SLC16A2 and PHEX gene mutant and application thereof

Abstract

The invention belongs to the technical field of gene diagnosis, and particularly discloses SLC16A2 and PHEX gene mutants and application thereof. On one hand, the invention relates to an SLC16A2 gene mutant and an application thereof, in particular to an application of SLC16A2c. 963964deinsAA mutation in a product for screening the Allan-Herdon-Dudley syndrome, and in particular relates to an application of SLC16A2c. 963964deinsAA mutation in a product for screening the Allan-Herdon-Dudley syndrome. On the other hand, the invention relates to a PHEX gene mutant and application thereof, in particular to application of PHEX c.112113insA mutation in products for screening X-linked hypophosphatemia vitamin D rickets. According to the invention, the pathogenic gene spectrum of Allan-Herdon-Dudley syndrome and X-linked hypophosphate vitamin D-resistant rickets is broadened, the understanding of clinical doctors on the disease is enhanced, experience is provided for clinical screening and diagnosis of the disease, and a basis is also provided for prenatal diagnosis.

Description

Technical Field

[0001] The invention belongs to the technical field of gene diagnosis, and in particular relates to SLC16A2 and PHEX gene mutants and applications thereof. Background Art

[0002] Allan-Herndon-Dudley syndrome (AHDS, OMIM 300523) is a rare X-linked recessive genetic disease. It is a rare endocrine disease caused by mutations in the thyroid hormone (TH) transporter SLC16A2 gene (OMIM 300095, formerly known as the MCT8 gene), which causes the inactivation of the monocarboxylate transporter 8 (MCT 8) it encodes, and the inability to mediate TH into target cells. AHDS can lead to damage to the nervous system and thyroid function. Its main clinical manifestations are hypotonia, feeding difficulties, and mild to severe developmental delay / intellectual disability in male children starting from infancy, which develop into pyramidal tract signs, delayed myelin development, extrapyramidal manifestations (dystonia, choreoathetosis, paroxysmal dyskinesia), motor function loss, thinness and elongated face, and drug-resistant epileptic seizures, easy coughing and respiratory infections, but growth disorders are generally not easily observed in early childhood. Other abnormal symptoms may include hypothyroidism and abnormal characteristic thyroid tests, namely, elevated triiodothyronine (FT3), decreased free tetraiodothyronine (FT4), and normal thyroid stimulating hormone (TSH). The disease mostly affects men, with a male prevalence of 1 / 70,000. Most heterozygous women may be asymptomatic or show only mild thyroid function abnormalities, but no symptoms of neurological damage.

[0003] The SLC16A2 gene is located on chromosome Xq13.2, with a total genome length of approximately 112.46 kb, including 6 exons and 5 introns. The exon length is approximately 4161 bp, encoding the 539 amino acid MCT8 protein. The MCT8 protein is composed of 12 transmembrane domains and is a specific transporter that mediates the entry of thyroid hormone T3 into cells, with the function of bidirectional transport of T3 and T4. MCT8 protein is mainly distributed on the membranes of endothelial cells, brain neurons, and oligodendrocytes that make up the human blood-brain barrier. The MCT8 protein on endothelial cells can transport thyroid hormone in the peripheral circulation to the central nervous system, and then the MCT8 protein on the membranes of brain neurons and oligodendrocytes is transported to the corresponding cells respectively. The active thyroid hormone (T3) that enters the cells or the active thyroid hormone converted from T4 binds to the nuclear receptors in the cells. The nuclear receptors bind to the thyroid hormone response element in the promoter region of the thyroid hormone target gene, guide the transcription and translation of the cells, and affect cell proliferation, neurogenesis, cell migration, cell differentiation, myelination, synaptogenesis, and cell apoptosis. At present, it is believed that the occurrence of AHDS is mainly caused by the partial or complete lack of MCT8 protein caused by SLC16A2 gene mutation, which causes the binding disorder of MCT8 protein to cell membrane and thyroid hormone, resulting in the decreased or complete lack of thyroid hormone distribution in neurons and oligodendrocytes in the brain, affecting the transcription and translation of brain neurons and oligodendrocytes, and further leading to the formation, migration, differentiation and maturation disorders of nerve cells and oligodendrocytes, resulting in a series of neurological symptoms, such as mental retardation, motor and language development retardation, convulsions, microcephaly, hearing abnormalities, dystonia, ataxia, etc. In addition, the defect of MCT8 protein can also lead to the increased expression of thyroid tissue deiodinase D1 and D2, resulting in the decrease of peripheral blood T4 and the increase of T3. The latter causes the peripheral tissue to show a state of increased metabolism, enhanced gluconeogenesis and glycolysis, enhanced fat synthesis and degradation, and accelerated protein decomposition in peripheral tissues and skeletal muscles. Patients may have myopathy-like facial features such as long face and emaciation. Friesema EC et al. (DOI:10.1016 / S0140-6736(04)17226-7) previously confirmed that AHDS is caused by mutations in the gene SLC16A2 encoding the MCT8 protein.

