Phex gene mutants and uses thereof

Abstract

The application belongs to the technical field of gene diagnosis, and specifically discloses a PHEX gene mutant and application thereof. The application mainly relates to the PHEX gene mutant and application thereof, and specifically relates to application of a PHEX c.112_113insA mutation in screening of a product of X-linked hypophosphatemic rickets resistant to vitamin D. The disclosure widens the pathogenic gene spectrum of X-linked hypophosphatemic rickets resistant to vitamin D, strengthens the understanding of the disease by clinical doctors, provides experience for screening and diagnosis of the disease in the clinic, and also provides a basis for prenatal diagnosis.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of genetic diagnosis, and particularly relates to a PHEX gene mutant and application thereof. BACKGROUND

[0002] Allan-Herndon-Dudley syndrome (AHDS, OMIM 300523) is a rare X-linked recessive genetic disease, which is caused by mutation of thyroid hormone (TH) transporter SLC16A2 gene (OMIM 300095, formerly known as MCT8 gene), resulting in inactivation of the encoded monocarboxylate transporter 8 (MCT 8) and inability to mediate TH into target cells, and is an endocrine rare disease. AHDS can cause damage to the nervous system and thyroid function, and 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 and drug-resistant seizures, easy to choke and develop respiratory tract infections, but growth retardation is not easy to observe in early childhood. Other abnormal symptoms can have hypothyroidism and characteristic thyroid test abnormalities, i.e. increased triiodothyronine (FT3), decreased free tetraiodothyronine (FT4) and no abnormality of thyroid stimulating hormone (TSH). The disease often involves males, and the male prevalence is 1 / 70000. Most heterozygous females can have no symptoms or only show mild thyroid function (thyroid function) abnormalities, but have no symptoms of nervous system damage.

[0003] SLC16A2 gene is located on chromosome Xq13.2, the full length of genome is about 112.46 kb, containing 6 exons and 5 introns, the length of exon is about 4161 bp, encoding 539 amino acid MCT8 protein. MCT8 protein is composed of 12 transmembrane domains, is a specific transporter to mediate thyroid hormone T3 into the cell, and has the function of bidirectional transport of T3 and T4. MCT8 protein is mainly distributed on the membrane of endothelial cells, brain neurons and oligodendrocytes in human blood brain barrier, and the MCT8 protein on endothelial cells can transport thyroid hormone in peripheral circulation to central nervous system, and then transported to the corresponding intracellular by MCT8 protein on the membrane of brain neurons and oligodendrocytes, and the active thyroid hormone (T3) or the active thyroid hormone converted from T4 in the cell binds to the nuclear receptor, the nuclear receptor binds to the thyroid hormone response element in the promoter region of the thyroid hormone target gene, guiding the transcription and translation of the cell, and affecting cell proliferation, neurogenesis, cell migration, cell differentiation, myelination, synapse formation and apoptosis. It is currently believed that the occurrence of AHDS is mainly caused by the partial deficiency or complete deletion of MCT8 protein caused by SLC16A2 gene variation, which causes the combination disorder of MCT8 protein and cell membrane and thyroid hormone, leading to the decrease or complete deletion of thyroid hormone in the brain neurons and oligodendrocytes, affecting the transcription and translation of brain neurons and oligodendrocytes, further leading to the formation, migration, differentiation and maturation disorder of neural cells and oligodendrocytes, and thus a series of nervous system symptoms such as mental retardation, motor and language development retardation, convulsions, microcephaly, hearing abnormalities, muscle tension disorders, ataxia, etc. In addition, the defect of MCT8 protein can also lead to the increased expression of thyroid deiodinase D1 and D2 in thyroid tissue, resulting in the decrease of T4 and the increase of T3 in peripheral blood, and the latter causes the peripheral tissues to show a state of increased metabolism, and the enhancement of gluconeogenesis and glycolysis, the enhancement of fat synthesis and degradation, and the accelerated protein decomposition of peripheral tissues and skeletal muscle, and the patients can have myopathic facial features 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 SLC16A2 gene encoding MCT8 protein.

