A screening method for primary ciliary dyskinesia gene mutation spectrum, product and application
By combining nasal nitric oxide testing with whole-exome sequencing, multiple gene mutations were screened out, solving the problem of incomplete screening for gene mutations in primary ciliary dyskinesia. This approach achieved highly sensitive and specific screening while reducing testing costs.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- SHANGHAI TONGJI HOSPITAL
- Filing Date
- 2025-08-06
- Publication Date
- 2026-07-31
AI Technical Summary
Current technologies for screening gene mutations in primary ciliary dyskinesia are not comprehensive enough, leading to diagnostic difficulties. In particular, they rely on high-cost transmission electron microscopy and whole-exome sequencing, which have low availability and are difficult to effectively screen and diagnose.
The nitric oxide content of individuals was detected using a nasal nitric oxide analyzer. Dual thresholds of 77 nl/min and 85.4 nl/min were set. Combined with whole-exome sequencing analysis, various related gene mutations were screened out, including DNAH1, DNAH11, and DNAH5, providing a method for screening gene mutation profiles.
It achieved high sensitivity (88.24%) and high specificity (97.05%) screening for primary ciliary dyskinesia, improving the diagnostic accuracy and accessibility of PCD and reducing testing costs.
Smart Images
Figure CN120700138B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biotechnology, specifically to a method, product, and application for screening gene mutation profiles of primary ciliary movement disorder. Background Technology
[0002] Primary ciliary dyskinesia (PCD) is a rare autosomal recessive genetic disorder caused by defects in the development of ciliary structure or function, resulting in impaired ciliary movement. Common manifestations of PCD include recurrent respiratory infections, sinusitis, otitis media, respiratory failure, infertility, and visceral displacement. Respiratory ciliary dyskinesia leads to impaired mucus clearance, manifesting as chronic sinusitis, bronchiectasis, and other chronic lung inflammations. Abnormal ciliary movement at the nodes during the embryonic period leads to impaired fluid flow at the nodes, affecting the asymmetrical distribution of organs, manifesting as situs inversus or ectopic organs. Fallopian tube ciliary dyskinesia causes impaired epithelial fluid flow, abnormal oocyte transport, and failure of the fertilized egg to implant normally, resulting in infertility. Abnormal sperm flagellar motility prevents normal fertilization, also resulting in sterility.
[0003] PCD is one of the important genetic causes of bronchiectasis. The diagnosis of PCD relies on the observation of abnormal ciliary ultrastructure by electron microscopy, or by referring to the diagnostic guidelines for PCD. That is, if a PCD patient has two of the following four characteristics, the sensitivity and specificity for diagnosing PCD are 80% and 72%, respectively. (1) Persistent cough and sputum production throughout the year shortly after birth (usually within 6 months) with poor response to anti-infective treatment; (2) Persistent sinusitis throughout the year within 6 months after birth with poor response to anti-infective treatment; (3) Unexplained respiratory distress syndrome in full-term newborns requiring airway support for more than 24 hours; (4) Clinical manifestations of situs inversus. However, due to the high cost and low availability of transmission electron microscopy (TEM) and whole exome sequencing (WES) for PCD diagnosis, the current screening for PCD is seriously insufficient.
[0004] Chinese Patent CN106905426A discloses the LRRC6 gene mutation forms in patients with primary ciliary dyskinesia and their applications. This invention protects the following proteins: (a1) a protein composed of amino acid residues 1-179 of LRRC6; and (a2) a protein composed of amino acid residues 1-249 of LRRC6. The invention also protects the use of substances for detecting mutations A and / or B in the preparation of a reagent kit; Mutation A: nucleotide 109 of sequence 3 in the human genome is mutated from C to T; Mutation B: nucleotide 96 of sequence 4 in the human genome is mutated from G to A; The functions of the reagent kit are: (c1) to evaluate the risk of the test subject having primary ciliary dyskinesia; (c2) to evaluate the risk of the test subject or the offspring of the test couple having primary ciliary dyskinesia; and (c3) to diagnose or assist in the diagnosis of whether the test subject has primary ciliary dyskinesia. This invention has significant application value for the diagnosis of patients with primary ciliary dyskinesia.