[0004] X-linked hypophosphatemic rickets (XLH, OMIM 307800) is a relatively rare X-linked dominant genetic disease with an incidence of about 1 / 20,000, and is more common in female patients. Its pathogenic gene is the phosphate regulating gene with homologies to endopeptidases on the X-chromosome (PHEX, OMIM 300550). The clinical manifestations of XLH are: children usually develop the disease at around 1 year old, mainly manifested by growth retardation, short stature, bone pain, walking weakness, bending deformity of both lower limbs, osteoporosis, multiple fractures and enamel dysplasia; adults mainly have rickets and bone and joint deformities. Laboratory tests can reveal 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 is located on chromosome Xp22.1, with a total length of 2861bp, consisting of 22 exons, encoding a protein composed of 749 amino acids, and has homology with a group of endopeptidase genes. The PHEX protein is composed of 749 amino acids and belongs to the zinc-binding endopeptidase family. The protein-encoding amino acid sequence is similar to that of neutral endopeptidase. It is a transmembrane protein, including a short cytoplasmic structure region composed of 20 amino acids, a transmembrane structure region composed of 25 amino acids, and an extracellular structure region containing zinc binding composed of 704 amino acids. The loss of PHEX protein function caused by mutation is the pathogenesis of XLH. On the one hand, the small peptides produced after the substrate extracellular matrix phosphoglycoprotein (MEPE) is hydrolyzed can inhibit the kidney's absorption of phosphate and affect bone mineralization. PHEX protein can protect MEPE from being hydrolyzed by hydrolases, thereby maintaining normal phosphorus metabolism. After the loss of PHEX protein function, it cannot play its role in protecting MEPE from hydrolysis. On the other hand, the inactivation of PHEX protein function will increase the circulating level of serum fibroblast growth factor 23 (FGF23). FGF23 acts on the kidneys and affects the reabsorption of phosphorus by the renal tubules, leading to increased urinary phosphorus excretion, decreased blood phosphorus levels and abnormal bone mineralization. Abnormal PHEX gene will lead to abnormal synthesis of the PHEX protein it encodes, which cannot protect MEPE protein from being hydrolyzed, thereby causing X-linked hypophosphatemic vitamin D-resistant rickets. Minamizaki T et al. (PMID: 32712387) have reported that MEPE protein deficiency can cause XLH. In addition, Kang Ke et al. (DOI:10.3969 / j.issn.1674-2591.2024.03.003) have also reported that the FGF23 level in the biochemical indicators of XLH patients was significantly increased.

[0006] At present, the research on the relationship between the above diseases and specific genes needs to be deepened, and the screening methods for the above diseases need to be improved to achieve early screening of potential susceptible diseases and provide new ideas for subsequent treatment and early intervention. Summary of the invention

[0007] In view of the above problems, the present invention provides SLC16A2 and PHEX gene mutants and their applications, mainly to provide new diagnostic methods for Allan-Herndon-Dudley syndrome and X-linked hypophosphatemic vitamin D-resistant rickets, and to further deepen the understanding of SLC16A2 and PHEX genes in the field.

[0008] In order to solve the above problems, the present invention adopts the following technical solutions:

[0009] The first aspect of the present invention provides SLC16A2 gene mutants and applications thereof, which not only provide a new pathogenic mutant, but also propose a scheme for the clinical application of the mutant.

[0010] With respect to the SLC16A2 gene mutant, the SLC16A2 gene mutant is any of the following:

[0011] A nucleic acid having a target fragment, and compared with the wild-type SLC16A2 gene of SEQ ID NO.1, the target fragment has a c.963_964delinsAA mutation, that is, the 963rd and 964th bases are deleted and two bases A are inserted;

[0012] A polypeptide, compared with the protein encoded by the wild-type SLC16A2 gene with a sequence of SEQ ID NO.2, the polypeptide has a p.Y321* mutation.

