CN115807078A - Capture probe and gene chip for screening recessive genetic diseases, and method and application thereof - Google Patents
Capture probe and gene chip for screening recessive genetic diseases, and method and application thereof Download PDFInfo
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- CN115807078A CN115807078A CN202310010197.6A CN202310010197A CN115807078A CN 115807078 A CN115807078 A CN 115807078A CN 202310010197 A CN202310010197 A CN 202310010197A CN 115807078 A CN115807078 A CN 115807078A
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Abstract
The invention discloses a capture probe and a gene chip of a recessive genetic disease pathogenic gene pathogenic mutation site and a pCNV detection region, and a method and application thereof. The gene chip comprises a recessive genetic disease pathogenic gene pathogenic mutation site and a capture probe of a pCNV detection region. The gene chip provided by the invention can screen pathogenic loci and pCNV of various high-incidence genetic diseases of Chinese population at one time, and compared with the traditional product which mainly screens single disease species, the gene chip has wide disease species coverage.
Description
Technical Field
The invention relates to the technical field of in-vitro gene detection, in particular to a capture probe and a gene chip of a recessive genetic disease pathogenic gene pathogenic mutation site and a pCNV detection region, and a method and application thereof.
Background
Birth defects refer to abnormalities in the body structure, function, and metabolism of an infant that occur before birth. Birth defects are of various types and complex etiology, and are mainly caused by genetic factors such as chromosome abnormality and gene mutation, and can also be caused by environmental factors. The comprehensive prevention and treatment of birth defects is mainly completed by three-level preventive measures. The first stage of prevention is to discover individuals carrying pathogenic variation through means of genetic counseling, gene detection and the like before marriage and pregnancy. The second stage is diagnosis during and before pregnancy to discover a birth defect fetus. The third prevention is the screening of the newborn after birth.
Carrier screening (carrier screening) refers to identifying healthy individuals carrying autosomal or X-chromosome linked recessive genetic disease related gene mutation through gene detection before or in the early stage of pregnancy, namely carriers, and is an important measure for primary prevention. The carrier usually has no obvious phenotype, but when the couples carry the same pathogenic variation, the risk of the carrier developing the diseased offspring is greatly increased. Therefore, the carrier screening method has great significance for the carrier screening of couples who have no family genetic history and have no birth of babies with monogenic diseases. The carrier screening starts in the 70 th 20 th century, and only a single disease and a single race are screened in the early stage, such as Tay-Sachs disease of Tajews; by this century, the range of international carrier screening has gradually expanded from single disease species to multiple disease species, and single ethnicity to multiple ethnicities. At present, the carrier screening is carried out clinically, the screening aiming at single diseases is common, such as deafness, thalassemia and the like, but the human genetic diseases are 9000, and the screening of the single diseases is far from achieving the effect of preventing high-incidence genetic diseases, which is also a problem faced by the whole international environment. In order to solve the problem, products capable of detecting multiple genetic diseases simultaneously appear on the market, for example, a whole exome sequencing method or a large Panel (hundreds of thousands of genetic diseases) combined with a second-generation sequencing method is adopted, and the products cover more types of diseases, but the adopted detection method inevitably has the defects of high sequencing cost, long period, overlarge data volume, insufficient coverage depth, detection omission and the like. Therefore, the detection method which can cover various high-incidence hereditary diseases of people and has accurate and reliable detection result, short period and low detection cost is a means for effectively preventing birth defects.