[0004] X-linked hypophosphatemic rickets (XLH, OMIM 307800) is a rare X-linked dominant disease with an incidence of about 1 / 20000, and female patients are more common. The pathogenic gene is phosphate regulating gene with homologies to endopeptidases on the X-chromosome (PHEX, OMIM 300550). The clinical manifestations of XLH include: children generally develop the disease at about 1 year old, and the main manifestations are growth retardation, short stature, bone pain, walking weakness, double lower limb flexion deformity, osteoporosis, multiple fractures, and hypoplastic dental enamel; adults mainly have cartilage disease and bone and joint deformity. Laboratory examination can find that the patient's blood phosphorus is significantly reduced, alkaline phosphatase is increased, blood calcium is normal, parathyroid hormone is normal or increased, and blood 1, 25-hydroxyvitamin D3 is normal or slightly decreased.

[0005] The PHEX gene, located on chromosome Xp22.1, is 2861 bp in length, consists of 22 exons, and encodes a 749-amino acid protein. It shares homology with a group of endopeptidase genes. The PHEX protein, composed of 749 amino acids, belongs to the zinc-binding endopeptidase family. Its encoded amino acid sequence is similar to that of neutral endopeptidases. It is a transmembrane protein comprising a short cytoplasmic region of 20 amino acids, a transmembrane region of 25 amino acids, and a zinc-binding extracellular region of 704 amino acids. Loss of PHEX protein function due to mutation is a pathogenesis of XLH. On one hand, the small peptides produced after the hydrolysis of the substrate matrix extracellular phosphoglycoprotein (MEPE) can inhibit renal reabsorption of phosphate and affect bone mineralization. PHEX protein protects MEPE from hydrolytic enzymes, thus maintaining normal phosphorus metabolism. Loss of PHEX protein function prevents it from protecting MEPE from hydrolysis. On the other hand, PHEX protein inactivation increases the circulating level of serum fibroblast growth factor 23 (FGF23). FGF23 acts on the kidneys, affecting renal tubular reabsorption of phosphorus, leading to increased urinary phosphorus excretion, decreased serum phosphorus levels, and abnormal bone mineralization. Abnormalities in the PHEX gene result in abnormal synthesis of the encoded PHEX protein, which fails to protect MEPE from hydrolysis, thus causing X-linked hypophosphatemia and vitamin D-resistant rickets. Minamizaki et al. (PMID: 32712387) have reported that MEPE protein deficiency causes XLH. In addition, Kang et al. (DOI:10.3969 / j.issn.1674-2591.2024.03.003) have also reported that FGF23 levels were significantly elevated in the biochemical indicators of XLH patients.

[0006] Research on the link between the aforementioned diseases and specific genes needs further investigation, and screening methods for these diseases need to be improved to enable early screening of potential predisposing diseases and provide new ideas for subsequent treatment and early intervention. Summary of the Invention

[0007] To address the aforementioned issues, this invention provides PHEX gene mutants and their applications, primarily to offer a new diagnostic method for X-linked hypophosphatemic rickets resistant to vitamin D, and to further deepen the understanding of the PHEX gene within the field.

[0008] To solve the above problems, the present invention adopts the following technical solution:

[0009] The present application provides a SLC16A2 gene mutant and application thereof, which not only provides a new pathogenic mutant, but also proposes a scheme for the application of the mutant in clinic.

[0010] As to the SLC16A2 gene mutant, the SLC16A2 gene mutant is any one of the following:

[0011] A nucleic acid having 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 the wild-type SLC16A2 gene with the sequence of SEQ ID NO. 1.

[0012] 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.

