Use of ptchd1 gene mutants and detection reagents

Abstract

The application discloses a PTCHD1 gene mutant and a detection reagent and belongs to the technical field of gene mutation detection. Compared with a wild type PTCHD1 gene, the mutation is a substitution of cytosine C to adenine A at a 1644th nucleotide in a coding sequence, which causes the protein translation to be terminated in advance at a 548th amino acid, and forms a truncated protein. It is proved that the c.1644C>A of the PTCHD1 gene is a pathogenic nonsense mutation causing AUTS4, new insights are provided for the genetic basis of the disease, and the blank of the mutation spectrum of the disease is filled.

Description

Technical Field

[0001] This invention belongs to the field of gene mutation detection technology, specifically relating to the application of a PTCHD1 gene mutant and detection reagent. Background Technology

[0002] Autism Spectrum Disorder (ASD) is a group of complex neurodevelopmental disorders with highly heterogeneous clinical presentations and genetic bases. Its core clinical features include persistent deficits in social communication and interaction, as well as restricted, repetitive patterns of behavior, interests, or activities. The etiology of ASD is extremely complex; currently, genetic factors are recognized as the main risk factor, but numerous genes are involved. Among various inheritance patterns, X-linked inheritance has attracted considerable attention due to its association with a higher prevalence of ASD in males and is an important direction of research in this field.

[0003] The PTCHD1 (Patched Domain Containing 1) gene is located on the X chromosome at p22.11. It encodes a transmembrane protein highly expressed in key brain regions, particularly the cerebellum, during nervous system development, and is presumed to play a crucial regulatory role in cell signaling pathways. Numerous clinical and functional studies have confirmed that loss-of-function (LoF) variants of the PTCHD1 gene, such as deletions of the entire gene or parts of exons, and truncation mutations leading to premature termination of protein translation, are direct causes of a range of neurodevelopmental disorders. These disorders have a broad clinical spectrum, often manifesting as varying degrees of intellectual disability, pervasive developmental delay, and typical symptoms of ASD. The disease caused by this gene variant has been formally named X-linked autism susceptibility 4 (AUTS4, MIM 300830).

[0004] For single-gene genetic diseases like AUTS4, molecular diagnosis via gene sequencing is the "gold standard" for definitive diagnosis. However, with the widespread adoption of sequencing technology, although new variants are constantly being discovered, the spectrum of pathogenic mutations is far from saturated. Many newly discovered rare variants, especially point mutations, have unclear clinical significance and are often classified as "variables of undetermined significance" (VUS), posing a significant challenge to clinical diagnosis and genetic counseling. Furthermore, current clinical practice primarily relies on large-scale gene package or whole-exome sequencing to screen for such mutations, which is costly and time-consuming, and lacks mature, convenient, and standardized molecular diagnostic tools for specific, validated pathogenic point mutations. Therefore, discovering and rigorously validating new pathogenic mutation sites, and developing diagnostic reagents and detection methods based on these sites that are highly specific, sensitive, cost-effective, and provide stable results, is of urgent practical need and significant clinical value for improving the diagnostic efficiency of this disease, providing clear genetic counseling for high-risk families, and implementing effective prenatal diagnosis and preimplantation genetic diagnosis (PGD) and other eugenic strategies. Summary of the Invention

[0005] The purpose of this invention is to provide a novel gene mutant clearly associated with AUTS4 and to develop corresponding detection reagents based on this mutant. This aims to address the shortcomings of existing technologies in detecting specific PTCHD1 gene mutations and the fact that the pathogenic mutation spectrum is far from saturated.

[0006] To achieve the above objectives, the present invention discloses the following technical solution:

[0007] A PTCHD1 gene mutant, compared to the wild-type PTCHD1 gene, has a substitution of cytosine C for adenine A at nucleotide 1644 of the coding sequence.

[0008] The PTCHD1 gene mutant mentioned above causes premature termination of protein translation at amino acid position 548, resulting in a truncated protein.