Disclosure of Invention
<xnotran> (pCNV, pathogenic copy number variants) , AGL, DBT, GBA, CRB1, USH2A, GJB3, KCNQ4, MMACHC, ACADM, HPS1, MYO3A, PCDH15, CDH23, POLR3A, PTS, MPZL2, SLC37A4, USH1C, OTOG, HBB, SMPD1, PYGM, MYO7A, TYR, PAH, ACADS, MSRB3, PCCA, GJB2, GJB6, ATP7B, OCA2, IVD, STRC, OTOA, HBA2, TBC1D24, SLC12A3, PMM2, MYO15A, TMEM132E, G6PC, ACADVL, USH1G, GAA, SGSH, NPC1, LOXHD1, LDLR, GCDH, BCKDHA, MYH14, PJVK, COL4A4, COL4A3, OTOF, TMPRSS3, MYH9, TRIOBP, MCM2, PCCB, GLB1, MMAA, ETFDH, GRXCR1, WFS1, SCD5, IDUA, PPIP5K2, SLC22A5, SMN1, ARSB, EYA4, CYP21A2, MMUT, MUT, PKHD1, MYO6, SLC26A4, CFTR, PDE1C, SLC25A13, TYRP1, ASS1, TJP2, TMC1, ABCD1, G6PD 12S rRNA . </xnotran>
Optionally, the pathogenic site comprises at least one selected from the group consisting of:
the pathogenic sites include 11:112099388, 11:112103901, 11:112103928, 11:112099026, 11:112101362, 16:56899326, 16:56914054, 1:155205043, 1:155207932, 1:155205620, 1:155208006, 1:155205497, 15:40710389, 2:227958870, 2:228128561, 4:83557920, 7:31890328, 11:118130890, 11:118133651, 11:112100933, 1:35250784, 1:35250860, 6:133834014, 22:36701983, 1:41284190, 1:41304146, 13:20763527, 13:20763138, 13:20763294, 13:20763395, 13:20763491, 13:20763582, 13:20763612, 13:20763712, 13:20766921, 19:50771562, 9:71852823, 4:6293031, 4:6302395, 4:6303195, 4:6303211, 4:6303521, 4:6303671, 4:6303692, 3:127318284, 5:102509657, 11:17574667, 11:17635243, 11:17655765, 11:17655766, 13:20797501, 16:21730783, 4:43032468, 6:76596598, 6:76599857, 9:75407236, 9:75435804, 9:75309494, 12:65856934, 16:2550465, 17:32959769, 16:56899395, 7:107303803, 7:107314608, 7:107329648, 7:107350576, 7:107303811, 7:107303845, 7:107314756, 7:107315495, 7:107315505, 7:107323796, 7:107323927, 7:107329575, 7:107330597, 7:107330644, 7:107330657, 7:107334920, 7:107335067, 7:107336429, 7:107336459, 7:107336462, 7:107338487, 7:107338528, 7:107338557, 7:107340599, 7:107344827, 1:197390534, 1:215848138, 1:215933077, 1:215990338, 1:215990440, 1:216051224, 1:216052410, 1:216246252, 1:216246634, 1:216251430, 10:55892719, 10:73550924, 11:17547982, 11:76886438, 11:76900471, 11:76901916, 17:72915838, 11:76901129, 10:73330641, 11:76867057, 11:76916643, 11:76919825, X:152991559, 9:12702411, 11:88911467, 11:88924445, 11:88960984, 11:89017955, 15:28096536, 10:100186986, 10:79769439, 12:103234270, 12:103234277, 12:103237449, 12:103240702, 12: 10324521, 12:103260411, 3:135975430, 3:136046081, 3:136046477, 3:136046480, 3:136046492, 13:100959516, 13:101167783, 15:43892272, 22:38119602, 22:38120925, 22:38121788, 10:26462691, 17:18025499, 17:18049349, 17:18054149, 17:18060523, 17:18075499, 17:18023292, 17:18025637, 2:179320828, 18:44114297, 18:44065090, 21:43808612, 21:43802210, 21:43809044, 2:26681086, 2:26683865, 2:26685039, 2:26685045, 2:26705384, 2:26706449, 19:11216105, 19:11216264, 19:11224008, 19:11226874, 6:51910887, 6:51924836, 19:41925187, 1:100706416, 13:52511706, 13:52513240, 13:52515330, 13:52523835, 13:52523908, 13:52524434, 13:52532469, 13:52532497, 13:52539170, 13:52539174, 13:52548830, 1:155207203, 1:155207932, 1:155208006, 1:155210420, 7:95751240 and 7:95813588, 7:95813688, 7:95818684, 7:95822344, 9:133342110, 17:7123518, 17:7127359, 4:146572222, 6:49399515, 6:49409685, 6:49415462, 6:49419231, 1:45966084, 1:45974001, 1:45974005, 7:117230498, 7:117232086, 7:117232300, 11:6412870, 11:6413142, 18:21113439, 18:21131617 and 3:33099712, 5:78135195, 17:78184631, 17:78187645, 4:995488, 4:996842, 4:997815, 4:998101, 1:100327076, 1:100345603, 1:100350260, 11:64517895, 11:118895981, 17:78078341, 17:78078931, 17:78082617, 17:78083828, 17:78086465, 17:78086721, 17:78086801, 17:78090815, 17:78092467, 17:78093083, 4:159603421, 19:13007763, 19:13008223, 19:13008590, 6:32006858, 6:32007203, 6:32007887, 