CN112708670A

CN112708670A - DNA library for detecting and diagnosing congenital thyroid disease pathogenic gene and application thereof

Info

Publication number: CN112708670A
Application number: CN202110152973.7A
Authority: CN
Inventors: 赵家军; 徐潮
Original assignee: Shandong Provincial Hospital Affiliated to Shandong First Medical University
Current assignee: Shandong Provincial Hospital Affiliated to Shandong First Medical University
Priority date: 2021-02-03
Filing date: 2021-02-03
Publication date: 2021-04-27

Abstract

The invention relates to a DNA library for detecting and diagnosing congenital thyroid disease pathogenic genes by a targeted high-throughput sequencing technology and application thereof, wherein the library comprises 362 congenital thyroid disease pathogenic genes. The invention preferably selects 362 congenital thyroid disease pathogenic genes, designs a probe pool, establishes a target region library aiming at the 362 congenital thyroid disease pathogenic genes, utilizes a high-throughput sequencing technology to sequence the library, searches pathogenic mutation and provides genetic and molecular biological basis for clinical diagnosis. The 362 genes have the characteristics of accuracy, rapidness, flexibility and low cost, and have important significance and clinical value for diagnosis, differential diagnosis and individualized treatment of congenital thyroid diseases aiming at almost all pathogenic genes of congenital thyroid diseases such as thyroid morphological abnormality (dysplasia, translocation and deficiency), thyroid dysfunction, familial thyroid tumors and the like.

Description

DNA library for detecting and diagnosing congenital thyroid disease pathogenic gene and application thereof

Technical Field

The invention belongs to the technical field of biomedicine, and particularly relates to a DNA library for detecting and diagnosing congenital thyroid disease pathogenic genes by a targeted high-throughput sequencing technology and application thereof.

Background

The thyroid gland is a very important gland of vertebrates and belongs to an endocrine organ. The mammal is located below the neck thyroid cartilage, on both sides of the trachea. The thyroid gland of human is like butterfly, so it is named as shield nail. The thyroid gland controls the rate of energy metabolism, produces proteins, and regulates the body's sensitivity to other hormones. Thyroid gland regulates metabolism, growth and development and regulates various organ systems of the body by producing thyroxine. The thyroid gland also produces calcitonin, which regulates the balance of calcium in the body.

The normal development and function of the thyroid gland are regulated by a plurality of genes, the dysfunction of the genes can cause thyroid morphological abnormality (dysplasia, translocation, deficiency), dysfunction (hypofunction, hyperfunction), familial thyroid tumor and the like, the onset of the thyroid diseases is obviously earlier than that of non-hereditary thyroid diseases and even occurs before or shortly after birth, so the thyroid diseases are collectively called congenital thyroid diseases, and the related genes are called congenital thyroid disease pathogenic genes.

Taking congenital adrenal cortical hyperplasia in congenital thyroid diseases as an example, the congenital adrenal cortical hyperplasia is one of the most common preventable causes of dull symptoms in the world, and is caused by insufficient secretion of thyroid hormone, and the incidence rate is 1:4000-1: 3000. Congenital adrenal cortical hyperplasia patients can cause permanent short stature and intellectual disability, namely, dull condition if the children cannot be treated by thyroid hormone in time in the early stage. 20 genes are found to be related to congenital adrenal cortical hyperplasia, and when a child patient finds a pathogenic gene in time within 3 months after birth and carries out targeted individualized treatment, the body development and intelligence can be basically close to normal people, so that the pathogenic gene of congenital thyroid diseases is found, early diagnosis and treatment are carried out, and the burden of the country and families can be effectively relieved.

It is known that the pathogenesis of congenital thyroid diseases is various, and one pathogenic gene can even cause completely opposite clinical manifestations, for example, pathogenic variation of TSHR gene can simultaneously cause hyperthyroidism, hypothyroidism and thyroid tumor. Accurate diagnosis of these congenital thyroid diseases can therefore only be achieved by means of genetic testing. At present, the traditional Sanger sequencing method is mostly adopted in clinical laboratories to detect gene mutation, and if a plurality of pathogenic genes of congenital thyroid diseases are detected simultaneously, the workload is huge, the detection efficiency is low, more importantly, precious DNA samples are wasted, the detection cost of gene diagnosis is obviously increased, and the large-scale application of the gene mutation in clinical molecular diagnosis is severely restricted. Therefore, there is a need to find a new method for detecting congenital thyroid disease pathogenic gene mutation, which can improve the diagnosis accuracy, reduce the cost and labor intensity, and improve the timeliness.