[0009] The newly discovered PTCHD1 gene mutant disclosed in this invention, compared to the wild-type PTCHD1 gene with GenBank accession number NM_173495.3, has a substitution of cytosine (C) for adenine (A) at nucleotide 1644 of the coding sequence (c.1644C>A) (located in exon 3). The PTCHD1 mutant protein encoded by the above gene mutant is a truncated protein with loss of function, and this mutation causes premature termination of protein translation at amino acid 548. .

[0010] This invention also provides the application of the detection reagent for the PTCHD1 gene mutant in the preparation of a detection kit for the diagnosis, screening, or risk assessment of AUTS4.

[0011] The above-mentioned testing reagents include those for Sanger sequencing, ARMS-PCR, HRM, dPCR, or CRISPR / Cas systems.

[0012] In addition to Sanger sequencing, the primer and target information disclosed in this invention can be used to develop other rapid, high-throughput detection technology platforms, such as: (1) ARMS-PCR (Amplification Arrest Mutation System): Design allele-specific primers, and determine the genotype based on the presence or absence of the product in a single PCR reaction; (2) High-resolution melting curve analysis (HRM): Different genotypes are distinguished by analyzing the differences in melting curves of PCR products; (3) Digital PCR (dPCR): can be used for absolute quantitative detection of trace samples (such as cell-free fetal DNA in non-invasive prenatal samples); (4) CRISPR / Cas system detection: Develop novel nucleic acid detection methods by utilizing the specific recognition capability of the CRISPR / Cas system for target sequences.

[0013] Integration in kit form: The primers and / or probes of this invention can be integrated into gene chips or high-throughput sequencing gene panels containing multiple neurodevelopmental disorder-related genes (such as MECP2, FMR1, etc.) to achieve one-time parallel screening of multiple related genetic diseases.

[0014] Furthermore, Sanger sequencing assays include PCR assays and sequencing assays.

[0015] Furthermore, the primer pair used for PCR amplification includes the upstream primer PTCHD1-F with the sequence shown in SEQ ID NO.1 and the downstream primer PTCHD1-R with the sequence shown in SEQ ID NO.2.

[0016] The primer sequences used for Sanger sequencing validation are identical to those used for PCR amplification.

[0017] This invention relates to a primer combination, a detection reagent comprising the primer combination, and an AUTS4 detection kit comprising the detection reagent. The kit may further comprise polymerase, nucleotides, reaction buffer, and positive and negative controls required for the detection.

[0018] The beneficial effects of this invention are:

[0019] (1) Enriched the pathogenic mutation spectrum: This invention is the first to discover and confirm the c.1644C>A mutation in the PTCHD1 gene. This pathogenic nonsense mutation that leads to AUTS4 provides new insights into the genetic basis of the disease and fills a gap in the mutation spectrum of the disease.

[0020] (2) Provide new research targets: Functional studies of this novel truncated protein will help elucidate the role of the PTCHD1 gene in neural development and provide new targets for the future development of potential therapeutic strategies (such as nonsense mutation readthrough drug screening).

[0021] (3) Provides precise diagnostic tools: The specific primers, reagents and kits provided by this invention can quickly and accurately detect target mutations, providing clear molecular diagnostic basis for AUTS4 patients and their families, and avoiding misdiagnosis and missed diagnosis.

[0022] (4) It has significant clinical application value: The diagnostic method of the present invention can be widely used in clinical practice, including the diagnosis of suspected patients, carrier screening of high-risk families, prenatal diagnosis and preimplantation genetic diagnosis (PGD), providing a reliable scientific basis for genetic counseling and reproductive choices. Attached Figure Description

[0023] Figure 1 : Genetic pedigree chart of family No. 1.

[0024] Figure 2 Peak diagram of Sanger sequencing results for family 1.

[0025] in Figure 2 A represents the sequencing results of the proband from family 1. Figure 2 B represents the sequencing results of the mother in family 1; Figure 2 C represents the sequencing result of the father in family 1.