[0005] Currently, the genes and gene mutation sites covered by primary ciliary dyskinesia are not comprehensive, and there is an urgent need to provide a screening method for the gene mutation spectrum of primary ciliary dyskinesia and a gene mutation spectrum of primary ciliary dyskinesia. Summary of the Invention
[0006] The purpose of this invention is to provide a method, product, and application for screening gene mutation profiles of primary ciliary movement disorder.
[0007] To achieve the above-mentioned objectives, the technical solution of the present invention is as follows: On the one hand, the present invention provides a method for screening gene mutation profiles of primary ciliary dyskinesia. The screening method involves using a nasal nitric oxide analyzer to detect and assess the nitric oxide content of an individual, setting dual thresholds of 77 nl / min and 85.4 nl / min, and analyzing the genes of assessed individuals that meet the threshold range using whole-exome sequencing to obtain the gene mutation profile of primary ciliary dyskinesia.
[0008] Specifically, the threshold range is less than 77 nl / min or greater than 85.4 nl / min.
[0009] Specifically, the gene mutation spectrum of primary ciliary dyskinesia includes: The DNAH1 gene has the following mutation sites: A1656V, D3927N, I190T, and K415R; The DNAH11 gene has the following mutation sites: Y2753H, C2794Y, R2997C, K4085*, V1211I, F2766L, Q3975*, P4287R, T2549R, G3039E, R2500S, V4339E, R2044*, pR3346*, R3625G, Q4390Tfs*6, R644*, Q3973*; The DNAH5 gene has the following mutation sites: R4158W, R819T, R4429Q, L3363Yfs*16, M366Rfs*8, R3885*, llel855N1s*6, D1300E, V209I, V3326L, G3008R, L3341P, D120N; The HYDIN gene has the following mutation sites: M2001T, S33G, G3472E, and H583P. The DNAH9 gene has the following mutation sites: S433C, R2272H, N1481D, G1581S, P2649R, R524*, L1152F, and R2472Q. The DNAH6 gene has the following mutation sites: S558L, G3147S; The CCDC40 gene has the following mutation sites: R301*, L1075P; The RSPH9 gene has the following mutation sites: E123*, E192*; The DNAH7 gene has the following mutation sites: Q3534R, R3204H; The DNAI2 gene has the following mutation site: T475Qfs*21; The TTC12 gene has the following mutation site: R374W; The ODAD4 gene has the following mutation sites: R252W, G587R; The RSPH1 gene has the following mutation sites: F82Yfs*9, G69R; The DNAH10 gene has the following mutation sites: Q3038K, R4196H; The CFAP74 gene has the following mutation sites: R638Q and T412M.
[0010] In another aspect, the present invention provides the application of reagents for detecting mutated genes in a risk assessment product for primary ciliary dyskinesia, wherein the genes include DNAH1, DNAH11, DNAH5, HYDIN, DNAH9, CCDC39, DNAH6, CCDC40, RSPH9, DNAH7, DNAI2, TTC12, ODAD4, RSPH1, DNAH10, CFAP74, and DNAAF4.