Disclosure of Invention

The invention aims to provide a DNA library for detecting and diagnosing congenital thyroid disease pathogenic genes by a targeted high-throughput sequencing technology, which can improve the diagnosis accuracy and reduce the cost and labor intensity.

The second object of the present invention is to provide the use of said DNA library.

The purpose of the invention is realized by the following technical scheme: a DNA library for diagnosing congenital thyroid disease based on high-throughput sequencing technology, the library comprising 362 congenital thyroid disease pathogenic genes, wherein the 362 congenital thyroid disease pathogenic genes are as follows:

ABCC6、ACP5、ADA、ADAMTSL1、ADAR、ADAT3、ADNP、AFF4、AIP、AIRE、AKT1、ALG8、ALMS1、ALX4、APC、APOE、ARL6IP6、ARNT2、ARVCF、ATRX、B3GLCT、BAZ1B、BCL10、BCOR、BIRC3、BMP4、BMPR1A、BRAF、BTNL2、BUB1、BUB1B、BUB3、C1QBP、C1S、CACNA1C、CACNA1S、CASP10、CASR、CCBE1、CDC73、CDH23、CDKN1A、CDKN1B、CDKN2B、CDKN2C、CDON、CEP128、CEP57、CHD7、CLCNKB、CLIP2、CLPB、COL7A1、COMT、CP、CTLA4、CTNNB1、CTNS、DACT1、DCAF17、DCLRE1C、DDOST、DEAF1、DICER1、DIO1、DIO2、DISP1、DLL1、DMXL2、DNAH1、DNAJC19、DNM1L、DUOX1、DUOX2、DUOXA1、DUOXA2、ECE1、EDN3、EDNRB、EFEMP2、EIF2AK3、ELN、ENPP1、EPCAM、ERCC1、EXOSC2、EXT2、EYA1、FAN1、FANCI、FAS、FASLG、FBLN5、FDX2、FGF8、FGFR1、FLCN、FLII、FMR1、FOXA2、FOXD3、FOXE1、FOXH1、FOXI1、FOXP1、FOXP3、FUCA1、FUT8、GABRA3、GABRD、GAS1、GATA1、GATA6、GCH1、GCM2、GDNF、GLI2、GLI3、GLIS3、GNAS、GNB1、GNE、GP1BB、GPC1、GPR161、GPR35、GREM1、GTF2I、GTF2IRD1、HABP2、HBB、HESX1、HHEX、HIRA、HNF1B、HNF4A、HPD、HR、HSD17B3、IDH1、IDH2、IFIH1、IFNG、IGSF1、IL12A、IL12RB1、IL2RA、IL2RG、IL7R、INSR、IQSEC2、IRF5、ITCH、IYD、JAG1、JMJD1C、KAT6B、KCNAB2、KCNJ10、KCNJ18、KDM6A、KEAP1、KIAA0556、KISS1R、KLLN、KMT2D、KRAS、LEP、LEPR、LHX3、LHX4、LIFR、LIG4、LIMK1、LMNA、LRBA、LRP4、MALT1、MARS、MC2R、MCM8、MEN1、MINPP1、MLH1、MLH3、MLXIPL、MMEL1、MMP14、MMP2、MRAP、MSH2、MSH3、MSH6、MST1、MSTO1、NF2、NIN、NKX2-1、NKX2-5、NLRP1、NNT、NODAL、NPHS1、NR1D1、NRAS、NRTN、NSDHL、OPA1、OTX2、PAX8、PCSK1、PDE4D、PDGFB、PDGFRB、PHF21A、PIEZO1、PIK3C2A、PIK3CA、PLAA、PLCG2、PLVAP、PMM2、PMS1、PMS2、POGZ、POLG、POLG2、POLR1C、POLR1D、POLR3A、POMC、POU1F1、POU2AF1、POU3F4、PPP1R15B、PRDM16、PRKAR1A、PRKCD、PROKR2、PROP1、PTCH1、PTEN、PTGS1、PTGS2、PTH2、PTPN22、PTRH2、RAG1、RAG2、RAI1、RASGRP1、RBM28、RCBTB1、RERE、RET、RFC2、RMRP、RNASEH2A、RNASEH2B、RNASEH2C、RNF4、ROBO1、RPS20、RREB1、RRM2B、SAA1、SALL1、SAMHD1、SASH1、SCN4A、SDHB、SDHC、SDHD、SEC23B、SEC24C、SECISBP2、SEMA3C、SEMA3D、SEMA3E、SEMA4A、SETBP1、SGPL1、SHH、SIX1、SIX3、SKI、SKIV2L、SLA、SLC12A3、SLC16A2、SLC25A4、SLC26A4、SLC35A2、SLC5A5、SLC6A17、SMARCAL1、SMARCB1、SOX3、SPIB、SQSTM1、SRD5A3、SRGAP1、SRY、STAR、STAT1、STAT3、STEAP3、STUB1、STX16、SUFU、SUGCT、TANGO2、TBC1D24、TBCK、TBL2、TBX1、TBX2、TCF4、TCOF1、TDGF1、TF、TG、TGFBR2、TGIF1、TH、THRA、THRB、TMEM67、TNFSF15、TNPO3、TONSL、TPO、TRAPPC9、TREX1、TRH、TRHR、TRIP13、TRMT10A、TSC1、TSC2、TSHB、TSHR、TTC37、TTC7A、TTF2、TWNK、TXNRD2、UBR1、UFD1、USF3、USP9X、VPS13A、WBP1、WDR11、WDR4、WFS1、WRN、XRCC4、YY1、ZBTB20、ZFAT、ZIC2。