[0013] As to the influence of the SLC16A2 gene mutant on cells, tissues or organs of the body, the protein encoded by the SLC16A2 gene is specifically the MCT8 protein. If the combination of the MCT8 protein and the cell membrane and thyroid hormone is impaired, it will lead to a decrease or complete absence of thyroid hormone distribution in neurons and oligodendrocytes in the brain, and the transcription and translation of brain neurons and oligodendrocytes are affected, leading to the formation, migration, differentiation and maturation of neural cells and oligodendrocytes. A series of nervous system symptoms such as low intelligence, motor and language development delay, convulsions, microcephaly, hearing abnormalities, muscle tone disorders, ataxia, etc. are caused. In addition, the defect of the MCT8 protein can also lead to the expression of thyroid deiodinase D1 and D2 in the thyroid tissue to increase, resulting in a decrease in peripheral blood T4 and an increase in T3, and the latter causes peripheral tissues to exhibit a state of increased metabolism, enhanced glycolysis and gluconeogenesis, enhanced fat synthesis and degradation, and accelerated protein degradation in peripheral tissues and skeletal muscle, which may cause the patient to have myopathic facial features (such as long face, emaciation, etc.).

[0014] 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 detecting whether the aforementioned SLC16A2 gene mutant exists in a sample (such as peripheral blood), it can be determined whether the sample is from an Allan-Herndon-Dudley syndrome patient or a risk population (both formed and unformed fetuses are temporarily listed as patients or risk populations), because the presence of the aforementioned SLC16A2 gene mutant in the to-be-tested population will inevitably lead to the occurrence of Allan-Herndon-Dudley syndrome or the risk of the population. More specifically, the reagent for detecting the aforementioned SLC16A2 gene mutant is at least one of a probe or primer specific to the SLC16A2 gene mutant, or other means such as Sanger, NGS sequencing (all within the scope of the invention). Further, the primer is at least a primer pair: F-GTGGTGTCTGCTGGGAGTAG, R-GCAGGTTGAGGGTAGCTTCT; and the probe is at least AACGCACTTACCGCATCTGGG. In order to more clearly introduce the reasons and principles why the aforementioned SLC16A2 c.963_964delinsAA mutation can be used for the diagnostic analysis of Allan-Herndon-Dudley syndrome, the SLC16A2 gene mutant can be used for the screening of Allan-Herndon-Dudley syndrome patients or risk populations: Since the SLC16A2 gene is located on the X chromosome, when a mutation occurs in the SLC16A2 gene on one X chromosome and there is no normal allele to mask it, disease symptoms will occur; in terms of men, since men only have one X chromosome, the SLC16A2 c.963_964delinsAA mutation is a hemizygous mutation. In a hemizygous state, 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 additive effects of gene dosage deficiency and loss of function, the total activity of the gene will be below the critical threshold, leading to cell or organ dysfunction and thus inducing disease. The only copy with a loss-of-function mutation directly induces the occurrence of disease; in terms of women, since women have two X chromosomes, if it is an X-linked recessive genetic disease (such as Allan-Herndon-Dudley syndrome), one normal chromosome in women is an asymptomatic carrier, but if she gives birth to a son, it may lead to a high risk of disease in her son (at least a 50% probability of disease). Further, Allan-Herndon-Dudley syndrome is an X-linked recessive genetic disease.Since male has only one X chromosome, when male has the mutation, there is no normal allele to complement, resulting in abnormal function of the encoded MCT8 protein, further leading to symptoms of Allan-Herndon-Dudley syndrome in male. Since female has two X chromosomes, when female has the aforementioned mutation, if the mutation only exists in one X chromosome, the SLC16A2 gene can normally encode MCT8 protein due to the complementation of the normal allele in the other X chromosome, and the function of MCT8 protein is maintained, so that the disease symptoms do not appear; when both X chromosomes of female have the aforementioned mutation, the SLC16A2 gene cannot normally encode MCT8 protein, and the disease symptoms appear. In addition, whether female is a carrier or a patient can be determined by detecting whether the SLC16A2 gene in both X chromosomes of female has the aforementioned mutation through Sanger, NGS sequencing and other means.