6:32008198, 16:8905010, 16:8905018, 16:8906904, 16:8941632, 5:131714072, 5:131719847, 5:131721062, 1:76211508, 1:76215238, 1:76227050, 12:103249009, 12:103288534, 12:103237555, 12:103260442, 12:103237426, 12:103288655, 12:103260385, 12:103246653, 12:103246591, 12:103234192, 12:103246665, 12:103246695, 13:52520505, 13:52524252, 13:52523859, 5:70220951, 5:70240540, 5:70240546, 5:70247796, 5:70238311, 5:70247763, 5:70238374, 1:45974647, 1:45974694, 7:117246728, 7:117199645, 18:21116700, 17:41063017, 17:41055965, 11:118898391, 11:118898518, 11:88924446, 11:88924382, 11:88924475, 5:131721127, 19:13010280, 17:7127303, 17:7127350, 17:7123443, 6:49425427, 6:49419405, 6:49421467, 6:49412365, 7:117149186, 7:117227874, 7:117171005, 12:121176944, 12:121164946, 5:131728257, 5:131705715, 5:131714104, 5:131706002, 1:76226846, 1:76198409, 1:76200534, 1:76226946, 1:76205776, 20:10653348, 11:6411832, 11:6413134, 11:6413037, 11:6415243, 11:6415506, 11:6411935, 7:107303857, 7:107314782, 7:107323898, 7:107330593, 7:107330645, 7:107330648, 7:107340625, 7:107342443, 7:107342495, 7:107350571, 7:107350577, 16:223539, 16:223547, 16:223597, 11:5247153, 11:5247802, 11:5247904, 11:5247992, 11:5247993, 11:5247994, 11:5247995, 11:5247996, 11:5248008, 11:5248009, 11:5248012, 11:5248028, 11:5248155, 11:5248159, 11:5248161, 11:5248166, 11:5248172, 11:5248173, 11:5248198, 11:5248200, 11:5248203, 11:5248206, 11:5248250, 11:5248259, 11:5248301, 11:5248329, 11:5248330, 11:5248331, 11:5248332, 11:5248333, 11:5248351, 11:5248374, 11:5248391, 12:103234255, 12:103246680, 12:103246701, 12:103246707, 12:103246714, 1:35250901, 1:35250910, 13:20763209, 13:20763421, 13:20763486, 13:20763530, 13:20763686, X:153760472, X:153760473, X:153760479, X:153760484, X:153760605, X:153760654, X:153761184, X:153761204, X:153761337, X:153762605, X:153762680, X:153762704, X:153762710, X:153763476, X:153763485, X:153774276, MT: 1095. MT:1494 and MT:1555.
the pCNV detection region includes at least one of: 16:199800-234700, X:31645789-31645979, X:31676106-31676261, X:31697491-31697703, X:31747747-31747865, X:31792076-31792309, X:31838091-31838200, X:31854834-31854939, X:31893307-31893490, X:31947712-31947862, X:31950196-31950344, X:31986455-31986631, X:32235032-32235180, X:32490280-32490426, X:32503035-32503216, X:32509393-32509635, X:32519871-32519959, X:32536124-32536248, X: 3256275-3256451, X:32583818-32583998, X:32591646-32591754, X:32591861-32591963, X:32613873-32613993, X:32632419-32632570, X:32662248-32662430, X:32663080-32663269, X:32715986-32716115, X:32717228-32717410, X:32827609-32827728, X:32834584-32834757, X:32841411-32841504, X:32862899-32862977 and X:32867844-32867937.
For example, the gene, disease, chromosome, position, reference base (i.e., wild-type base) sequence and variant base sequence of the pathogenic site are shown in FIGS. 1A to 1K, and the disease and chromosome position of the pCNV are shown in FIGS. 2A to 2D.
Optionally, the capture probe is according to the above 1) or 2):
1) Consists of one or more probes shown in SEQ ID No.1 to SEQ D No.1109 in a sequence table;
2) The probe consists of one or more of derivatives of each probe in 1), wherein the derivatives of each probe are the probes which are obtained by substituting and/or deleting and/or adding one or more nucleotides in each probe in 1) and have the same functions.
The substitution and/or deletion and/or addition of one or several nucleotides is a substitution and/or deletion and/or addition of not more than 5 nucleotides, such as not more than 1, 2, 3, 4 or 5 nucleotides.
The information of the disease, chromosome position, etc. corresponding to the capture probe can be shown in FIGS. 1A-1K and FIGS. 2A-2D.
The invention also provides a gene chip comprising the capture probe.
The application of the capture probe and the gene detection chip also belong to the protection scope of the invention. Specifically, the application is the application of the capture probe or the gene detection chip in the preparation of products for screening and/or predicting and/or assisting in diagnosing and/or diagnosing recessive genetic diseases.