the DNA library provided by the invention covers almost all pathogenic genes of congenital thyroid diseases such as thyroid morphological abnormality (dysplasia, translocation and deficiency), thyroid dysfunction (hypofunction and hyperfunction), familial thyroid tumor and the like, and comprises 362 congenital thyroid disease pathogenic genes. This 362 gene selection was based on the internationally reported and recognized clinical disease gene database (OMIM database, HGMD database, ClinVar database). The 362 gene pathogenic gene variation can cause diseases alone or in combination to cause more serious and complex clinical phenotypes, and the invention can comprehensively sequence the pathogenic gene variation at one time for the first time.

The invention designs a probe pool which can cover the exons of the genes and the adjacent +/-20 bp intron regions according to 362 congenital thyroid disease pathogenic genes, utilizes the probe to target and capture to establish a target region library containing 362 congenital thyroid disease pathogenic genes, utilizes a high-throughput sequencing technology to sequence the library, searches pathogenic mutation, determines the genetic cause of congenital thyroid disease, and provides a theoretical basis of genetics and molecular biology for clinical diagnosis.

The invention also provides an application of the DNA library in preparing a diagnostic kit, and the kit is used for searching pathogenic genes of congenital thyroid diseases and diagnosing the congenital thyroid diseases.

Further, the application comprises the following steps:

1) collecting clinical data and clinical biological samples of patients with congenital thyroid diseases, preferably, the clinical biological samples refer to various samples derived from human bodies, including but not limited to peripheral blood, body fluid, tissue and organ samples from subjects, such as saliva, hair or oral mucosa of the subjects.

2) Extracting the genome DNA of the sample, preferably, the extraction method comprises a DNA extraction kit or various manual extraction methods;

3) quantifying the extracted genomic DNA and constructing a library, preferably, the quantification methods include, but are not limited to, fluorescence quantification methods and electrophoresis; wherein, the library construction comprises the following steps:

a) fragmenting the genomic DNA, preferably by methods including but not limited to ultrasonication, transposase cleavage, and restriction endonuclease cleavage;

b) carrying out end repair on the fragmented genomic DNA and simultaneously carrying out 3' end addition of A;

c) ligating the product of adding A at the 3' end to a linker for amplification of the ligation-effective product, preferably from a high throughput sequencing library kit;

d) performing PCR amplification on the ligation product by using a universal primer, and adding a complete joint, wherein the universal primer is preferably from a high-throughput sequencing and library building kit;

e) the target area is captured by using the probes aiming at the 362 congenital thyroid disease pathogenic genes, and preferably, the capture method comprises but is not limited to liquid-phase probe capture and solid-phase chip hybridization capture;

f) washing off the uncaptured library, and only reserving the target area library;

j) obtaining a target region capture library.