[0013] As for the effects of SLC16A2 gene mutants on the body's cell tissues or organs, the protein encoded by the SLC16A2 gene is specifically the MCT8 protein. If the binding of the MCT8 protein to the cell membrane and thyroid hormone is impaired, the distribution of thyroid hormone in neurons and oligodendrocytes in the brain will be reduced or completely absent, and the transcription and translation of brain neurons and oligodendrocytes will be affected, leading to the formation, migration, differentiation and maturation of nerve cells and oligodendrocytes, resulting in a series of neurological symptoms, such as mental retardation, motor and language development retardation, convulsions, microcephaly, hearing abnormalities, dystonia, ataxia, etc.; in addition, the defect of MCT8 protein can also lead to the increased expression of thyroid tissue deiodinase D1 and D2, resulting in a decrease in peripheral blood T4 and an increase in T3. The latter causes peripheral tissues to show a state of increased metabolism, enhanced gluconeogenesis and glycolysis, enhanced fat synthesis and degradation, and accelerated protein decomposition in peripheral tissues and skeletal muscle, which may cause patients to have a myopathy-like facies (such as long face, emaciation, etc.).

[0014] As for 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 comes from an Allan-Herndon-Dudley syndrome patient or a risk group (formed and unformed fetuses are temporarily listed as patients or risk groups), because when the aforementioned SLC16A2 gene mutant is present, the population to be tested will inevitably suffer from Allan-Herndon-Dudley syndrome or be a risk group. More specifically, the reagent for detecting the aforementioned SLC16A2 gene mutant is at least one of a probe or primer specific for the SLC16A2 gene mutant, and may also be other methods such as Sanger, NGS sequencing, etc. (all within the scope of the rights claimed by the present invention). Further, the primer is at least a primer pair: F-GTGGTGTCTGCTGGGAGTAG, R-GCAGGTTGAGGGTAGCTTCT; the probe is at least AACGCACTTACCGCATCTGGG. In order to more clearly explain the reasons and principles for the aforementioned SLC16A2 c.963_964delinsAA mutation to be used for Allan-Herndon-Dudley syndrome diagnosis and analysis, the SLC16A2 gene mutant can be used for Allan-Herndon-Dudley syndrome patients or risk population screening to explain: Since the SLC16A2 gene is located on the X chromosome, when the SLC16A2 gene on one X chromosome mutates and there is no normal allele to cover it, it will cause disease symptoms; for males, since males have only one X chromosome, SLC16A2 The c.963_964delinsAA mutation is a hemizygous mutation. In the hemizygous state, if the only copy has a loss-of-function mutation (such as a nonsense mutation or a frameshift mutation (including a base deletion or insertion)), due to the combined effect of insufficient gene dosage and loss of function, the total activity of the gene will be lower than the critical threshold, leading to cell or organ dysfunction and inducing disease. The loss-of-function mutation in the only copy directly induces the occurrence of the disease. For women, since women have two X chromosomes, if it is an X-linked recessive genetic disease (such as Allan-Herndon-Dudley syndrome), if one of the chromosomes of a woman is normal, she is a carrier without the disease, but if she gives birth to a son, it may cause her son to have an extremely high risk of disease (at least 50% probability of disease). Further, Allan-Herndon-Dudley syndrome is an X-linked recessive genetic disease.Since men only have one X chromosome, when men have this mutation, there is no normal allele complement, resulting in abnormal function of the encoded MCT8 protein, which further causes symptoms of Allan-Herndon-Dudley syndrome in men. Since women have two X chromosomes, when women have the aforementioned mutation, if the mutation only exists in one X chromosome, due to the normal allele complement function in the other X chromosome, the SLC16A2 gene can normally encode the MCT8 protein, and the MCT8 protein function will not appear if the disease symptoms are maintained; when both X chromosomes of women have the aforementioned mutation, the MCT8 protein cannot be encoded normally, and disease symptoms will appear. In addition, Sanger, NGS sequencing and other methods can be used to detect whether the SLC16A2 gene in the two X chromosomes of women has the aforementioned mutation to clarify whether they are carriers or patients.

[0015] As for the application of screening for Allan-Herndon-Dudley syndrome, screening for Allan-Herndon-Dudley syndrome includes screening for diseased and risk groups, as well as further examination of specific targets as an auxiliary basis for diagnosis. In addition, when used for prenatal diagnosis in clinical practice, genetic testing and imaging examinations are combined on the basis of genetic counseling to make a clear 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 originates from a male, the male can be determined to be a diseased population; when the mutation originates from a female, the female is a risk population, who may be a carrier or a patient, but it still has clinical application significance, specifically: when a female is a carrier, if she gives birth to a male baby, the male baby has at least a 50% probability of being sick, and further, if the female is a patient with Allan-Herndon-Dudley syndrome, the male baby she gives birth to must also be a patient with Allan-Herndon-Dudley syndrome.

