagnosis and effective treatment of X-linked hypophosphatemic rickets, and also provides new molecular targets for the development of specific drugs for the treatment of X-linked hypophosphatemic rickets in practice. Therefore, targeted genetic research on the above diseases can provide potential clinical significance for genetic counseling and individualized prevention and treatment of patients with the above diseases, provide research direction and new theoretical basis for early diagnosis and effective treatment of the above diseases, and also provide new molecular targets for the development of specific drugs for the treatment of the above diseases in practice.

[0051] II. Introduction of mutants and application cases

[0052] As an example of the application of such products in clinical practice, a brief introduction is provided. Such products can generally be used to analyze the obtained samples to be tested, determine whether the sample is from a person with a certain disease or a high-risk population by analyzing whether the sample has a specific mutation, and provide a reference for clinical diagnosis and treatment, especially in pre-pregnancy screening, which provides better guidance for screening potential severe diseases in fetuses during pregnancy to provide accurate recommendations.

[0053] In clinical application, the general steps can be: S1, extracting nucleic acid sample from biological sample (the sample in this step can also be directly provided by the detection party): the type of biological sample is not particularly limited, as long as the nucleic acid sample reflecting whether the gene in the biological sample has a mutation can be extracted from the biological sample; the biological sample can be at least one selected from human blood, skin, subcutaneous tissue, and preferably peripheral blood. It should be noted that the term "nucleic acid sample" used in this part should be interpreted broadly, which can be any sample that can reflect whether the gene in the biological sample has a mutation, for example, it can be whole genome DNA extracted directly from the biological sample, it can be a part of the whole genome containing the coding sequence of the gene, it can be total RNA extracted from the biological sample, or it can be mRNA extracted from the biological sample. S2, after obtaining the nucleic acid sample, analyzing the nucleic acid sample to determine the nucleic acid sequence (mainly the target region of the mutation) of the obtained nucleic acid sample: the method and device for determining the nucleic acid sequence of the obtained nucleic acid sample are not particularly limited, and the nucleic acid sequence of the nucleic acid sample can be determined by sequencing method. The method and device for sequencing are not particularly limited, and at least one selected from BGISEQ500, MGISEQ-200, MGISEQ-2000, MGISEQ-T7, HISEQ2000, SOLID, 454, ABI3730XL and single molecule sequencing device can be used to sequence the nucleic acid sequence; it should be noted that the term "nucleic acid sequence" used in this part should be interpreted broadly, which can be the complete nucleic acid sequence information obtained after assembling the sequencing data obtained by sequencing the nucleic acid sample, or it can be the sequencing data (reads) obtained by directly using the sequencing data obtained by sequencing the nucleic acid sample as the nucleic acid sequence, as long as these nucleic acid sequences contain the coding sequence of the corresponding gene. S3, after determining the nucleic acid sequence of the nucleic acid sample, comparing the obtained nucleic acid sequence of the nucleic acid sample with the wild type sequence: if the obtained nucleic acid sequence has the specific mutation involved in the present disclosure, it indicates that the biological sample source is susceptible to the corresponding disease.

[0054] Taking the application of SLC16A2 c.963_964delinsAA mutation in the diagnostic analysis of Allan-Herndon-Dudley syndrome as an example (only in terms of the pathogenicity of this mutation, without considering other pathogenic factors), at least two analysis methods can be used:

[0055] Firstly, the Sanger sequencing or NGS can be directly used to analyze the sample to be tested to determine whether the SLC16A2 gene in the X chromosome has the c.963_964delinsAA mutation. If the subject is male, he will be definitely ill if the mutation exists. If the subject is female, she will be a carrier if the SLC16A2 gene in one of her X chromosomes has the c.963_964delinsAA mutation, and she will be definitely ill if the SLC16A2 gene in both of her X chromosomes has the c.963_964delinsAA mutation. This method is faster and more convenient, and has less dependence on family investigation.

[0056] Secondly, for the male to be tested, he will be definitely ill if he has the SLC16A2 c.963_964delinsAA mutation. For the female to be tested, if it is not clear whether the mutation exists in both of her X chromosomes, family analysis is still needed. In actual application, an analysis model can be constructed by the following logic, and the risk of the subject to be tested can be obtained after inputting the corresponding variables, and the risk of the subject to be tested to be ill or to have offspring can be known according to the relevant circumstances. The analysis model at least includes the following main situations:

[0057] Situation 1: If the parents of the female to be tested are both patients with Allan-Herndon-Dudley syndrome, the two X chromosomes obtained from the parents should both have the SLC16A2 gene c.963_964delinsAA mutation, and the female to be tested will be definitely ill;

[0058] Situation 2: If the mother of the female to be tested is a patient with Allan-Herndon-Dudley syndrome and the father is healthy, the female to be tested obtains a healthy X chromosome from the father and an abnormal X chromosome from the mother, and the female to be tested is a carrier;

[0059] Situation 3: If the father of the female to be tested is a patient with Allan-Herndon-Dudley syndrome and the mother is healthy (and is not a carrier), the female to be tested obtains an abnormal X chromosome from the father and a healthy X chromosome from the mother, and the female to be tested is a carrier;

[0060] Situation 4: If the father of the female to be tested is a patient with Allan-Herndon-Dudley syndrome and the mother is healthy (is a carrier), the female to be tested obtains an abnormal X chromosome from the father and an X chromosome from the mother, and the female to be tested is a carrier. If the female to be tested obtains a healthy X chromosome from the mother, the female to be tested is a carrier. If the female to be tested obtains an abnormal X chromosome from the mother, the female to be tested is definitely ill.