4) Performing quantitative operation on the library, preferably, the quantitative method includes but is not limited to fluorescence quantitative method and electrophoresis;

5) performing high throughput sequencing of the library using a sequencing device, preferably, the sequencing device includes, but is not limited to, a Novaseq series, a Hiseq series, a Nexeseq series, a BGIseq series second generation nucleic acid sequencer;

6) and comparing the obtained sequencing data by bioinformatics, and obtaining the related information of the pathogenic site by mutation interpretation.

A diagnostic kit comprising said DNA library.

Compared with the prior art, the invention has the advantages that:

1. congenital thyroid disorders encompass a variety of genetic disorders, although these disorders share common features: the congenital genetic mutation causes different degrees of disease, has various clinical manifestations, and is caused by primordial diseases, adult-onset hyperthyroidism, familial thyroid cancer susceptibility and the like. In particular, there is a need for differential diagnosis of other thyroid disorders associated with infectivity, autoimmunity, iodine uptake, and thus, accurate etiological diagnosis is difficult. The invention aims to establish a new method for quickly, accurately and high-flux detecting the pathogenic gene of the congenital thyroid disease, thereby helping to understand the pathogenesis and laying a foundation for assisting clinical diagnosis, prognosis judgment, prenatal diagnosis and accurate treatment.

2. The gene diagnosis is helpful for clinical genetic counseling, prenatal diagnosis and the like. Through clinical genetic consultation, family members can know the possibility of suffering from diseases; parents or the patients can guide the breeding through gene detection, and the health of future born offspring is ensured. Also helps to find the carriers of the young pathogenetic genes which are not symptomatic in families, so as to make family medical treatment and rehabilitation plans earlier.

3. The DNA library of the application refers to a large number of documents by the inventor on the basis of developing high-throughput gene detection service for many years and accumulating a large number of clinical cases of the congenital thyroid diseases in China, and finally, the 362 congenital thyroid disease pathogenic genes are preferably selected by adopting a disease-gene correlation screening method provided by an authorized Wei database such as ClinGen and the like, so that the comprehensive property, the accuracy and the scientificity are considered, most of the genes are pathogenic genes discovered by the inventor in Han people, and the DNA library is particularly suitable for detecting yellow race patients including Chinese. The invention preferably selects 362 congenital thyroid disease pathogenic genes, designs a probe pool, establishes a target region library aiming at the 362 congenital thyroid disease pathogenic genes, utilizes a high-throughput sequencing technology to sequence the library, searches pathogenic mutation and provides genetic and molecular biological basis for clinical diagnosis. The 362 gene detection regions can detect almost all pathogenic genes of congenital thyroid diseases such as thyroid morphological abnormality (dysplasia, translocation and deficiency), thyroid dysfunction (hypofunction and hyperfunction), familial thyroid tumor and the like, and have important significance and clinical value for diagnosis and differential diagnosis of the congenital thyroid diseases.

4. The method adopts a high-throughput sequencing technology to sequence the target region capture library, and can simultaneously detect all exons and adjacent regions of 362 congenital thyroid disease pathogenic genes related to the method in one sequencing reaction. Compared with the traditional sequencing technology, the method has the advantages of obviously improved detection efficiency and obviously reduced cost, has great advantages in the aspects of congenital thyroid disease gene excavation and pathogenic gene screening, and is an efficient, reliable and economic congenital thyroid disease pathogenic gene detection technology.

5. In conclusion, the DNA library and the application thereof have the characteristics of accuracy, flexibility, rapidness and low cost; through clinical evaluation, the invention has good auxiliary diagnosis value on congenital thyroid diseases.

Drawings

FIG. 1 is the quality control information for creating a target region-targeting DNA library for 362 congenital thyroid disease-causing genes of proband samples in example 1;

FIG. 2 is data coverage information for sequencing a target region of 362 congenital thyroid disease-causing genes of proband samples in example 1;

fig. 3 is a family map of the family of congenital thyroid diseases described in example 1, in which arrows indicate probands, solid icons indicate patients, open icons indicate no disease, and dots inside the open icons indicate a carrying state.

Detailed Description

The invention provides a DNA library for detecting and diagnosing congenital thyroid disease pathogenic genes and application thereof, which are further described in the following with reference to specific embodiments.

Unless otherwise indicated, the techniques used in the examples are conventional and well known to those skilled in the art, and may be performed according to the fourth edition of the molecular cloning, laboratory Manual, or related information, and the reagents and products used are also commercially available. Various procedures and methods not described in detail are conventional methods well known in the art, and the sources, trade names, and components of the reagents used are indicated at the time of first appearance, and the same reagents used thereafter are the same as those indicated at the first appearance, unless otherwise specified.