[0016] The second aspect of the present invention provides PHEX gene mutants and applications thereof, which not only provide a new pathogenic mutant, but also propose a solution for the clinical application of the mutant.

[0017] With respect to the PHEX gene mutant, the PHEX gene mutant is any of the following:

[0018] A nucleic acid having a target fragment, and compared with the wild-type PHEX gene of SEQ ID NO.3, the target fragment has a c.112_113insA mutation, that is, a base A is inserted between the 112th and 113th bases;

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

[0020] As for the effect of the protein encoded by the PHEX gene on the body's cell tissues or organs, the protein encoded by the PHEX gene is the PHEX protein, and the loss of PHEX protein function caused by mutation is the pathogenesis of XLH. On the one hand, after the loss of PHEX protein function, it cannot protect MEPE from being hydrolyzed by hydrolases, resulting in the small peptides produced after the hydrolysis of MEPE inhibiting the kidney's absorption of phosphate and affecting bone mineralization, and thus failing to maintain normal phosphorus metabolism; on the other hand, the inactivation of PHEX protein function increases the circulating level of FGF23, and more circulating FGF23 acts on the kidneys, affecting the reabsorption of phosphorus by the renal tubules, leading to increased urinary phosphorus excretion, decreased blood phosphorus levels and abnormal bone mineralization.

[0021] As for the use of reagents for detecting PHEX gene mutants in the preparation of products for screening X-linked hypophosphatemic vitamin D resistant rickets; by detecting whether the aforementioned PHEX gene mutants exist in a sample (such as peripheral blood), it can be determined whether the sample comes from a patient with X-linked hypophosphatemic vitamin D resistant rickets (formed and unformed fetuses are temporarily listed as patients), because PHEX gene mutations will lead to abnormal function of PHEX protein and will inevitably induce X-linked hypophosphatemic vitamin D resistant rickets. Among them, the reagent for detecting the aforementioned PHEX gene mutant is at least one of the probes and primers specific for 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; the probe is at least ACGATCCTCTTTCTAGTGAGT. In order to more clearly introduce the reasons and principles why the aforementioned PHEX c.112_113insA mutation can be used for the diagnosis and analysis of X-linked hypophosphatemic vitamin D-resistant rickets, it is explained that the PHEX gene mutant can be used for screening of patients or risk groups with X-linked hypophosphatemic vitamin D-resistant rickets: the PHEX c.112_113insA mutation is a hemizygous / heterozygous mutation. The pathogenicity of hemizygous mutations is mainly due to the superposition effect of insufficient gene dosage and loss of function. In this case, if the only copy undergoes a loss-of-function mutation (such as a nonsense mutation, a frameshift mutation (including the deletion or insertion of bases)), the total activity of the gene will be lower than the critical threshold, leading to cell or organ dysfunction and thus inducing the disease; the pathogenicity of heterozygous mutations is that, in addition to the aforementioned mutations leading to insufficient gene dosage, it may also be due to the deletion or insertion of bases at a certain site of the DNA molecule (this mutant is a new insertion of an adenine), causing changes in the reading frame, resulting in a series of downstream code changes, so that the gene that originally encodes a certain peptide chain becomes one that encodes another completely different peptide chain sequence, resulting in abnormal protein function and activity and further leading to cell or organ dysfunction and thus inducing the disease.

[0022] As for the application of screening X-linked hypophosphatemic antivitamin D rickets, screening X-linked hypophosphatemic antivitamin D rickets includes screening risk groups and further examination of specific targets as auxiliary basis for diagnosis. In addition, when used for prenatal diagnosis in clinical practice, genetic testing and imaging examination are combined on the basis of genetic counseling to make a clear diagnosis of high-risk fetuses, thereby reducing the birth defect rate and improving the quality of eugenics and population quality. Since X-linked hypophosphatemic antivitamin D rickets is an X-linked dominant genetic disease, when the PHEX c.112_113insA mutation occurs, both males and females have X chromosomes, and as long as the PHEX gene function of any X chromosome is abnormal, X-linked hypophosphatemic antivitamin D rickets will occur, so as long as the sample has the PHEX c.112_113insA mutation, it must come from an X-linked hypophosphatemic antivitamin D rickets patient.