[0011] Specifically, the DNAH1 gene has the following mutation sites: A1656V, D3927N, I190T, and K415R; The DNAH11 gene has the following mutation sites: Y2753H, C2794Y, R2997C, K4085*, V1211I, F2766L, Q3975*, P4287R, T2549R, G3039E, R2500S, V4339E, R2044*, pR3346*, R3625G, Q4390Tfs*6, R644*, Q3973*; The DNAH5 gene has the following mutation sites: R4158W, R819T, R4429Q, L3363Yfs*16, M366Rfs*8, R3885*, llel855N1s*6, D1300E, V209I, V3326L, G3008R, L3341P, D120N; The HYDIN gene has the following mutation sites: M2001T, S33G, G3472E, and H583P. The DNAH9 gene has the following mutation sites: S433C, R2272H, N1481D, G1581S, P2649R, R524*, L1152F, and R2472Q. The DNAH6 gene has the following mutation sites: S558L, G3147S; The CCDC40 gene has the following mutation sites: R301*, L1075P; The RSPH9 gene has the following mutation sites: E123*, E192*; The DNAH7 gene has the following mutation sites: Q3534R, R3204H; The DNAI2 gene has the following mutation site: T475Qfs*21; The TTC12 gene has the following mutation site: R374W; The ODAD4 gene has the following mutation sites: R252W, G587R; The RSPH1 gene has the following mutation sites: F82Yfs*9, G69R; The DNAH10 gene has the following mutation sites: Q3038K, R4196H; The CFAP74 gene has the following mutation sites: R638Q and T412M.
[0012] Furthermore, the genes mentioned include the DNAH5 gene, DNAH11 gene, DNAH9 gene, DNAH1 gene, HYDIN gene, and DNAAF4 gene.
[0013] Furthermore, the genes mentioned include the DNAH5 gene, the DNAH11 gene, and the DNAH9 gene.
[0014] Furthermore, the DNAH5 gene includes the following mutation sites: R4158W, R4429Q, R3885*, and I855N1s*6.
[0015] Furthermore, the DNAH9 gene includes the following mutation sites: S433C and R2472Q.
[0016] In another aspect, the present invention provides a risk assessment product for primary ciliary dyskinesia, the product comprising reagents for detecting mutated genes, the genes including DNAH1, DNAH11, DNAH5, HYDIN, DNAH9, CCDC39, DNAH6, CCDC40, RSPH9, DNAH7, DNAI2, TTC12, ODAD4, RSPH1, DNAH10, CFAP74, and DNAAF4.
[0017] Specifically, the DNAH1 gene has the following mutation sites: A1656V, D3927N, I190T, and K415R; The DNAH11 gene has the following mutation sites: Y2753H, C2794Y, R2997C, K4085*, V1211I, F2766L, Q3975*, P4287R, T2549R, G3039E, R2500S, V4339E, R2044*, pR3346*, R3625G, Q4390Tfs*6, R644*, Q3973*; The DNAH5 gene has the following mutation sites: R4158W, R819T, R4429Q, L3363Yfs*16, M366Rfs*8, R3885*, llel855N1s*6, D1300E, V209I, V3326L, G3008R, L3341P, D120N; The HYDIN gene has the following mutation sites: M2001T, S33G, G3472E, and H583P. The DNAH9 gene has the following mutation sites: S433C, R2272H, N1481D, G1581S, P2649R, R524*, L1152F, and R2472Q. The DNAH6 gene has the following mutation sites: S558L, G3147S; The CCDC40 gene has the following mutation sites: R301*, L1075P; The RSPH9 gene has the following mutation sites: E123*, E192*; The DNAH7 gene has the following mutation sites: Q3534R, R3204H; The DNAI2 gene has the following mutation site: T475Qfs*21; The TTC12 gene has the following mutation site: R374W; The ODAD4 gene has the following mutation sites: R252W, G587R; The RSPH1 gene has the following mutation sites: F82Yfs*9, G69R; The DNAH10 gene has the following mutation sites: Q3038K, R4196H; The CFAP74 gene has the following mutation sites: R638Q and T412M.
[0018] Specifically, the products mentioned include, but are not limited to, reagent kits.
[0019] Specifically, the test samples for the product include, but are not limited to, blood, tissue, and saliva.
[0020] The beneficial effects of this invention are as follows: (1) This invention provides a method for screening gene mutation spectrum of primary ciliary movement disorder. The PCD-related gene mutation spectrum obtained by screening provides experience and reference for clinical PCD screening.