[0015] In terms of application for screening Allan-Herndon-Dudley syndrome, screening Allan-Herndon-Dudley syndrome includes screening both the patient population and the risk population, and further checking specific targets as an auxiliary basis for diagnosis. In addition, when used for prenatal diagnosis in clinic, it is combined with genetic testing and imaging examination on the basis of genetic counseling to make a definite diagnosis of high-risk fetuses, thereby reducing the birth defect rate and improving the quality of eugenics and population quality. When the SLC16A2 c.963_964delinsAA mutation is derived from male, the male can be determined as a patient population; when the mutation is derived from female, the female is a risk population, who can be a carrier or a patient, but still has application significance in clinic. Specifically, when female is a carrier, if she gives birth to a male baby, the male baby has at least a 50% probability of being ill, and further, if the female is an Allan-Herndon-Dudley syndrome patient, the male baby she produces is necessarily an Allan-Herndon-Dudley syndrome patient.

[0016] The present application provides a PHEX gene mutant and application thereof , which not only provides a new pathogenic mutant, but also proposes a scheme for the application of the mutant in clinic.

[0017] In terms of PHEX gene mutant, the PHEX gene mutant is any one of the following:

[0018] A nucleic acid having a target fragment, and the target fragment has a c.112_113insA mutation, that is, 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;

[0019] A polypeptide having a p.F38Yfs*13 mutation compared with a protein encoded by a wild-type PHEX gene with a sequence of SEQ ID NO. 4.

[0020] In terms of the effect of the PHEX gene-encoded protein on the body's cells, tissues, organs, etc., the PHEX gene-encoded protein is the PHEX protein, and the loss of function of the PHEX protein caused by the variation is the pathogenesis of XLH. On the one hand, after the loss of function of the PHEX protein, MEPE cannot be protected from being hydrolyzed by a hydrolytic enzyme, resulting in small peptides produced after MEPE is hydrolyzed 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, resulting in increased urinary phosphate excretion, decreased blood phosphate level, and abnormal bone mineralization.

[0021] The reagent for detecting the PHEX gene mutant is used in the preparation of the product for screening X-linked hypophosphatemia resistant to vitamin D rickets. By detecting whether the aforementioned PHEX gene mutant exists in the sample (such as peripheral blood), it can be determined whether the sample is from an X-linked hypophosphatemia patient resistant to vitamin D rickets (both formed and unformed fetuses are temporarily listed as patients). The reason is that the PHEX gene mutation can cause abnormal function of the PHEX protein and inevitably induce X-linked hypophosphatemia resistant to vitamin D rickets. The reagent for detecting the aforementioned PHEX gene mutant is at least one of the probes and primers specific to the PHEX gene mutant, and can also be other means such as Sanger sequencing. The primers are at least the 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 aforementioned PHEX c.112_113insA mutation can be used for the diagnosis and analysis of X-linked hypophosphatemia resistant to vitamin D rickets, the PHEX gene mutant is explained as follows: 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 aforementioned mutation causing gene dosage deficiency, 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, causing abnormal protein function and activity and further causing cell or organ dysfunction and further inducing diseases.

[0022] In terms of the application of screening X-linked hypophosphatemia rickets, screening X-linked hypophosphatemia rickets includes screening the risk population and further checking the specific target as an auxiliary basis for diagnosis. In addition, when used for prenatal diagnosis in the clinic, the diagnosis of high-risk fetuses is made on the basis of genetic counseling combined with genetic testing and imaging examination, so as to reduce the birth defect rate and improve the quality of eugenics and population quality. Since X-linked hypophosphatemia rickets is an X-linked dominant genetic disease, when PHEX c.112_113insA mutation occurs, both males and females have X chromosomes, and as long as the PHEX gene of any X chromosome is abnormal, X-linked hypophosphatemia rickets will occur. Therefore, as long as the sample has PHEX c.112_113insA mutation, it must come from an X-linked hypophosphatemia rickets patient.