[0023] The present disclosure broadens the pathogenic gene spectrum of Allan-Herndon-Dudley syndrome and X-linked hypophosphatemic vitamin D-resistant rickets, enhances clinicians' understanding of the disease, provides experience for clinical screening and diagnosis of the above diseases, and also provides a basis for prenatal diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the pedigree chart of family 1;

[0025] Figure 2 The following are the imaging results of proband 1 in family 1. (A) myelination is significantly delayed, (B) perivascular spaces are prominent, (C) corpus callosum is slightly thinner, and (D) MR corpus callosum of a normal infant.

[0026] Figure 3 This is the next-generation sequencing map of proband 1 in family 1;

[0027] Figure 4 This is the first generation sequencing map of the family 1 member;

[0028] Figure 5 This is the pedigree chart of family 2;

[0029] Figure 6 The imaging results of proband 2 in family 2 are as follows: (A) (B) the distal femoral and tibia metaphysis is enlarged, and the diaphysis is curved; (C) the left wrist ulna and radius metaphysis is enlarged, and the edge is brush-like; (D) the proximal left humerus metaphysis is enlarged, and the bone density is reduced;

[0030] Figure 7 This is the next-generation sequencing map of proband 2 in family 2;

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

[0032] The above contents of the present invention are 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 invention being limited to the following embodiments.

[0033] 1. Research on pathogenic genes and mutation sites

[0034] 1. Sample collection subjects: The probands and their families in this study signed the informed consent.

[0035] Family 1: Allan-Herndon-Dudley syndrome family was collected. □ represents normal males, ○ represents normal females, ■ represents diseased males, ● represents diseased females, and ☉ represents female carriers of XR trait (such as Figure 1 Proband 1: growth retardation, unstable limb muscle tone; cranial MRI showed: bilateral cerebral hemispheres were basically symmetrical, gray and white matter contrast was blurred, central anterior and posterior gyri T2WI low signal was blurred, DWI showed no abnormal signal; the perivascular space in the right basal ganglia was prominent, bilateral lateral ventricles were slightly full, the size and shape of the remaining ventricles and cisterns were normal, and no obvious widening was found; the midline structure was centered, the cerebellum and brainstem were normal, and the corpus callosum was slightly thin (such as Figure 2 ); thyroid function test results showed that free triiodothyronine was elevated (10.15pmol / L) and free thyroxine was low (7.93pmol / L). The mother of proband 1 had no related abnormalities and was considered a heterozygous carrier of the variant. The eldest brother of proband 1 was a child with cerebral palsy and was also diagnosed with Allan-Herndon-Dudley syndrome. In family 1: through family analysis, it was found that the X chromosome of proband 1's second brother was inherited from his mother's healthy X chromosome, while the X chromosomes of proband 1 and his eldest brother were inherited from their mother's abnormal X chromosome.

[0036] Family 2: X-linked hypophosphatemic and vitamin D-resistant rickets family was collected. □ represents normal males, ○ represents normal females, ■ represents diseased males, and ● represents diseased females (such as Figure 5). Proband 2: At 2 years and 4 months old, height was 84.3cm (P10), weight was 11.99kg (P25); O-shaped legs, stumbled when walking, fell easily, and swayed from side to side when walking; low blood phosphorus (0.81mmol / L), high urine phosphorus (55.18mmol / L), abnormally elevated alkaline phosphatase (489U / L); bone DR showed: the shape, size, and bone density of the bones constituting the pelvis were normal, and the bone structure was intact; there was no abnormal position of the bilateral femoral epiphysis, Shen Tong's lines were continuous, and the acetabulum shape was acceptable; the distal femur and proximal and distal tibia metaphysis were enlarged, the diaphysis was bent, and the bilateral knee joint space was acceptable. Two carpal bones were visible in the left wrist, the early calcification band of the distal metaphysis of the left ulna and radius was blurred, the metaphysis of the ulna and radius was enlarged, and the edge had 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 had congenital dysplasia of bones, low bone density, multiple joint inflexibility, bow legs (corrective surgery has been performed), height 145 cm (adult); low blood phosphorus (0.76 mmol / L). The mother of proband 2 was 145 cm tall and had the same clinical phenotype as her son. The grandmother of proband 2 had similar symptoms and is now deceased.

[0037] 2. Sample collection: Take 5 ml of peripheral blood from the physical examination vein, add EDTA anticoagulation, and use 2 ml of it Genomic DNA was extracted using DNABlood Mini Kit (QIAGEN). The concentration was determined by dsDNA HS Assay Kit (Invitrogen) and then stored at -20°C for future use.