Example 1:

in this embodiment, a Hiseq sequencing platform of Illumina corporation is used to detect genomic DNA of peripheral blood of a human subject, and the specific implementation steps are as follows:

1. sample source

The syndrome of the first patient is a patient with dwarfism from Shandong province in China, 4 years old, female, and the syndrome of the first patient mainly complains about dwarfism and short height of the young. Vomiting, which is the gastric content, occurs repeatedly in recent times. Laboratory tests show that: growth hormone deficiency (partial), thyroid dysfunction (central hypothyroidism). Denying a family genetic or congenital history. The pedigree of the pedigree is shown in fig. 3, the arrow points to the proband, the solid icon indicates the diseased state, the open icon indicates the non-diseased state, and the dot inside the open icon indicates the carried state. A total of 3 family members were tested, including proband (diseased), proband mother, and proband father, to obtain informed consent of proband father and mother, and 10mL of venous blood (EDTA anticoagulated) was collected from the elbow veins of the family members in this example for testing.

2. Extraction of specimen genomic DNA:

a genomic DNA extraction kit (HiPure Blood) from magenta was used according to the instructions provided by the trade company&Tissue DNA Kit) genomic DNA was extracted from peripheral blood samples, the purity of the DNA was measured using Nanodrop one, and OD of the genomic DNA obtained_260nm/OD_280nmAll are located between 1.7 and 2.0, and the concentration of the DNA is measured by using Nanodrop one, and the concentration of the obtained genomic DNA is 50 to 100 ng/. mu.L, and the total amount is 5 to 10. mu.g.

3. Establishing a genome amplification library:

according to the instructions provided by the trade company, the Kit of KAPA company (KAPA Hyperplus Library Preparation Kit) is used for carrying out enzyme digestion and fragmentation, end repair, 3' end A addition, linker ligation and PCR amplification on genomic DNA, and finally, the obtained Library is quantified and quality checked, and the specific implementation steps are as follows:

1) and (3) carrying out genome DNA fragmentation reaction, wherein the reaction system is as follows:

name of reagent	Dosage (mu L)
		Genomic DNA (100ng)	1
NF H₂O	16.5
		KAPA Frag Buffer	2.5
KAPA Frag Enzyme	5
		Total volume	25

Reaction conditions are as follows: the reaction was carried out at 37 ℃ for 12 min.

2) End repairing and 3' end adding A reaction, wherein the reaction system is as follows:

reaction conditions are as follows: reacting at 20 ℃ for 1 min; reacting at 65 ℃ for 30 min; constant temperature of 20 DEG C

3) Linker linking reaction, the reaction system is as follows:

name of reagent	Dosage (mu L)
		DNA obtained in step 2)	30
NF H₂O	2.5
		KAPA Ligation Buffer	15
Adaptor(15μM)	2.5
		KAPA DNA Ligase	5
Total volume	55

Reaction conditions are as follows: the reaction was carried out at 20 ℃ for 20 min.

4) The first PCR amplification reaction comprises the following reaction systems:

name of reagent	Dosage (mu L)
		DNA obtained in step 3)	16
KAPA HiFi HotStart Ready Mix(2×)	20
		T5*Primer(10μM)	1.5
T8*Primer(10μM)	1.5
		Total volume	39

Reaction conditions are as follows: 45s at 98 ℃; (98 ℃ for 15s,60 ℃ for 30s,72 ℃ for 30 s). times.6 cycles; 1min at 72 ℃; storing at 12 deg.C.

5) DNA quantification and quality control:

measuring the concentration and the strip distribution condition of the PCR amplification product by using Nanodrop one; preparing 2% agarose gel, mixing the obtained PCR amplification product with the sample loading buffer solution, and observing the position, brightness and uniformity of the band by electrophoresis, wherein the size of the band is qualified between 200 and 800 bp.