[0023] In the present disclosure, the pathogenic gene spectrum of X-linked hypophosphatemia rickets is broadened, the understanding of the disease by clinicians is strengthened, experience is provided for the screening and diagnosis of the disease in the clinic, and a basis is also provided for prenatal diagnosis. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 It is a pedigree chart of family 1;

[0025] Figure 2 It is the image examination result 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;

[0026] Figure 3 It is the second-generation sequencing chart of proband 1 of family 1;

[0027] Figure 4 It is the first-generation sequencing chart of members of family 1;

[0028] Figure 5 It is a pedigree chart of family 2;

[0029] Figure 6 It is the image examination result of proband 2 of family 2; wherein, (A) (B) distal metaphyseal expansion of femur and tibia, curved diaphysis, (C) left distal metaphyseal expansion of ulna and radius, edge brush-like change, (D) left proximal metaphyseal expansion of humerus, reduced bone density;

[0030] Figure 7 It is the second-generation sequencing chart of proband 2 of family 2;

[0031] Figure 8 It is the first-generation sequencing chart of members of family 2. DETAILED DESCRIPTION

[0032] The above content of the present application will be further described in detail below by combining specific research examples. However, it should not be understood that the above subject matter of the present application is limited to the following examples only.

[0033] I. Research on pathogenic genes and mutation sites

[0034] 1. Sample collection object: The proband and his family members of the present study signed the informed consent form.

[0035] Family 1: A Allan-Herndon-Dudley syndrome family was collected, □ represents a normal male, o represents a normal female, ■ represents a male patient, ● represents a female patient, and ☉ represents an XR trait female carrier (such as Figure 1 ). Proband 1: growth and development retardation, unstable muscle tension in limbs; brain MRI shows that the bilateral cerebral hemispheres are basically symmetrical, the gray-white matter contrast is blurred, the central pre-posterior gyrus T2WI low signal is blurred, and no abnormal signal is found on DWI; the right basal ganglia perivascular space is obvious, the bilateral lateral ventricles are slightly full, the remaining ventricles and cisterns are normal in size and shape, 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 ); the thyroid function test results show that the free triiodothyronine is slightly high (10.15 pmol / L) and the free thyroxine is slightly low (7.93 pmol / L). The mother of the proband 1 is a heterozygous carrier of the variation, and the proband 1's older brother is a child with cerebral palsy and is also diagnosed as Allan-Herndon-Dudley syndrome. In family 1: through family analysis, it is considered that the X chromosome of the proband 1's second brother is inherited from his mother's healthy X chromosome, and the X chromosomes of the proband 1 and his older brother are inherited from their mother's abnormal X chromosome.

[0036] Family 2: A X-linked hypophosphatemic rickets family was collected, □ represents a normal male, o represents a normal female, ■ represents a male patient, and ● represents a female patient (such as Figure 5 ). Proband 2: height 84.3 cm (P10) and weight 11.99 kg (P25) at 2 years and 4 months; 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 shows that the shape, size, and bone density of each bone constituting the pelvis are normal, and the bone structure is complete; the position of bilateral femoral head epiphysis is normal, the Shenton line is continuous, and the acetabular shape is acceptable; the distal femur and the proximal and distal tibia metaphysis of both sides are enlarged, the bone shaft is curved, and the bilateral knee joint space is acceptable. Two wrist bones can be seen in the left wrist, the distal metaphysis of the left ulna and radius is blurred, the metaphysis of the ulna and radius is enlarged, and the edge is changed to a brush-like shape; the proximal metaphysis of the left humerus is slightly enlarged, and the bone density of the humerus is reducedFigure 6 Proband 2's brother: congenital bone dysplasia, low bone density, multiple joint stiffness, O-leg (orthopedic surgery has been performed), height 145 cm (adult); low blood phosphorus (0.76 mmol / L). The mother of the proband 2 is 145 cm tall, and has the same clinical phenotype as her son. The maternal grandmother of the proband 2 has similar symptoms and is deceased.