The recessive genetic disorder can include a disorder selected from glycogen accumulating disease, maple syrup urine disease, gaucher disease, usher syndrome, autosomal dominant hereditary deafness type 2B, autosomal dominant hereditary deafness type 2A, methylmalonic acidemia, medium-chain acyl-CoA dehydrogenase deficiency, albino (syndromic) HPS-1, autosomal recessive hereditary deafness type 30, hypomyelinating white matter dystrophy 7 with or without hypodentate and/or gonadotropic hypogonadism, BH4 deficiency homophenylalaninemia, autosomal recessive deafness type 111, autosomal recessive hereditary deafness type 18B, beta thalassemia, niemann pick's disease, albino (non-syndromic) OCA-1, phenylketonuria, short-chain acyl-CoA dehydrogenase deficiency, autosomal recessive deafness type 74, propionic acidemia, autosomal hereditary deafness type 3A, autosomal recessive hereditary deafness type 1A, autosomal recessive hereditary deafness type 1B, hepatolenticular degeneration, albinism (non-integrative) OCA-2, isovaleric acidemia, autosomal recessive hereditary deafness type 16, autosomal recessive hereditary deafness type 22, alpha-type thalassemia, autosomal recessive hereditary deafness type 86, gitelman syndrome, congenital glycosylation disorder, autosomal recessive hereditary deafness type 3, autosomal recessive hereditary deafness type 99, very long chain acyl coenzyme A dehydrogenase deficiency, mucopolysaccharidosis storage disorder, autosomal recessive hereditary deafness type 77, homozygotic familial hypercholesterolemia, glutaremia, autosomal dominant hereditary deafness type 4A, autosomal recessive hereditary deafness type 59, alport syndrome, autosomal recessive hereditary deafness type 9, autosomal recessive hereditary deafness type 8/10, autosomal dominant hereditary deafness type 17, autosomal recessive hereditary deafness type 28, autosomal dominant hereditary deafness type 70, autosomal recessive hereditary deafness type 25, autosomal dominant hereditary deafness type 6/14/38, autosomal dominant hereditary deafness type 79, autosomal recessive hereditary deafness type 100, primary carnitine deficiency, spinal muscular atrophy, autosomal dominant hereditary deafness type 10, congenital adrenal hyperplasia, polycystic kidney, autosomal recessive hereditary deafness type 37, pendred syndrome/autosomal recessive deafness type 4, cystic fibrosis, autosomal dominant hereditary deafness type 74, cucurbitemia, OCA (non-amineismic) disease OCA-3, autosomal dominant hereditary deafness type 51, albino autosomal recessive hereditary deafness type 7/11, autosomal hereditary deafness, X-linked adrenoleukodystrophy, progressive myodystrophy [ 6-G-glucose dehydrogenase ] or a variety of drug-deficient PDs.
The invention also provides a method for capturing the pathogenic site of the recessive genetic disease pathogenic gene and the pCNV detection region in a sample to be detected, which comprises the following steps:
the capture probe is used for capturing genome DNA of a sample to be detected, single base extension and fluorescence labeling are carried out, and a target site is obtained, wherein the target site is a pathogenic site of a recessive genetic disease pathogenic gene in the sample to be detected or a related site in a pCNV detection area.
The invention also provides a method for detecting the pathogenic site and pCNV of the recessive genetic disease pathogenic gene in a sample to be detected, which comprises the following steps:
1) Capturing the genome DNA of a sample to be detected by adopting the capture probe to obtain a target region fragment;
2) Performing single base extension and fluorescence labeling on the target region segment;
3) Scanning the fluorescent marker to obtain fluorescent marker data;
4) And analyzing the fluorescence labeling data to obtain a detection result of the pathogenic site of the recessive genetic disease pathogenic gene and the pCNV in the sample to be detected.
In the above method, the step 1) may further comprise nucleic acid extraction and/or library construction. The nucleic acid extraction comprises the step of performing nucleic acid extraction by using peripheral blood or saliva of a detected person (namely a sample to be detected) to obtain genome DNA of the sample to be detected. The library construction comprises the steps of carrying out isothermal displacement amplification on genome DNA of a sample to be detected by using a random primer and polymerase, carrying out fragmentation treatment on an amplification product, and purifying the fragmentation product to obtain a DNA library, wherein the step 1) comprises the step of capturing the DNA library by using the capture probe to obtain a target region fragment.
Hereinbefore, the reference bases/sequences (i.e. wild type bases/sequences) are from the reference genome of the human hg19 version of the Ensembl database (download path: ftp:// ftp. Ensemble. Org/pub/grch37/current/fasta/Homo _ sapiens/dna/Homo _ sapiens. GRCh37.Dna. Topevel. Fa. Gz.).
In the above, in the pathogenic locus and pCNV detection region, the number or letter before the colon indicates the chromosome where the pathogenic locus or pCNV detection region is located, and the number after the colon indicates the position of the pathogenic locus or pCNV detection region on the chromosome, for example, "11:112099388 "indicates position 112099388 on chromosome 11.
The gene chip provided by the invention can screen pathogenic loci and pCNV of various high-incidence genetic diseases of Chinese population at one time, and compared with the traditional product which mainly screens single disease species, the gene chip has wide disease species coverage.
The method uses a chip platform to detect the pathogenic locus variation and pCNV, has high flux, low cost, accessibility of large-scale screening, good specificity, high accuracy, rapidness and convenience, and avoids the defects of high platform sequencing cost, long period, overlarge data volume, insufficient coverage depth, detection omission and the like of whole exome sequencing and the like.
The invention provides a method for detecting a pathogenic site of a recessive genetic disease pathogenic gene and pCNV in a sample to be detected, and avoids the defects of inaccurate result, long time consumption and the like caused by sample transfer and other operations.
Drawings
FIGS. 1A-1K are diagrams illustrating the information related to the disease, gene, chromosome, position, reference sequence, variant sequence, etc. corresponding to the pathogenic site of the recessive genetic disease pathogenic gene of the capture probe according to an embodiment of the present invention.