[0026] Figure 3 : Genetic pedigree chart of family No. 2.

[0027] Figure 4 Peak diagram of Sanger sequencing results for family 2.

[0028] in Figure 4 A represents the sequencing results of the proband from family 2. Figure 4 B represents the sequencing results of the father in family 2; Figure 4 C represents the sequencing result of the mother in family 2; Figure 4 D represents the sequencing results of a normal person outside the family. Detailed Implementation

[0029] The following examples are intended to further illustrate the present invention, but not to limit it.

[0030] Example 1: Discovery and verification of pathogenic mutants:

[0031] 1. Clinical Data and Sample Collection: One AUTS4 family (family 1) was used as the study subject; its clinical information is shown in Table 1. This project was approved by the hospital's ethics committee, and all subjects or their guardians signed informed consent forms. Peripheral blood samples of 3-5 mL were collected from the proband and their parents, and genomic DNA was extracted using a commercially available kit.

[0032] Diagnostic criteria: Please refer to:

[0033] Expert consensus on diagnostic strategies for the causes of intellectual disability or global developmental delay in children [J]. Chinese Journal of Pediatrics, 2018, 56(11):806-810.

[0034] Or, the consensus of experts on early diagnosis of autism spectrum disorder in young children in China, published by the Developmental and Behavioral Group of the Pediatrics Branch of the Chinese Medical Association and the Child Health Care Group of the Pediatrics Branch of the Chinese Medical Doctor Association [J]. Chinese Journal of Pediatrics, 2022, 60(07):640-646.

[0035] ;

[0036] 2. Experimental Instruments and Reagents

[0037] Main instruments: high-throughput sequencer (Illumina NextSeq550Dx), PCR instrument (Bio-Rad T100), gene analyzer (ABI 3500 Dx), nucleic acid quantification instrument (Qubit), centrifuge, etc.

[0038] Main reagents: Whole exon capture kit (FreySeq), DNA extraction kit (QIAGEN), Taq polymerase, dNTPs, BigDye Terminator v3.1 sequencing kit (Thermo Fisher).

[0039] 3. WES and Sanger sequencing validation

[0040] To determine the genetic cause of the proband's illness in family 1, whole-exome sequencing (WES) was performed on the core members of the family. Analysis revealed a hemizygous nonsense mutation in the proband's PTCHD1 gene (NM_173495.3): c.1644C>A. Family pedigree verification showed that the mother was a heterozygous carrier at this locus, and the father was wild-type, consistent with an X-linked inheritance pattern.

[0041] To verify the mutation, PCR amplification and Sanger sequencing were performed using the primers provided in this invention.

[0042] PCR amplification: Upstream primer PTCHD1-F (SEQ ID NO:1) and downstream primer PTCHD1-R (SEQ ID NO:2) were used. Reaction system (25 μL): 10×PCR Buffer 2.5 μL, dNTPs (2.5 mM) 2.0 μL, PTCHD1-F (10 μM) 1.0 μL, PTCHD1-R (10 μM) 1.0 μL, genomic DNA (50 ng / μL) 1.0 μL, Taq enzyme (5 U / μL) 0.25 μL, ddH2O 17.25 μL.

[0043] PCR program: 95℃ pre-denaturation for 5 minutes; 95℃ for 30 seconds, 58℃ for 30 seconds, 72℃ for 45 seconds, for a total of 35 cycles; 72℃ extension for 7 minutes.

[0044] Sequencing: After purification of the PCR product, the amplification primers were used as sequencing primers for bidirectional Sanger sequencing.

[0045] Results: Sanger sequencing results ( Figure 2 This confirms that the proband is hemizygous A at c.1644, his mother is C / A heterozygous, and his father is wild-type C, which is completely consistent with the WES results.

[0046] Figure 1 The genetic relationships of family 1 are shown. Boxes represent males, and circles represent females. Fully black symbols represent patients (II-1), half-black symbols represent carriers (I-2), and blank symbols represent normal individuals (I-1). The genotype at the c.1644 locus is indicated below each symbol.