[0037] 2. Sample collection: 5 ml of venous peripheral blood for physical examination was taken, and 2 ml of EDTA anticoagulant was added. The genomic DNA was extracted by using DNABlood Mini Kit (QIAGEN) kit, and the concentration was determined by Qubit (dsDNA HS Assay Kit, Invitrogen) before being stored at -20℃ for standby use.

[0038] 3. Whole exome sequencing and result analysis

[0039] 3.1 Whole exome sequencing: First, the Covaris ultrasonic crusher was used to fragment the genomic DNA, and the broken product was subjected to end repair, A addition, adapter addition and amplification operation by using the library construction kit to complete the pre-library construction. Then, the human whole exome capture kit was used to process the above pre-library, 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 automatic nucleic acid analysis system, and the qualified library was quantified by using the quantitative kit. Finally, the sequencing reaction was completed on the MGISEQ-T7 gene sequencer of Huada.

[0040] The capture probe sequence used:

[0041] SLC16A2 c.963_964delinsAA: AACGCACTTACCGCATCTGGG.

[0042] PHEX c.112_113insA: ACGATCCTCTTTCTAGTGAGT.

[0043] ​​3.2 Data processing: After sequencing is completed, the sequences passing quality control are aligned to the human genome reference sequence using BWA software; GATK software is used to identify mutation sites in the target sequence, and Annovar annotation software is used to annotate the mutation sites to the public mutation database. 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 influence degree of the mutation on the protein function is predicted; then combined with the sample clinical phenotype, the pathogenicity of the mutation is interpreted according to the ACMG (The American College of Medical Genetics and Genomics) variation classification standard and guide.

[0044] 3.3 Proband whole exome sequencing results:

[0045] Proband 1: The analysis of this sample found that there was a semi-hybrid variation in exon 3 of SLC16A2 gene: c.963_964delinsAA (NM_006517.5), that is, the 963th and 964th cytosine nucleotides were deleted and two adenines were inserted, which finally caused the 321th amino acid to change from tyrosine to a stop codon (p.Y321*) (PVS1); this variation has not been reported in the normal population gene database (allele frequency (%): gnomeAD:.) (PM2_PP); the clinical symptoms of this case are consistent with Allan-Herndon-Dudley syndrome (PP4).

[0046] Proband 2: The analysis of this sample found that there was a heterozygous variation in exon 1 of PHEX gene: c.112_113insA (NM_000444.6), that is, an adenine was inserted between the 112th and 113th thymine nucleotides, which finally caused the 38th amino acid to mutate from phenylalanine to tyrosine and caused the appearance of a stop codon in advance (p.F38Yfs*13) (PVS1); this variation has not been reported in the normal population gene database (allele frequency (%): gnomeAD:.) (PM2_PP); the clinical manifestations of this case are consistent with X-linked hypophosphatemia and vitamin D-resistant rickets (PP4).

[0047] 3.4 Bioinformatics prediction analysis:

[0048] (1) Harmfulness and pathogenicity analysis

[0049] Proband 1: SLC16A2 c.963_964delinsAA (p.Y321*), which is a pathogenic variant according to the American College of Medical Genetics and Genomics (ACMG) variant classification guidelines (ACMG: PVS+2PP).

[0050] Proband 2: PHEX c.112_113insA (p.F38Yfs*13), which is a pathogenic variant according to the American College of Medical Genetics and Genomics (ACMG) variant classification guidelines (ACMG: PVS+2PP).