[0038] 3. Whole exome sequencing and result analysis

[0039] 3.1 Whole exome sequencing: First, the genomic DNA was fragmented using a Covaris ultrasonic crusher, and the fragmented products were end-repaired, A-added, adapter-added, and amplified using a library construction kit to complete the pre-library construction; then, the human whole exome sequence capture kit was used to process the above pre-library, and the target area was targeted and enriched by the probe hybridization capture method to obtain the final library. The library was analyzed for concentration and fragment distribution using the Qubit and QIAGEN QIAxcelAdvanced fully automatic nucleic acid analysis systems, and the qualified library was quantified using a quantitative kit; finally, the sequencing reaction was completed on the BGI MGISEQ-T7 gene sequencer.

[0040] Capture probe sequences used:

[0041] SLC16A2 c.963_964delinsAA:AACGCACTTACCGCATCTGGG.

[0042] PHEX c.112_113insA:ACGATCCCTCTTTCTAGTGAGT.

[0043] 3.2 Data processing: After sequencing is completed, the sequences that have passed quality control are aligned to the human genome reference sequence using BWA software; GATK software is used to identify the mutation sites in the target sequence, and Annovar annotation software is used to annotate the mutation sites to the public mutation database. The impact of the mutation on protein function is predicted based on the frequency of the mutation site in the normal population, sequence conservation, amino acid changes caused by the mutation, and the position in the protein structure; then, combined with the clinical phenotype of the sample, the pathogenicity of the mutation is interpreted according to the ACMG (The American College of Medical Genetics and Genomics) variation classification standards and guidelines.

[0044] 3.3 Whole exome sequencing results of the proband:

[0045] Proband 1: The sample analysis revealed a hemizygous mutation in exon 3 of the SLC16A2 gene: c.963_964delinsAA (NM_006517.5), i.e., the 963rd and 964th cytosine nucleotides were deleted and two adenines were inserted, which eventually caused the 321st amino acid to change from tyrosine to a stop codon (p.Y321*) (PVS1); this mutation 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 sample analysis revealed a heterozygous mutation in exon 1 of the PHEX gene: c.112_113insA (NM_000444.6), which means an adenine is inserted between the 112th and 113th thymine nucleotides, eventually causing the 38th amino acid to mutate from phenylalanine to tyrosine and causing the premature appearance of the stop codon (p.F38Yfs*13) (PVS1); this mutation 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 hypophosphatemic 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*), according to the ACMG (The American College of Medical Genetics and Genomics) variant classification guidelines, this variant is a pathogenic variant (ACMG: PVS+2PP).

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

[0051] (2) Protein function and conservation analysis

[0052] SLC16A2 c.963_964delinsAA (p.Y321*): This mutation is the deletion of two cytosine nucleotides at 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, thereby making the MCT8 protein encoded by the SLC16A2 gene abnormal and leading to the occurrence of Allan-Herndon-Dudley syndrome; the MCT8 protein encoded by the SLC16A2 gene is highly conserved. If the MCT8 protein encoded by the SLC16A2 gene is abnormal, it will lead to MCT8 protein deficiency and induce Allan-Herndon-Dudley syndrome.

[0053] PHEX c.112_113insA (p.F38Yfs*13): This mutation inserts an adenine between the two thymine nucleotides 112 and 113, causing the 38th amino acid of the encoded protein to mutate from phenylalanine to tyrosine, affecting the normal translation of the protein, thereby making the PHEX protein encoded by the PHEX gene abnormal and leading to the occurrence of X-linked hypophosphatemic vitamin D-resistant rickets; the PHEX protein encoded by the PHEX gene is highly conserved. If the PHEX protein encoded by the PHEX gene is abnormal, it will lead to PHEX protein deficiency, and then the PHEX protein will be unable to effectively protect MEPE from being hydrolyzed by hydrolases, inducing X-linked hypophosphatemic vitamin D-resistant rickets.

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

[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 the family members who participated in the validation test were analyzed): (1) In family 1, the mother and eldest 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 situation of the previous suspected carrier. The father and second brother did not have the mutation. (2) In family 2, the elder brother and mother of proband 2 also had the PHEX c.112_113insA mutation, while the father did not have the mutation. The proband's mutation was a heterozygous mutation (consistent with the father not having the mutation), and the mother also had a heterozygous mutation. The grandmother of proband 2 was deceased and could not be tested.