FIGS. 2A to 2D are diagrams illustrating the information related to the disease, chromosome position, etc. corresponding to the recessive genetic disease-causing gene pCNV corresponding to the capture probe according to an embodiment of the present invention.
FIG. 3 shows the detection of the causative mutation in example 1.
FIG. 4 shows the detection of the causative mutation in example 2.
Wherein "-" represents a base deletion.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Reagents for hybrid Capture and extension labeling used in the following examples were kits of Illumina
HTS Assay Kit.
Example 1 discovery of high-risk couples and avoidance of birth defects Using the screening method of the present invention
(1) Collecting samples: peripheral blood of a couple of couples in the preparatory pregnancy stage (both couples have no family history of genetic diseases and have no child with the genetic disease) is collected, 2mL of the peripheral blood is collected for each couple, and the peripheral blood is stored by using an EDTA anticoagulant tube.
(2) Experimental and analytical procedures:
DNA extraction was performed using a blood sample of the subject, template amplification was performed using 2. Mu.g of DNA, and the amplified product was subjected to fragmentation treatment to obtain a DNA library.
The gene chip provided by the invention is used for carrying out hybridization capture and extension labeling on target fragments in the DNA library. The method mainly comprises the following steps: placing the gene chip in
In the hybridization cassette of HTS Assay Kit, use
Carrying out hybridization capture on the DNA library by using related reagents in the HTS Assay Kit and 330ml of absolute ethyl alcohol, and incubating overnight at 20 ℃; use of
Relevant reagents in the HTS Assay Kit and 95% formamide/1 mM EDTA were extension-labeled, and the gene chip with the extension-labeled was placed in a vacuum pump and dried at 0.9bar for 50min.
And scanning the chip by using an iScan scanner for about 40min to obtain fluorescence labeling data.
And finally, converting the idat signal file (namely fluorescence labeling data) into a file in a gtc format and a genotype file in a plink format (plus/. Map) respectively by using matched data analysis software according to the BPM annotation file and the EGT clustering file of the chip. Where gtc formatted files will be used for pCNV analysis and plink formatted files will be used for disease-causing site analysis.
The genotype of the SNP site in the genotype file in the plink format was converted into the genotype of the sense strand of the genome. And (3) extracting the locus genotype of each sample according to the predefined carrier screening related locus (namely the pathogenic locus detected by the gene chip), and obtaining the data of all the carrier screening related loci of each sample. Since the wild-type locus is not pathogenic, only homozygous or heterozygous loci for variation in each sample are reported. And finally, performing disease annotation, genetic mode annotation, population frequency annotation, protein damage prediction, ACMG/AMP pathogenicity rating annotation and the like on the loci respectively.
Extracting BAF (B allow Frequency) and LRR (Log R Ratio) information of all sites of the sample from a file in gtc format, identifying CNV (copy number variation) according to the BAF and LRR of the sample, reserving a CNV detection region at least comprising 10 probes, and prompting the risk of disease to the subject if the CNV detection region is pCNV.
(3) After the pathogenic loci and pCNV of the couple are detected by using the screening method, the wild type loci are filtered out, and the pathogenic mutation detection condition is shown in figure 3.
In this example, both couples tested all the pathogenic loci and pCNV contained in the genechip, and both tested 1 pathogenic mutant loci, both heterozygous pathogenic mutations of SLC26A4 gene: c.919-2A > G, which is a splicing variation, and the other sites are wild type. The variation is the pathogenic variation of Pendred syndrome or autosomal recessive hereditary deafness type 4 with vestibular aqueduct enlargement (both belonging to hereditary deafness). Through the screening method, both couples and couples are determined to be carriers of hereditary deafness pathogenic variation, and although the two couples have no deafness related phenotype and family history, the probability of breeding descendants of hereditary deafness is 25%. Through gene screening, the method is helpful for timely effective prevention measures of the couple, and the good prenatal and postnatal care is realized.
Example 2 screening of carriers of recessive genetic diseases in a region Using the screening method of the present invention
(1) Collecting samples: peripheral blood was collected from 1000 healthy individuals in a region, 2mL per person, and stored using EDTA anticoagulant tubes.
(2) Following the experimental and analytical procedures of example 1, nucleic acid extraction-library construction-hybrid capture and extension tag-chip scanning were performed sequentially on each sample, and the scan data was finally analyzed.
(3) The screening results are shown in FIG. 4.
In this example, heterozygous variations, i.e., pathogenic variations of PAH and G6PD genes, were detected in two healthy individuals, and no pathogenic variation contained in the gene chip was detected in the remaining 998 subjects.
Heterozygous variation of PAH gene was detected in blood samples from subject 1 (male, 30 years, maiden): c.331C > Tp.R111 nonsense mutation, is pathogenic variation of phenylketonuria (autosomal recessive inheritance), and other sites are wild type. By the screening method, the detected person 1 is a phenylketonuria pathogenic variation carrier, and the carrier is recommended to be screened for phenylketonuria in a marriage or a pre-pregnancy spouse in the future so as to determine the risk of developing the sick offspring.