[0051] (2) Protein function, conservation analysis

[0052] SLC16A2 c.963_964delinsAA (p.Y321*): This mutation is the deletion of two cytosine nucleotides at positions 963 and 964 and the insertion of two adenines, which causes the 321st amino acid of the encoded protein to change from tyrosine to a stop codon, affecting the normal translation of the protein, and thus causing the abnormality of the MCT8 protein encoded by the SLC16A2 gene and leading to the occurrence of Allan-Herndon-Dudley syndrome; the MCT8 protein encoded by the SLC16A2 gene has high conservation, and if the MCT8 protein encoded by the SLC16A2 gene is abnormal, it will cause a lack of MCT8 protein, inducing Allan-Herndon-Dudley syndrome.

[0053] PHEX c.112_113insA (p.F38Yfs*13): This mutation is the insertion of an adenine between two thymine nucleotides at positions 112 and 113, which causes the 38th amino acid of the encoded protein to change from phenylalanine to tyrosine, affecting the normal translation of the protein, and thus causing the abnormality of the PHEX protein encoded by the PHEX gene and leading to the occurrence of X-linked hypophosphatemic rickets; the PHEX protein encoded by the PHEX gene has high conservation, and if the PHEX protein encoded by the PHEX gene is abnormal, it will cause a lack of PHEX protein, and thus the PHEX protein cannot effectively protect MEPE from being hydrolyzed by hydrolytic enzymes, inducing X-linked hypophosphatemic rickets.

[0054] 4. Sanger sequencing verification: Sanger sequencing was used to verify the site of the mutation found. Take 20 ng of DNA (such as peripheral blood genomic DNA) from family members, use primers, and follow the TaKaRa LA PCR TM Kit Ver. 2.1 (TaKaRa) operation procedure to perform PCR reaction;

[0055] Primer sequence 1: F-GTGGTGTCTGCTGGGAGTAG, R-GCAGGTTGAGGGTAGCTTCT.

[0056] Primer sequence 2: F-CAGCCACCAAACCACGAAA, R-ATGAACGCAGGCAAACAGC.

[0057] Sequencing analysis was performed on the ABI3730XL (Applied Biosystems) platform, and the presence of each mutation in the gene was finally confirmed (only for the family members involved in the verification and detection analysis): (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 had only one X chromosome with the SLC16A2 c.963_964delinsAA mutation, which was consistent with the previous suspected carrier, and the father and the 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 proband's mutation was heterozygous (consistent with the father not having the mutation), the mother was also heterozygous, and the proband's grandmother was dead and could not be detected.

[0058] 5. Comprehensive conclusion

[0059] (1) Based on the conclusion of the mutation analysis of proband 1: The detection of SLC16A2 gene c.963_964delinsAA hemizygous mutation by whole exon sequencing technology is the cause of this Allan-Herndon-Dudley syndrome family; The results of this study expand the gene spectrum of Allan-Herndon-Dudley syndrome, provide experience for clinical screening and diagnosis of Allan-Herndon-Dudley syndrome, and also provide a basis for prenatal diagnosis; Therefore, targeted genetic research on Allan-Herndon-Dudley syndrome can provide research direction and new theoretical basis for early diagnosis and effective treatment of Allan-Herndon-Dudley syndrome, and also provide new molecular targets for the development of specific drugs for the treatment of Allan-Herndon-Dudley syndrome in practice.

[0060] (II) Based on the conclusion of the analysis of the mutation of the proband 2: the detection of the c.112_113insA hemizygous / heterozygous mutation in the PHEX gene by whole exome sequencing technology is the cause of the X-linked hypophosphatemia and vitamin D-resistant rickets family. The research results broaden the gene spectrum of X-linked hypophosphatemia and vitamin D-resistant rickets, provide experience for the screening and diagnosis of X-linked hypophosphatemia and vitamin D-resistant rickets in clinic, and also provide a basis for prenatal diagnosis. Therefore, targeted genetic research on X-linked hypophosphatemia and vitamin D-resistant rickets can provide research direction and new theoretical basis for early diagnosis and effective treatment of X-linked hypophosphatemia and vitamin D-resistant rickets, and also provide new molecular targets for the development of specific drugs for the treatment of X-linked hypophosphatemia and vitamin D-resistant 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.