[0021] (2) Based on the existing electrochemical nasal nitric oxide (nNO) analyzer, this invention achieves highly sensitive (88.24%) and highly specific (97.05%) screening of primary ciliary dyskinesia (PCD) in bronchiectasis patients by setting dual thresholds of 77 nl / min and 85.4 nl / min. Attached Figure Description
[0022] Figure 1 The receiver operating characteristic (ROC) curve for the diagnostic efficacy of nasal nitric oxide (nNO).
[0023] Figure 2 ROC curve analysis was performed to assess the diagnostic efficacy of primary ciliary dyskinesia (PCD) in the test set using a critical value of nNO=77 nl / min.
[0024] Figure 3 To verify the diagnostic efficacy of PCD based on the international standard threshold (nNO=77 nl / min).
[0025] Figure 4 To verify the effectiveness of PCD hierarchical screening with a centrally optimized threshold (nNO=85.4 nl / min).
[0026] Figure 5 This is a distribution map of the PCD gene mutation frequency. Detailed Implementation
[0027] To make the technical means, creative features, and achieved objectives and effects of this invention easier to understand, the invention is further illustrated below with specific embodiments. However, the following embodiments are merely preferred embodiments of this invention and not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments described herein without creative effort are all within the protection scope of this invention. Unless otherwise specified, the operating methods and equipment used in the following embodiments are conventional operating methods, and the materials and equipment used in each embodiment are the same.
[0028] Manufacturer of experimental equipment: The electrochemical nasal nitric oxide (nNO) analyzer was purchased from Zhejiang Yiliankang Medical Technology Co., Ltd., specifically the Youhuxi BA200 exhalation analyzer.
[0029] Example 1 A total of 173 patients diagnosed with bronchiectasis by high-resolution CT were included. All subjects completed the primary ciliary dyskinesia (PCD) screening process based on medical history, clinical symptoms and prior assessment, and underwent nasal nitric oxide (nNO) level testing.
[0030] The method for detecting nasal nitric oxide (nNO) levels is as follows: Instrument preparation: Ensure the instrument is connected to a power source and powered on; wait for the instrument to complete its warm-up and self-test; check the calibration status; prepare a disposable nasal breathing sampling kit (model TF731, including a whistle and nasal adapter); ensure the instrument display is working properly and that the battery is fully charged.
[0031] Patient preparation: Explain the purpose and procedure of the test to the patient to gain their cooperation. Inform the patient to avoid the following activities or substances for a period of time before the test (usually 1-2 hours) to avoid affecting the results: strenuous exercise; smoking or exposure to secondhand smoke; use of nasal spray. The patient should adopt a comfortable sitting posture; ensure the patient's nasal passages are relatively clear.
[0032] Testing Procedure: On the instrument's main interface, select the "Nasal Nitric Oxide (nNO)" detection mode. Enter patient information (patient's ID card, name, age, and gender); connect the disposable nasal adapter to the sampling port. Plug one tip of the nasal adapter into one of the patient's nostrils and instruct the patient to hold a whistle in their mouth. Instruct the patient to inhale deeply and then exhale gently and continuously through their nose while whistling (this creates pressure at the back of the mouth, helping to close the soft palate and prevent air from the lungs from entering the nasal cavity, thus ensuring that the collected gas primarily comes from the nasal cavity and sinuses). Once the patient is ready and begins the specified breathing / humming action, click "Start" or the corresponding button on the instrument to initiate gas sampling. The instrument will begin drawing a gas sample from the nasal adapter. The patient needs to continue nasal exhalation and humming as instructed until the instrument completes sampling (10 seconds). After sampling, the instrument screen will immediately display the nasal NO measurement result and sampling curve.
[0033] In the initial screening, 15 patients (8.67%, 15 / 173) were found to have nNO levels below the internationally recommended diagnostic threshold of 77 nl / min. Figure 2 Sixteen patients with PCD were ultimately diagnosed through comprehensive clinical diagnosis, accounting for 9.25% (16 / 173) of the total screened population. ROC curve analysis determined that the optimal threshold for nNO in this population to diagnose PCD was 85.4 nl / min. Figure 1 ).