[0047] Figure 2 The sequencing results around the c.1644 site of the PTCHD1 gene are shown for the proband (II-1) and his mother (I-2). The arrow indicates the c.1644 site. Figure 2 A (the proband) is shown as a single green "A" peak at the arrow. Figure 2 B (mother) is shown as an overlapping peak of blue "C" and green "A" at the arrow. Figure 2 C (father) is shown as a single blue "C" peak at the arrow.

[0048] In this invention, when the sample originates from a male, the genotype at the c.1644 locus of the PTCHD1 gene is specifically either "c.1644C>A hemizygote" or "c.1644C hemizygote". If the genotype at this locus is "c.1644C>A hemizygote", it is determined that the PTCHD1 gene has a mutation, and the male individual is a patient; if the genotype at this locus is "c.1644C hemizygote", it is determined that the mutation does not exist, and the male individual is a normal person.

[0049] When the sample is from a female, the genotype at the c.1644 locus of the PTCHD1 gene can be one of three types: "c.1644A / A homozygous", "c.1644C>A heterozygous", or "c.1644C / C homozygous". If the genotype at this locus is "c.1644A / A homozygous", a homozygous mutation is identified, and the female individual is a patient. If the genotype at this locus is "c.1644C>A heterozygous", a heterozygous mutation is identified, and the female individual is a carrier. If the genotype at this locus is "c.1644C / C homozygous", no mutation is identified, and the female individual is normal.

[0050] Example 2: Primer design, validation, and optimization:

[0051] To ensure the specificity and stability of the detection, three pairs of candidate amplification primers were designed for the c.1644 site and optimized experimentally. Evaluation indicators included primer dimer, non-specific amplification, and target band clarity. The results are shown in Table 2.

[0052] ;

[0053] According to the verification results, primer pair 1 (i.e., SEQ ID NO:1 and SEQ ID NO:2 of the present invention) performed the best and was selected as the final amplification primer.

[0054] Example 3: Composition of the detection kit and its application in another family and 100 healthy individuals:

[0055] 1. Kit Components

[0056] PTCHD1 Amplification Premix: Contains optimized PCR buffer, dNTPs, and Mg. 2+ Upstream primer PTCHD1-F (SEQ ID NO:1) and downstream primer PTCHD1-R (SEQ ID NO:2).

[0057] Taq polymerase (5 U / μL).

[0058] Positive control: Synthetic DNA fragments containing the PTCHD1 c.1644C>A hemizygous mutation or known patient DNA.

[0059] Negative control: Wild-type human genomic DNA.

[0060] Instruction manual.

[0061] 2. Application in family lineage 2 and 100 normal individuals outside the family lineage.

[0062] Simultaneously, testing was conducted on another family clinically diagnosed with ASD (family 2) and 100 non-family normal individuals; their clinical information is shown in Table 3. The proband of family 2 was a 4-year-old male, and his mother had a normal phenotype. DNA was extracted and tested using the kit of this invention according to the instructions. The results are as follows: Figure 4 As shown, the proband of family 2 also carries the PTCHD1 c.1644C>A hemizygous mutation, his mother is a heterozygous carrier, while his father and 100 normal individuals outside the family did not test positive for this mutation. This result provides a clear etiological diagnosis for family 2.

[0063] ;

[0064] Figure 3 The genetic relationships of family 2 are shown. Boxes represent males, and circles represent females. Fully black symbols represent patients (II-1), half-black symbols represent carriers (I-1), and blank symbols represent normal individuals (I-2). The genotype at locus c.1644 is indicated below each symbol.