[0058] 5. Summary

[0059] (I) Based on the conclusion of the mutation analysis of proband 1: The hemizygous mutation c.963_964delinsAA in the SLC16A2 gene detected by whole exome sequencing is the cause of the Allan-Herndon-Dudley syndrome in this family; the results of this study broaden the genetic spectrum of Allan-Herndon-Dudley syndrome, provide experience for the 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 will provide research directions and new theoretical basis for the early diagnosis and effective treatment of Allan-Herndon-Dudley syndrome, and will also provide new molecular targets for the development of specific drugs for the treatment of Allan-Herndon-Dudley syndrome in practice.

[0060] (II) Conclusions based on the analysis of the proband 2 mutation study: Whole exome sequencing technology detected that the hemizygous / heterozygous mutation of c.112_113insA in the PHEX gene was the cause of the disease in this X-linked hypophosphatemia and vitamin D resistant rickets family; the results of this study broadened the genetic spectrum of X-linked hypophosphatemia and vitamin D resistant rickets, provided experience for the clinical screening and diagnosis of X-linked hypophosphatemia and vitamin D resistant rickets, and also provided a basis for prenatal diagnosis; therefore, targeted genetic research on X-linked hypophosphatemia and vitamin D resistant rickets will provide research directions and new theoretical basis for the early diagnosis and effective treatment of X-linked hypophosphatemia and vitamin D resistant rickets, and will also provide new molecular targets for the development of specific drugs for the treatment of X-linked hypophosphatemia and vitamin D rickets in practice. Therefore, conducting targeted genetic research on the above-mentioned diseases and identifying the pathogenic genes and their pathogenic mechanisms have potential clinical significance for genetic counseling and individualized prevention and treatment of patients with the above-mentioned diseases, providing research directions and new theoretical basis for the early diagnosis and effective treatment of the above-mentioned diseases, and will also provide new molecular targets for the development of specific drugs for the treatment of the above-mentioned diseases in practice.

[0061] 2. Introduction and application cases of mutants

[0062] Here is a brief introduction to the clinical application examples of this type of product. It can generally be used to analyze the samples obtained for testing. By analyzing whether the samples contain specific mutations, it can be determined whether the source of the samples suffers from a certain disease or is a high-risk population. This can provide a reference for clinical diagnosis and treatment, especially in pre-pregnancy screening. It can screen for potential serious diseases in the fetus during pregnancy and provide accurate advice.

[0063] When clinically applied, the general steps are as follows: S1. Extracting nucleic acid samples from biological samples (the samples in this step can also be directly provided by the testing party): The type of the biological sample is not particularly limited, as long as a nucleic acid sample reflecting whether there is a mutation in the gene of the biological sample can be extracted from the biological sample; the biological sample can be at least one selected from human blood, skin, and subcutaneous tissue, preferably peripheral blood. It should be noted that the term "nucleic acid sample" used in this section should be understood in a broad sense, and it can be any sample that can reflect whether there is a mutation in the gene in the biological sample, for example, it can be the whole genome DNA directly extracted from the biological sample, or it can be a part of the whole genome containing the gene coding sequence, it can be the total RNA extracted from the biological sample, or it can be the mRNA extracted from the biological sample. S2. After obtaining the nucleic acid sample, the nucleic acid sample is analyzed to determine the nucleic acid sequence of the obtained nucleic acid sample (mainly the target region where the mutation is located): The method and equipment 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. The method and equipment for sequencing are not particularly limited. The second generation sequencing technology, the third generation and the fourth generation or more advanced sequencing technology can be used, and the nucleic acid sequence is sequenced using at least one selected from BGISEQ500, MGISEQ-200, MGISEQ-2000, MGISEQ-T7, HISEQ2000, SOLID, 454, ABI3730XL and a single molecule sequencing device; it should be noted that the term "nucleic acid sequence" used in this section should be understood in a broad sense, which can be the complete nucleic acid sequence information obtained after assembling the sequencing data obtained by sequencing the nucleic acid sample, or the sequencing data (reads) obtained by sequencing the nucleic acid sample can be directly used 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, the nucleic acid sequence of the obtained nucleic acid sample is compared with the sequence of the wild type: if the obtained nucleic acid sequence has a specific mutation involved in the present disclosure, it indicates that the source of the biological sample is susceptible to the corresponding disease.