[0034] When 77 nl / min was used as the diagnostic cutoff for nasal nitric oxide (nNO) screening for primary ciliary dyskinesia (PCD) in the validation set, the sensitivity of the detection was 88% and the specificity was 93%. Figure 3 Further receiver operating characteristic (ROC) curve analysis showed that 85.4 nl / min was the optimal threshold for maximizing diagnostic accuracy, with a sensitivity of 92% and a specificity of 93%, indicating that it can serve as an important supplement to auxiliary judgment and graded screening. Figure 4 ).
[0035] Patients carrying DNAH5 or DNAH9 mutations generally have nNO levels higher than 77 nl / min (Table 1), suggesting that using 77 nl / min as the sole screening threshold for these specific genotypes may lead to missed diagnoses. Therefore, this invention proposes using dual thresholds (77 nl / min and 85.4 nl / min) for tiered identification, which helps improve overall screening accuracy and population adaptability.
[0036] Table 1. nNO levels and genotypes in PCD patients
[0037] By performing whole-exome sequencing (WES) analysis on patients diagnosed with primary ciliary dyskinesia (PCD), this invention identified multiple known or potential pathogenic gene variants (Table 2, ...). Figure 5 ).
[0038] Whole exome sequencing provides BGI with whole exome sequencing (WES) services based on its DNBSEQ™ sequencing technology.
[0039] Variant detection frequency corresponds Figure 5 Gene detection frequency was calculated as the ratio of single-gene detection counts to total gene detection counts. The genes with the highest mutation detection frequency were DNAH5 (23.1% = 9 / 38) and DNAH11 (20.5%), followed by DNAH9 (12.8%). Furthermore, mutations in HYDIN, DNAH1, and DNAAF4 each accounted for 5.1% of the samples. Other low-frequency mutated genes included CCDC40, CCDC39, DNAH6, RSPH1, RSPH9, DNAH7, DNAI2, TTC12, ODAD4, and DNAH10, all of which were detected only in single cases.
[0040] Among specific mutation sites, some of the more common ones include: DNAH5 gene: R4158W (16.67%, 3 / 18), R4429Q (11.11%, 2 / 18), R3885* (11.11%, 2 / 18), I855N1s*6 (11.11%, 2 / 18). DNAH9 gene: S433C and R2472Q each account for 20% (2 / 10); DNAAF4 gene: c.784-1G>A variant appeared in all tested samples (2 / 2), with a detection rate of 100%.
[0041] It is worth noting that the two patients carrying the DNAAF4 c.784-1G>A variant exhibited different visceral localization phenotypes: case 33 showed normal visceral location, while case 34 showed visceral inversion (Table 2). This phenomenon suggests that significant phenotypic differences may still exist even under the same gene mutation background, and also supports the value of the strategy proposed in this invention, which uses graded nNO thresholds for screening combined with genotypic supplementary judgment.
[0042] Table 2. Inheritance of PCD in the queue
[0043] Table 2 (continued 1)
[0044] Table 2 (continued 2)
[0045] Table 2 (continued 3)
[0046] Table 2 (continued 4)
[0047] Note: cHGVS: HGVS (Human Genome Variation Society) refers to the HGVS nomenclature for coding DNA sequences; pHGVS: HGVS nomenclature for protein sequences; Ens CondelPred: Ensembl database Condel harmfulness prediction; NA: No data / Not Available; P: Pathogenic; Polymorphism.
[0048] Example 2 Forty-three patients underwent transmission electron microscopy (TEM) examination of nasal epithelial cells.