Heterozygous variation of the G6PD gene was detected in blood samples of subject 2 (female, 25 years old, maiden): c.95A > G p.H32R missense mutation, is pathogenic variation of glucose 6-phosphate dehydrogenase deficiency anemia (X chromosome recessive inheritance), and other sites are all wild types. The disease is recessive inheritance of an x chromosome, and a detected person 2 is mated with a healthy male to breed male offspring with 50% of the risk of the disease and breed female offspring which are all healthy phenotypes.
Screening recessive genetic disease carriers to screen out one carrier of the pathogenic variation carriers of phenylketonuria and glucose 6-phosphate dehydrogenase deficiency anemia in the area, giving reasonable suggestions to future marriage and childbirth plans, helping the carriers to take effective intervention measures in time, helping to improve the population quality of the area and promoting the development of prenatal and postnatal care in the area.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific examples, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.
Claims (8)
1. A group of recessive genetic disease pathogenic gene pathogenic locus and pathogenic copy number variation pCNV detection area capture probes, characterized in that: the recessive genetic disease pathogenic gene comprises AGL, DBT, GBA, CRB1, USH2A, GJB3, KCNQ4, MMACHC, ACADM, HPS1, MYO3A, PCDH15, CDH23, POLR3A, PTS, MPZL2, SLC37A4, USH1C, OTOG, HBB, SMPD1, PYGM, MYO7A, TYR, PAH, ACARDS, MSRB3, PCCA, GJB2, GJB6, ATP7B, OCA2, IVD, STRC, OTOA, HBA2, TBC1D24, SLC12A3, PMM2, MYO15A, TMEM132E, G6PC, ACALDVUSH 1G, GAA one or more of SGSH, NPC1, LOXHD1, LDLR, GCDH, BCKDHA, MYH14, PJVK, COL4A4, COL4A3, OTOF, TMPRSS3, MYH9, TRIOBP, MCM2, PCCB, GLB1, MMAA, ETFDH, GRXCR1, WFS1, SCD5, IDUA, PPIP5K2, SLC22A5, SMN1, ARSB, EYA4, CYP21A2, MMUT, MUT, PKHD1, MYO6, SLC26A4, CFTR, PDE1C, SLC25A13, TYRP1, ASS1, TJP2, TMC1, ABCD1, G6PD, and 12S rRNA.
2. The capture probe of claim 1, wherein: the pathogenic site comprises at least one selected from the group consisting of:
11:112099388, 11:112103901, 11:112103928, 11:112099026, 11:112101362, 16:56899326, 16:56914054, 1:155205043, 1:155207932, 1:155205620, 1:155208006, 1:155205497, 15:40710389, 2:227958870, 2:228128561, 4:83557920, 7:31890328, 11:118130890, 11:118133651, 11:112100933, 1:35250784, 1:35250860, 6:133834014, 22:36701983, 1:41284190, 1:41304146, 13:20763527, 13:20763138, 13:20763294, 13:20763395, 13:20763491, 13:20763582, 13:20763612, 13:20763712, 13:20766921, 19:50771562, 9:71852823, 4:6293031, 4:6302395, 4:6303195, 4:6303211, 4:6303521, 4:6303671, 4:6303692, 3:127318284, 5:102509657, 11:17574667, 11:17635243, 11:17655765, 11:17655766, 13:20797501, 16:21730783, 4:43032468, 6:76596598, 6:76599857, 9:75407236, 9:75435804, 9:75309494, 12:65856934, 16:2550465, 17:32959769, 16:56899395, 7:107303803, 7:107314608, 7:107329648, 7:107350576, 7:107303811, 7:107303845, 7:107314756, 7:107315495, 7:107315505, 7:107323796, 7:107323927, 7:107329575, 7:107330597, 7:107330644, 7:107330657, 7:107334920, 7:107335067, 7:107336429, 7:107336459, 7:107336462, 7:107338487, 7:107338528, 7:107338557, 7:107340599, 7:107344827, 1:197390534, 1:215848138, 1:215933077, 1:215990338, 1:215990440, 1:216051224, 1:216052410, 1:216246252, 1:216246634, 1:216251430, 10:55892719, 10:73550924, 11:17547982, 11:76886438, 11:76900471, 11:76901916, 17:72915838, 11:76901129, 10:73330641, 11:76867057, 11:76916643, 11:76919825, X:152991559, 9:12702411, 11:88911467, 11:88924445, 11:88960984, 11:89017955, 15:28096536, 10:100186986, 10:79769439, 12:103234270, 12:103234277, 12:103237449, 12:103240702, 12: 10324521, 12:103260411, 3:135975430, 3:136046081, 3:136046477, 3:136046480, 3:136046492, 13:100959516, 13:101167783, 15:43892272, 22:38119602, 22:38120925, 22:38121788, 10:26462691, 17:18025499, 