[0064] Taking the application of SLC16A2 c.963_964delinsAA mutation in the diagnosis and analysis of Allan-Herndon-Dudley syndrome as an example (only considering the pathogenicity of this mutation, without considering other pathogenic factors), at least two analysis methods can be used:

[0065] First, the sample to be tested can be directly analyzed through Sanger sequencing and NGS to determine whether the SLC16A2 gene in the X chromosome has the c.963_964delinsAA mutation. If the subject is a male, then as long as it exists, the patient must be ill. If the subject is a female, if SLC16A2 in one X chromosome has the c.963_964delinsAA mutation, then the patient is a carrier. If SLC16A2 in both X chromosomes has the c.963_964delinsAA mutation, then the patient must be ill. This method is faster and more convenient, and has less reliance on family investigation of patients.

[0066] Second, for males to be tested, it is the same as the first item above. As long as they have the SLC16A2 c.963_964delinsAA mutation, they will definitely have the disease. For females to be tested, if it is unclear whether both X chromosomes have the mutation, family analysis is still needed. In practical application, the analysis model can be constructed by the following logic. After inputting the corresponding variables, the disease risk of the subject to be tested can be obtained, and the disease risk or fertility risk can be obtained based on relevant circumstances. The analysis model at least includes the following main situations:

[0067] Scenario 1: If both parents of the woman to be tested are patients with Allan-Herndon-Dudley syndrome, then the two X chromosomes obtained from her parents should both have the SLC16A2 c.963_964delinsAA mutation, and the woman must be sick;

[0068] Scenario 2: If the mother of the woman to be tested is a patient with Allan-Herndon-Dudley syndrome and the father is healthy, then she has a healthy X chromosome from her father and an abnormal X chromosome from her mother, and the woman is a carrier;

[0069] Scenario 3: If the father of the woman to be tested is an Allan-Herndon-Dudley syndrome patient and the mother is healthy (and not a carrier), then she has an abnormal X chromosome from her father and a healthy X chromosome from her mother, and the woman is a carrier;

[0070] Situation 4: If the father of the woman to be tested is a patient with Allan-Herndon-Dudley syndrome and the mother is healthy (a carrier), then the X chromosome obtained from the father is abnormal, and the X chromosome obtained from the mother is to be determined (at this time, it is generally necessary to further analyze the situation of the woman's grandparents). If the X chromosome obtained from the mother is healthy, the woman is a carrier, and if the X chromosome obtained from the mother is abnormal, the woman is sick.

[0071] The method and equipment for comparing the detected nucleic acid sequence with the wild type are not particularly limited, and any conventional software can be used for operation. Unless otherwise specified, the technical means used in the embodiments are conventional means familiar to those skilled in the art, and the reagents and products used are also commercially available. Various processes and methods not described in detail are conventional methods known in the art, and the sources, trade names, and components of the reagents used are indicated when they first appear, and the same reagents used thereafter are the same as those indicated for the first time unless otherwise specified.

[0072] It will be clear to those skilled in the art that various modifications to the above embodiments may be made without departing from the overall spirit and concept of the present invention. All of these modifications fall within the scope of protection of the present invention. The protection scheme of the present invention shall be subject to the claims attached to the present invention.

Claims

1. A SLC16A2 gene mutant, characterized in that: The SLC16A2 gene mutant is any 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 of SEQ ID NO.1; A polypeptide, compared with the protein encoded by the wild-type SLC16A2 gene with a sequence of SEQ ID NO.2, the polypeptide has a p.Y321* mutation.

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 of claim 1 in preparing a product for screening Allan-Herndon-Dudley syndrome.

4. The use according to claim 3; wherein: Products for screening Allan-Herndon-Dudley syndrome are products for screening patients or risk groups 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 a probe and a primer specific to the SLC16A2 gene mutant.

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

7. A PHEX gene mutant, characterized in that: The PHEX gene mutant is any of the following: A nucleic acid having a target fragment, and the target fragment has a c.112_113insA mutation compared with the wild-type PHEX gene with a sequence of SEQ ID NO.3; A polypeptide, compared with the protein encoded by the wild-type PHEX gene with a sequence of SEQ ID NO.4, the polypeptide has a p.F38Yfs*13 mutation.

8. Use of a reagent for detecting the PHEX gene mutant according to claim 7 in the preparation of a product for screening X-linked hypophosphatemic vitamin D-resistant rickets.

9. The use according to claim 8; wherein: The reagent for detecting the PHEX gene mutant in claim 7 is at least one of a probe and a primer specific to the PHEX gene mutant.

10. The use according to claim 9; wherein: The primer is at least a primer pair: F-CAGCCACCAAACCACGAAA, R-ATGAACGCAGGCAAACAGC; the probe is at least ACGATCCTCTTTCTAGTGAGT.

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