4. Constructing a target region targeting DNA library based on probe hybridization capture:

the library is constructed by adopting a library construction kit of IGT company according to the operation of the instruction provided by a merchant, a probe is designed according to 362 candidate congenital thyroid disease pathogenic gene sequences, and is synthesized and labeled by biotin, and the specific implementation steps are as follows:

1) and (3) probe hybridization:

mixing 5 mu g of PCR product obtained in the step 3 with 5 mu L of Cot-1human DNA, and carrying out vortex oscillation; adding 2.5 × Ampure XP beads according to the total volume, uniformly mixing and centrifuging, and standing for 5min at room temperature; the magnetic beads are centrifugally washed for 2 times by 80% ethanol, and are eluted by a hybridization solution for hybridization reaction, wherein the system of the hybridization solution is as follows:

name of reagent	Dosage (mu L)
		IDT 2×Hybridization Buffer	8.5
Hybridization Enhancer	2.75
		NF H₂O	1.75
Universal Blockers-TS Mix	2
		Biotin-labeled probe (362 genes)	4
Total volume	19

Hybridization conditions: reacting at 95 ℃ for 10 min; at 65 ℃ overnight

2) The probes hybridized with the sample target sequences were captured on magnetic beads by binding biotin to Streptavidin using 80. mu.L of Streptavidin-labeled magnetic beads (M-270Streptavidin beads). Washing with 200 μ L of 1 × Bead Wash Buffer at 65 deg.C and room temperature for three times, each for 3min, and washing the magnetic beads with 20 μ L of NF H₂And (4) resuspending the solution.

3) And carrying out a second PCR amplification reaction on the captured target sequence, wherein the reaction system is as follows:

name of reagent	Dosage (mu L)
		KAPA HiFi Hot start Readymix(2×)	25
X Gen Library Amplification primer-Ts Mix	5
		DNA with magnetic beads obtained in step 2)	20
Total volume	50

Reaction conditions are as follows: 45s at 98 ℃; (98 ℃ for 15s,60 ℃ for 30s,72 ℃ for 30 s). times.7 cycles; 1min at 72 ℃; storing at 4 ℃.

5) And (3) PCR product quantification and quality inspection:

5. Generating information analysis and variant interpretation of sequencing data:

NGS sequencing results were aligned to the human reference genome UCSC NCBI37/hg19 using Novocraft Novoalign to obtain a unique aligned sequence aligned to the genome. The VarScan mpileup2snp and VarScan mpileup2indel detection is used for determining the variation of 362 congenital thyroid disease pathogenic gene regions. Common variations in dbSNP and ExAC databases were removed using Remove Run Common Variants and Remove Global Common Variants software. The variants were then annotated using Interactive Biosoftware Alamut Batch. The database used for annotation includes: dbSNP, ExAC, 1000g, ClinVar, OMIM, etc., and utilizes the software FATHMM, FATHMMMKL, METALR, METASVM, MUTATIONASSESSOR, MUTATIONTASTERAGGGD, AGVGD, LRT, PROVEAN, SIFT to predict the variant function. According to ACMG genetic variation classification standard and guideline, mutation sites which are meaningful for diagnosing congenital thyroid diseases are obtained by analysis.

6. Sequencing results interpretation and analysis

362 congenital thyroid disease pathogenic genes of 3 examinees are sequenced at one time by the DNA library for detecting and diagnosing congenital thyroid disease pathogenic genes provided by the invention. Taking prover as an example, through quality control analysis, the size of the obtained DNA library is mainly distributed in 200-800bp, the average length is 374bp, and the concentration and the fragment size meet the sequencing requirement, thereby prompting that the quality of the library construction is qualified (as shown in FIG. 1). The DNA library comprises 362 genes, wherein the length of a coding region is 896544bp, the length of a non-coding region is 191162bp, and the total length is 1087706 bp. Wherein the 10 × coverage of the coding region is 99.36%, the 20 × coverage is 99.15%, and the 50 × coverage is 90.76% (see fig. 2). The sequencing result meets the data requirement of variation interpretation. Through the analysis of the sequencing data, the pathogenic gene variation of the congenital thyroid disease related to the clinical phenotype of the tested person is found, and is shown in the following table:

two heterozygous variations of the TSHB gene were detected from the proband whole blood genomic DNA: c.28del (p.Leu 10Phefs. 31) and c.94G > A (p.Glu32Lys), and the results of parental detection show that the two variants are respectively derived from mother and father.

Autosomal recessive variants of the TSHB gene are associated with the development of autosomal recessive non-nodular congenital hypothyroidism type 4. The autosomal recessive non-nodular congenital hypothyroidism type 4 is mainly characterized by severe growth retardation, non-nodular hypothyroidism, reduced TSH level, low potassium and the like, and serious mental retardation can occur without treatment. Facial abnormalities include: low nasal bridge, abnormal fontanel and anterior and posterior fontanel.