[0061] II. Introduction of mutants and application cases

[0062] As an example of the application of such products in clinical practice, a brief introduction is made. They can generally be used to analyze the obtained samples to be tested, determine whether the sample is from a patient 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. It can better provide guidance for screening potential severe diseases in fetuses during pregnancy to provide accurate recommendations.

[0063] 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 nucleic acid sample reflecting whether the gene in the biological sample has mutation can be extracted from the biological sample; the biological sample can be at least one selected from human blood, skin, subcutaneous tissue, preferably peripheral blood. It should be noted that the term "nucleic acid sample" used in this part should be understood broadly, which can be any sample that can reflect whether the gene in the biological sample has mutation, for example, it can be whole genome DNA directly extracted from the biological sample, it can be a part of the whole genome containing gene coding sequence, 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 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 understood broadly, which can be complete nucleic acid sequence information obtained after assembling the sequencing data obtained by sequencing the nucleic acid sample, or it can be directly using the sequencing data (reads) obtained by sequencing the nucleic acid sample as the nucleic acid sequence, as long as the nucleic acid sequence contains 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 disclosure, it indicates that the biological sample source is susceptible to the corresponding disease.

[0064] 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:

[0065] 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 sick as long as the mutation exists. If the subject is female, she is a carrier if the SLC16A2 gene in one of her X chromosomes has the c.963_964delinsAA mutation. If the SLC16A2 gene in both of her X chromosomes has the c.963_964delinsAA mutation, she will be sick. This method is faster and more convenient, and has less dependence on family investigation.

[0066] Secondly, for the male to be tested, he will be sick as long as he has the SLC16A2 c.963_964delinsAA mutation. For the female to be tested, if it is not clear whether both of her X chromosomes have the mutation, 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. According to the relevant circumstances, the risk of the subject to be tested to be sick or to have children can be known. The analysis model at least includes the following main situations:

[0067] 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 c.963_964delinsAA mutation. The female to be tested will be sick.

[0068] 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 X chromosome obtained from the father is healthy and the X chromosome obtained from the mother is abnormal. The female to be tested is a carrier.

[0069] 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 not a carrier), the X chromosome obtained from the father is abnormal and the X chromosome obtained from the mother is healthy. The female to be tested is a carrier.

[0070] Situation 4: If the father of the female to be tested is a patient with Allan-Herndon-Dudley syndrome and the mother is healthy (and a carrier), the X chromosome obtained from the father is abnormal and the X chromosome obtained from the mother is pending (at this time, further analysis of the maternal grandparents of the female to be tested is generally needed). If the X chromosome obtained from the mother is healthy, the female to be tested is a carrier. If the X chromosome obtained from the mother is abnormal, the female to be tested is sick.

[0071] The method and apparatus for aligning the detected nucleic acid sequence with the wild type are not particularly limited and any conventional software can be used. If not otherwise 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. The 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 components 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.

[0072] Those skilled in the art can clearly understand that various modifications can be made to the above examples without departing from the overall spirit and concept of the present application. All of them 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 the PHEX gene, characterized in that, The PHEX gene mutant is a nucleic acid as follows: The nucleic acid has an adenine inserted between the 112th and 113th thymine nucleotides compared with the wild-type PHEX gene with the sequence of SEQ ID NO.

3.

2. Use of a reagent for detecting the PHEX gene mutant of claim 1 in the preparation of a product for screening X-linked hypophosphatemic rickets resistant to vitamin D.

3. Use according to claim 2; wherein, The reagent for detecting the PHEX gene mutant of claim 1 is at least one of a probe and a primer specific to the PHEX gene mutant.

4. Use according to claim 3; wherein, The primer is at least a primer pair: F-CAGCCACCAAACCACGAAA, R-ATGAACGCAGGCAAACAGC; and the probe is at least ACGATCCTCTTTCTAGTGAGT.

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