17:18049349, 17:18054149, 17:18060523, 17:18075499, 17:18023292, 17:18025637, 2:179320828, 18:44114297, 18:44065090, 21:43808612, 21:43802210, 21:43809044, 2:26681086, 2:26683865, 2:26685039, 2:26685045, 2:26705384, 2:26706449, 19:11216105, 19:11216264, 19:11224008, 19:11226874, 6:51910887, 6:51924836, 19:41925187, 1:100706416, 13:52511706, 13:52513240, 13:52515330, 13:52523835, 13:52523908, 13:52524434, 13:52532469, 13:52532497, 13:52539170, 13:52539174, 13:52548830, 1:155207203, 1:155207932, 1:155208006, 1:155210420, 7:95751240 and 7:95813588, 7:95813688, 7:95818684, 7:95822344, 9:133342110, 17:7123518, 17:7127359, 4:146572222, 6:49399515, 6:49409685, 6:49415462, 6:49419231, 1:45966084, 1:45974001, 1:45974005, 7:117230498, 7:117232086, 7:117232300, 11:6412870, 11:6413142, 18:21113439, 18:21131617 and 3:33099712, 5:78135195, 17:78184631, 17:78187645, 4:995488, 4:996842, 4:997815, 4:998101, 1:100327076, 1:100345603, 1:100350260, 11:64517895, 11:118895981, 17:78078341, 17:78078931, 17:78082617, 17:78083828, 17:78086465, 17:78086721, 17:78086801, 17:78090815, 17:78092467, 17:78093083, 4:159603421, 19:13007763, 19:13008223, 19:13008590, 6:32006858, 6:32007203, 6:32007887, 6:32008198, 16:8905010, 16:8905018, 16:8906904, 16:8941632, 5:131714072, 5:131719847, 5:131721062, 1:76211508, 1:76215238, 1:76227050, 12:103249009, 12:103288534, 12:103237555, 12:103260442, 12:103237426, 12:103288655, 12:103260385, 12:103246653, 12:103246591, 12:103234192, 12:103246665, 12:103246695, 13:52520505, 13:52524252, 13:52523859, 5:70220951, 5:70240540, 5:70240546, 5:70247796, 5:70238311, 5:70247763, 5:70238374, 1:45974647, 1:45974694, 7:117246728, 7:117199645, 18:21116700, 17:41063017, 17:41055965, 11:118898391, 11:118898518, 11:88924446, 11:88924382, 11:88924475, 5:131721127, 19:13010280, 17:7127303, 17:7127350, 17:7123443, 6:49425427, 6:49419405, 6:49421467, 6:49412365, 7:117149186, 7:117227874, 7:117171005, 12:121176944, 12:121164946, 5:131728257, 5:131705715, 5:131714104, 5:131706002, 1:76226846, 1:76198409, 1:76200534, 1:76226946, 1:76205776, 20:10653348, 11:6411832, 11:6413134, 11:6413037, 11:6415243, 11:6415506, 11:6411935, 7:107303857, 7:107314782, 7:107323898, 7:107330593, 7:107330645, 7:107330648, 7:107340625, 7:107342443, 7:107342495, 7:107350571, 7:107350577, 16:223539, 16:223547, 16:223597, 11:5247153, 11:5247802, 11:5247904, 11:5247992, 11:5247993, 11:5247994, 11:5247995, 11:5247996, 11:5248008, 11:5248009, 11:5248012, 11:5248028, 11:5248155, 11:5248159, 11:5248161, 11:5248166, 11:5248172, 11:5248173, 11:5248198, 11:5248200, 11:5248203, 11:5248206, 11:5248250, 11:5248259, 11:5248301, 11:5248329, 11:5248330, 11:5248331, 11:5248332, 11:5248333, 11:5248351, 11:5248374, 11:5248391, 12:103234255, 12:103246680, 12:103246701, 12:103246707, 12:103246714, 1:35250901, 1:35250910, 13:20763209, 13:20763421, 13:20763486, 13:20763530, 13:20763686, X:153760472, X:153760473, X:153760479, X:153760484, X:153760605, X:153760654, X:153761184, X:153761204, X:153761337, X:153762605, X:153762680, X:153762704, X:153762710, X:153763476, X:153763485, X:153774276, MT: 1095. MT:1494 and MT:1555;
the pCNV detection region includes at least one of:
16:199800-234700, X:31645789-31645979, X:31676106-31676261, X:31697491-31697703, X:31747747-31747865, X:31792076-31792309, X:31838091-31838200, X:31854834-31854939, X:31893307-31893490, X:31947712-31947862, X:31950196-31950344, X:31986455-31986631, X:32235032-32235180, X:32490280-32490426, X:32503035-32503216, X:32509393-32509635, X:32519871-32519959, X:32536124-32536248, X: 3256275-3256451, X:32583818-32583998, X:32591646-32591754, X:32591861-32591963, X:32613873-32613993, X:32632419-32632570, X:32662248-32662430, X:32663080-32663269, X:32715986-32716115, X:32717228-32717410, X:32827609-32827728, X:32834584-32834757, X:32841411-32841504, X:32862899-32862977 and X:32867844-32867937.