[0061] The method and device for aligning the detected nucleic acid sequence with the wild type are not particularly limited, and any conventional software can be used. If not particularly specified, the technical means used in the examples are conventional means familiar to those skilled in the art, and the reagents and products used are all commercially available. Various processes and methods not described in detail are conventional methods known in the art, and the source of the reagents used, the trade name, and if necessary, the composition thereof are indicated at the first occurrence, and the same reagents used thereafter are the same as those indicated at the first occurrence unless otherwise specified.

[0062] The application introduces the PHEX gene mutant and its application, provides a new pathogenic mutant, and proposes a scheme for the application of the mutant in clinic.

[0063] As to the PHEX gene mutant, the PHEX gene mutant is any one of the following: a nucleic acid having a target fragment, and the target fragment has a c.112_113insA mutation, i.e., one base A is inserted between the 112th and 113th bases, compared with the wild-type PHEX gene with the sequence of SEQ ID NO. 3; a polypeptide having a p.F38Yfs*13 mutation, compared with the protein encoded by the wild-type PHEX gene with the sequence of SEQ ID NO. 4.

[0064] As to the effect of the protein encoded by the PHEX gene on the body cells, tissues or organs, the protein encoded by the PHEX gene is PHEX protein, and the loss of function of the PHEX protein caused by the variation is the pathogenesis of XLH. On the one hand, the PHEX protein loses its function and cannot protect MEPE from being hydrolyzed by hydrolytic enzymes, resulting in small peptides produced by the hydrolysis of MEPE inhibiting the absorption of phosphate by the kidney and affecting bone mineralization, and thus failing to maintain normal phosphorus metabolism; on the other hand, the inactivation of the PHEX protein increases the circulating level of FGF23, and more circulating FGF23 acts on the kidney, affecting the reabsorption of phosphate by the renal tubule, leading to increased urinary phosphate excretion, decreased blood phosphate level and abnormal bone mineralization.

[0065] The reagent for detecting the PHEX gene mutant is at least one of a probe and a primer specific to the PHEX gene mutant, and can also be other means such as Sanger sequencing. The primer is at least a primer pair: F-CAGCCACCAAACCACGAAA, R-ATGAACGCAGGCAAACAGC; and the probe is at least ACGATCCTCTTTCTAGTGAGT. In order to more clearly introduce the reason and principle why the PHEX c.112_113insA mutation can be used for the diagnosis and analysis of X-linked hypophosphatemic rickets, the PHEX gene mutant can be used for the screening of X-linked hypophosphatemic rickets patients or risk groups: the PHEX c.112_113insA mutation is a semi-heterozygous mutation. The pathogenic reason of the semi-heterozygous mutation mainly comes from the superimposed effect of gene dosage deficiency and functional loss. In this case, if the only copy has a loss-of-function mutation (such as a nonsense mutation, a frameshift mutation (including deletion or insertion of bases)), the total activity of the gene will be lower than the critical threshold, causing cell or organ dysfunction and further inducing diseases; the pathogenic reason of the heterozygous mutation is that in addition to the gene dosage deficiency caused by the aforementioned mutation, it can also be due to the deletion or insertion of a base at a certain site of the DNA molecule (this mutant is a new insertion of an adenine), causing a change in the reading frame, causing a series of changes in the downstream codons, making the gene originally encoding a certain peptide chain become a sequence encoding another completely different peptide chain, resulting in abnormal protein function and activity and further causing cell or organ dysfunction and further inducing diseases.

[0066] For the application of screening X-linked hypophosphatemia rickets, the screening of X-linked hypophosphatemia rickets not only involves the identification of high-risk population, but also includes in-depth examination of specific objects as an auxiliary means in the diagnosis process. In addition, in clinical practice, the prenatal diagnosis of the disease combines genetic counseling, genetic testing and imaging examination, aiming to make a clear diagnosis of high-risk fetuses, so as to reduce the occurrence of birth defects and improve the level of eugenics and population quality. Since X-linked hypophosphatemia rickets is an X-linked dominant genetic disease, once the PHEX c.112_113insA mutation occurs, whether male or female, as long as the PHEX gene on any X chromosome carried by the individual is abnormal, the disease will occur. Therefore, if the PHEX c.112_113insA mutation is detected in the sample, it can be determined that it comes from a patient with X-linked hypophosphatemia rickets.

[0067] Those skilled in the art can clearly make various modifications to the above embodiments without departing from the overall spirit and concept of the present application. All fall within the scope of the present application. The protection scheme of the present application is subject to the claims attached to the present application.

Claims

1. A mutant of SLC16A2 gene, characterized in that, The SLC16A2 gene mutant is any one of the following: a nucleic acid having a target fragment, and the target fragment has a c.963_964delinsAA mutation compared with the wild-type SLC16A2 gene with the sequence of SEQ ID NO. 1; a polypeptide having a p.Y321* mutation compared with the protein encoded by the wild-type SLC16A2 gene with the sequence of SEQ ID NO.

2.

2. The SLC16A2 gene mutant according to claim 1; wherein, The protein encoded by the SLC16A2 gene is MCT8 protein.

3. Use of a reagent for detecting the SLC16A2 gene mutant in claim 1 in the preparation of a product for screening Allan-Herndon-Dudley syndrome.

4. Use according to claim 3; wherein, The product for screening Allan-Herndon-Dudley syndrome is a product for screening patients or high-risk population of Allan-Herndon-Dudley syndrome.

5. The use according to claim 3; wherein, The reagent for detecting the SLC16A2 gene mutant in claim 1 is at least one of the following: a probe, a primer, or a combination thereof, which is specific to the SLC16A2 gene mutant.

6. Use according to claim 5; wherein, The primer is at least a primer pair: F-GTGGTGTCTGCTGGGAGTAG, R- GCAGGTTGAGGGTAGCTTCT.

7. The use according to claim 5; wherein, The probe is at least: AACGCACTTACCGCATCTGGG.

Images