[0049] The specific steps for transmission electron microscopy (TEM) examination are as follows: Nasal epithelial cells were collected from the bilateral inferior turbinates using a sterile cell brush and immediately fixed in 2.5% glutaraldehyde (pH 7.4, 4°C) under light-protected conditions. Samples were processed and photographed using a transmission electron microscope (TEM, Leica UC7, Germany) (performed by Wuhan Saiwei Biotechnology Co., Ltd.) to assess ciliary ultrastructure. The diagnostic criteria for primary ciliary dyskinesia (PCD) included: 1) isolated external dynein arm (ODA) defect; 2) combined ODA and internal dynein arm (IDA) defect; and 3) IDA defect with microtubule disintegration. All TEM images were independently evaluated by two specialists to ensure an unbiased interpretation of the ultrastructural abnormalities.
[0050] Transmission electron microscopy (TEM) was performed on nasal epithelial cell samples from 43 patients to assess changes in ciliary ultrastructure. Interpretable images were obtained from 19 samples. Of these 19, 8 showed significant ciliary structural abnormalities, including the following three types: (1) Outer Dynein Arm (ODA) defects: accounting for 50% (4 / 8); (2) Combined defects of inner and outer power arms (IDA / ODA): accounting for 25% (2 / 8); (3) Abnormal central microtubule structure or disordered microtubule arrangement: accounting for 25% (2 / 8).
[0051] It is noteworthy that phenotypic inconsistencies were observed even when genotypes were identical. For example, identical twin cases (numbers #12 and #13) both carried the same DNAH5 mutation, but differed in TEM test results and clinical presentation: only one case showed situs inversus, while the other did not.
[0052] Example 3 To further validate the optimal threshold of 85.4 nl / min, PCD screening and diagnosis were performed again on 218 patients with bronchiectasis. When 77 nl / min was used as the diagnostic threshold for nNO screening of PCD, the detection sensitivity was 88% and the specificity was 93%. Figure 3 When a diagnostic threshold of 85.4 nl / min is used, the detection sensitivity is 92% and the specificity is 93%. Figure 4 The above results indicate that when using nNO as an auxiliary diagnostic indicator, the proposed method of using 77 nl / min and 85.4 nl / min for graded screening can improve the accuracy and applicability of overall nNO screening.
[0053] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.
Claims
1. A method for screening gene mutation profiles of primary ciliary dyskinesia, characterized in that, The screening method involves using a nasal nitric oxide analyzer to detect and assess an individual's nitric oxide content, setting a threshold of 85.4 nl / min. The threshold range is less than 85.4 nl / min. Individuals meeting the threshold range are analyzed using whole-exome sequencing to obtain the gene mutation spectrum of primary ciliary dyskinesia. The aforementioned gene mutation spectrum of primary ciliary dyskinesia includes: The DNAH1 gene has the following mutation sites: A1656V, D3927N, I190T, and K415R; The DNAH11 gene has the following mutation sites: Y2753H, C2794Y, R2997C, K4085*, V1211I, F2766L, Q3975*, P4287R, T2549R, G3039E, R2500S, V4339E, R2044*, pR3346*, R3625G, Q4390Tfs*6, R644*, Q3973*; The DNAH5 gene has the following mutation sites: R4158W, R819T, R4429Q, L3363Yfs*16, M366Rfs*8, R3885*, llel855N1s*6, D1300E, V209I, V3326L, G3008R, L3341P, D120N; The HYDIN gene has the following mutation sites: M2001T, S33G, G3472E, and H583P. The DNAH9 gene has the following mutation sites: S433C, R2272H, N1481D, G1581S, P2649R, R524*, L1152F, and R2472Q. The DNAH6 gene has the following mutation sites: S558L, G3147S; The CCDC40 gene has the following mutation sites: R301*, L1075P; The RSPH9 gene has the following mutation sites: E123*, E192*; The DNAH7 gene has the following mutation sites: Q3534R, R3204H; The DNAI2 gene has the following mutation site: T475Qfs*21; The TTC12 gene has the following mutation site: R374W; The ODAD4 gene has the following mutation sites: R252W, G587R; The RSPH1 gene has the following mutation sites: F82Yfs*9, G69R; The DNAH10 gene has the following mutation sites: Q3038K, R4196H; The CFAP74 gene has the following mutation sites: R638Q and T412M.