3. The capture probe of claim 1 or 2, wherein: the capture probe is 1) or 2) as follows:
1) Consists of at least one of the probes shown in SEQ ID No.1 to SEQ ID No.1109 in the sequence table;
2) Consists of one or more of derivatives of each probe in 1), wherein the derivatives of each probe are the probes which have the same function and are obtained by substituting and/or deleting and/or adding one or more nucleotides in each probe in 1).
4. A gene chip, which is characterized in that: comprising the capture probe of any one of claims 1-3.
5. The application is characterized in that: the application is the application of the capture probe of any one of claims 1 to 3 or the gene detection chip of claim 4 in the preparation of products for screening and/or predicting and/or assisting in diagnosing and/or diagnosing recessive genetic diseases.
6. Use according to claim 5, characterized in that: the recessive genetic disease is selected from glycogen accumulating disease, maple syrup urine disease, gaucher disease, usher syndrome, autosomal dominant hereditary deafness type 2B, autosomal dominant hereditary deafness type 2A, methylmalonemia, medium-chain acyl-CoA dehydrogenase deficiency, albinism (combined) HPS-1, autosomal recessive hereditary deafness type 30, hypomyelinating white matter dystrophy type 7 with or without teeth and/or gonadotropic hypogonadism, BH4 deficiency hyperphenylalaninemia, autosomal recessive hereditary deafness type 111, autosomal recessive hereditary deafness type 18B, beta thalassemia, niemann pick's disease, albinism (non-combined) OCA-1, phenylketonuria, short-chain acyl-CoA dehydrogenase deficiency, autosomal recessive deafness type 74, propionemia, thyroidism, achromosomal hereditary deafness type 2B, autosomal dominant deafness type 2A, autosomal dominant deafness type 7, hypo-deficiency type H-CoA deficiency, and gonadotropin autosomal dominant hereditary hearing loss type 3A, autosomal recessive hereditary hearing loss type 1B, hepatolenticular degeneration, albinism (non-integrative) OCA-2, isovaleric acidemia, autosomal recessive hereditary hearing loss type 16, autosomal recessive hereditary hearing loss type 22, alpha-thalassemia, autosomal recessive hereditary hearing loss type 86, gitelman's syndrome, congenital glycosylation disorder, autosomal recessive hereditary hearing loss type 3, autosomal recessive hereditary hearing loss type 99, very long chain acyl-CoA dehydrogenase deficiency, mucopolysaccharidosis storage disease, autosomal recessive hereditary hearing loss type 77, homozygotic familial hypercholesterolemia, glutaremia, autosomal dominant hereditary hearing loss type 4A, autosomal recessive hereditary hearing loss type 59, alport syndrome, autosomal recessive hereditary deafness type 9, autosomal recessive hereditary deafness type 8/10, autosomal dominant hereditary deafness type 17, autosomal recessive hereditary deafness type 28, autosomal dominant hereditary deafness type 70, autosomal recessive hereditary deafness type 25, autosomal dominant hereditary deafness type 6/14/38, autosomal dominant hereditary deafness type 79, autosomal recessive hereditary deafness type 100, primary carnitine deficiency, spinal muscular atrophy, autosomal dominant hereditary deafness type 10, congenital adrenal hyperplasia, polycystic kidney, autosomal recessive hereditary deafness type 37, pendred syndrome/autosomal recessive deafness type 4, cystic fibrosis, autosomal dominant hereditary deafness type 74, cucurbitemia, OCA (non-amineismic) disease OCA-3, autosomal dominant hereditary deafness type 51, albino autosomal recessive hereditary deafness type 7/11, drug-induced deafness, X-linked adrenoleukodystrophy, progressive dystrophia [ 6-glucose-6 ] phosphate dehydrogenase, at least one of PD 6-deficiency of G-phosphate dehydrogenase.
7. A method for capturing a recessive genetic disease pathogenic gene pathogenic site and a pCNV detection region in a sample to be detected is characterized in that: the method comprises the following steps:
capturing the genome DNA of a sample to be detected by using the capture probe of any one of claims 1-3, performing single base extension and fluorescence labeling to obtain a target site, wherein the target site is a pathogenic site of a recessive genetic disease pathogenic gene or a pCNV detection region in the sample to be detected.
8. A method for detecting a recessive genetic disease pathogenic gene pathogenic site and pCNV in a sample to be detected is characterized in that: the method comprises the following steps:
1) Capturing the genomic DNA of a sample to be tested by using the capture probe of claim 3 to obtain a target region fragment;
2) Performing single base extension and fluorescent labeling on the target region fragment;
3) Scanning the fluorescent marker to obtain fluorescent marker data;
4) And analyzing the fluorescence labeling data to obtain the detection result of the pathogenic site of the recessive genetic disease pathogenic gene and the pCNV in the sample to be detected.
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