No relevant literature reports exist for the detected c.28del (p.Leu10Phefs 31) mutation of the subject. This variation has not been included in the gnomAD and HGMD databases. The variation is located in exon 2 (total exon 3) of the TSHB gene transcript NM-000549.4, the variation is located in the signal peptide region (the first 1-18 amino acids are signal peptide regions), and multiple nonsense variations, frameshift variations downstream of the variation are reported as pathogenic variations. The effect of this variation on protein function is unknown, and may result in loss of function of the TSHB gene. According to the American ACMG variation classification guidelines, the variation is classified as a pathogenic variation.

The subject detected c.94g > a (p.glu32lys) variation was reported in homozygous form in 2 patients with congenital hypothyroidism. The trans position of this mutation detected a pathogenic mutation in the subject c.28del. The gnomAD database showed that the allele frequency of this variation is currently 0.001% in the general population (3/251294), the highest in the latin american population is 0.003% (1/34574), and there are no homozygotes. According to ACMG guidelines, the variant is classified as a pathogenic variant.

Through detection, the syndrome of the first patient is diagnosed as non-nodular congenital hypothyroidism type 4, and a foundation is laid for subsequent individualized treatment. It is suggested that the first patient can apply exogenous thyroid hormone to prevent the disease before the serious complications such as mental retardation, sleep disorder, etc. occur. Levothyroxine replacement therapy is the treatment of choice. Concomitant adrenocorticotropic hormone deficiency should be excluded before starting treatment to avoid adrenal crisis.

For genetic counseling, since the disease is autosomal recessive inheritance, the recurrence risk of procreation parents to give rise to non-nodular congenital hypothyroidism type 4 offspring is 1/4, and the risk of procreation disease carriers is 1/2. The patients and the children who have born the birth are all carriers of pathogenic variation. (see fig. 3)

Reference throughout this specification to the description of "one embodiment," "this embodiment," "an embodiment," or the like, means that a particular feature, structure, material, or characteristic described in connection with the embodiment is included in at least one embodiment of the invention and is not specifically referred to.

It should be noted that, although the above embodiments have been described herein, the invention is not limited thereto. Therefore, based on the innovative concepts of the present invention, the technical solutions of the present invention can be directly or indirectly applied to other related technical fields by making changes and modifications to the embodiments described herein, or by using equivalent structures or equivalent processes performed in the content of the present specification and the attached drawings, which are included in the scope of the present invention.

Claims

1. A DNA library for diagnosing congenital thyroid diseases based on high-throughput sequencing technology, wherein the library comprises 362 causative genes of congenital thyroid diseases, wherein the 362 causative genes of congenital thyroid diseases are as follows:

2. use of a DNA library according to claim 1 for the preparation of a diagnostic kit, characterized in that: the kit is used for diagnosing congenital thyroid diseases.

3. The application according to claim 2, characterized in that it comprises the following steps:

1) extracting genomic DNA of a sample of a subject;

2) quantifying the extracted genomic DNA and constructing a library according to the following steps:

a) fragmenting the genomic DNA;

b) adding a base A to the 3' end of the fragmented genomic DNA while performing end repair;

c) connecting a product with a base A added at the 3' end with a joint;

d) performing PCR amplification on the ligation product, and adding a complete linker;

e) targeting a capture target region using a probe against 362 congenital thyroid disease-causing genes of claim 1;

j) obtaining a target region capture library;

3) performing quantitative operation on the library;

4) high-throughput sequencing;

5) and (5) analyzing the data to obtain the related information of the pathogenic site.

4. Use according to claim 3, characterized in that: the sample in the step 1) is from peripheral blood, body fluid and a tissue organ sample of a subject.

5. Use according to claim 3, characterized in that: the quantitative method in the step 2) comprises a fluorescence quantitative method and electrophoresis.

6. Use according to claim 3, characterized in that: the fragmentation method in the step a) comprises ultrasonic crushing, transposase enzyme digestion and restriction enzyme digestion.

7. Use according to claim 3, characterized in that: the quantitative method in the step 3) comprises a fluorescence quantitative method and electrophoresis.

8. A diagnostic kit comprising the DNA library of claim 1.