[0065] Figure 4 The sequencing results around the c.1644 site of the PTCHD1 gene are shown for the proband (II-1) and his mother (I-1). The arrow indicates the c.1644 site. Figure 4 A (the proband) is shown as a single green "A" peak at the arrow. Figure 4 B (father) is shown as a single blue "C" peak at the arrow. Figure 4 C (mother) is shown as an overlapping peak of blue "C" and green "A" at the arrow. Figure 4 D (non-familial normal individuals) is shown as a single blue "C" peak at the arrow.

[0066] Example 4: Gene Mutation Rating and Pathogenicity Interpretation

[0067] To clarify the clinical significance of the PTCHD1 gene c.1644C>A variant discovered in this invention, its pathogenicity was assessed according to the "Standards and Guidelines for Interpreting Sequence Variations" published by the American College of Medical Genetics and Genomics (ACMG).

[0068] 1. ACMG variant interpretation criteria and scoring rules

[0069] The ACMG guidelines are an internationally recognized classification system for the pathogenicity of variants. It categorizes variants into five levels: "Pathogenic" (P), "Likely Pathogenic" (LP), "Violent Significance" (VUS), "Likely Benign" (LB), and "Benign" (B). This rating relies on combinations of evidence of varying strengths, with evidence divided into two main categories: pathogenic and benign.

[0070] Evidence of pathogenicity:

[0071] (1) PVS1 (Very strong)

[0072] (2) PS (strong, divided into PS1-PS4)

[0073] (3) PM (moderate, divided into PM1-PM6)

[0074] (4) PP (support, divided into PP1-PP5)

[0075] Benign evidence:

[0076] (1) BA1 (independent)

[0077] (2) BS (Strong, divided into BS1-BS4)

[0078] According to the guidelines, variants can be rated according to the following rules (only those related to disease incidence are listed):

[0079] Pathogenicity:

[0080] (1) 1 PVS1 + ≥ 1 PS

[0081] (2) 1 PVS1 + ≥ 2 PM

[0082] (3) ≥ 2 PS

[0083] (4) 1 PS + ≥ 3 PM

[0084] Likely Pathogenic:

[0085] (1) 1 PVS1 + 1 PM

[0086] (2) 1 PS + 1-2 PM

[0087] (3) ≥ 3 PM

[0088] (4) 1 PM + ≥ 2 PP

[0089] 2. Evidence rating for the PTCHD1 c.1644C>A variant

[0090] Using the above criteria, the specific evidence for the c.1644C>A variant in this invention is graded, as shown in Table 4.

[0091] ;

[0092] 3. Overall Conclusion

[0093] The PTCHD1 gene c.1644C>A variant in this invention meets two pieces of pathogenic evidence:

[0094] (1) PVS1 (very pathogenic)

[0095] (2) PM2_Supporting (moderate pathogenicity)

[0096] According to the ACMG scoring rules listed in Section 1, one of the criteria for determining "likely pathogenic" is (1 PVS1 + 1 PM).

[0097] The evidence combination of this variant (PVS1 + PM2_Supporting) contains one “very strong” piece of evidence (PVS1) and one “moderately strong” piece of evidence (PM2_Supporting), which fully meets the rating rule of (1 PVS1 + 1 PM).

[0098] Conclusion: Based on the above evidence (PVS1 + PM2_Supporting), according to the ACMG guidelines, the c.1644C>A variant of the PTCHD1 gene can be classified as a "likely pathogenic" variant.

Claims

1. A PTCHD1 gene mutant, characterized in that, Compared to the wild-type PTCHD1 gene with GenBank accession number NM_173495.3, a substitution of cytosine C for adenine A occurs at nucleotide 1644 of the coding sequence.

2. Application of the detection reagent for PTCHD1 gene mutants in the preparation of AUTS4 diagnostic, screening or risk assessment kits, wherein the PTCHD1 gene mutant, compared with the wild-type PTCHD1 gene (GenBank accession number: NM_173495.3), has a substitution of cytosine C for adenine A at nucleotide position 1644 of the coding sequence.

3. The application according to claim 2, characterized in that, The detection reagents include ARMS PCR, dPCR, or CRISPR / Cas system detection reagents.

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