CN113564247B - Primer group and kit for simultaneously detecting multiple mutations of 9 genes related to congenital adrenal cortical hyperplasia - Google Patents

Abstract

The invention relates to a primer group, a kit and a method for simultaneously detecting multiple mutations of 9 genes related to congenital adrenal cortical hyperplasia. Wherein the kit comprises the following reagents: (1) reagents for multiplex PCR amplification; and (2) reagents for constructing a three-generation sequencing library. Wherein the method comprises the steps of: (1) preparing a sample of a subject; (2) multiplex PCR amplification of 9 genes in the sample: (CYP21A2、CYP21A1P、CYP11B1、CYP11B2、CYP17A1、HSD3B2、StAR、PORAndCYP11A1) (ii) a (3) Constructing a third generation sequencing library; (4) sequencing and analyzing the gene mutation types.

Description

Primer group and kit for simultaneously detecting multiple mutations of 9 genes related to congenital adrenal cortical hyperplasia

Technical Field

The invention relates to a primer group and a kit for simultaneously detecting multiple mutations of 9 genes related to congenital adrenal cortical hyperplasia by using a third-generation long-reading sequencing platform, and a related method.

Background

Congenital Adrenal cortical Hyperplasia (CAH) is a group of autosomal recessive genetic diseases caused by enzyme defects in the synthesis of adrenocortical hormone. According to the type of enzyme deficiency, CAH can be classified into 21-Hydroxylase deficiency (21-Hydroxylase, 21-OHD), 11 beta-Hydroxylase deficiency (11 beta-Hydroxylase, 11 beta-OHD), 17 alpha-Hydroxylase deficiency (17 alpha-Hydroxylase, 17 alpha-OHD), 3 beta-Hydroxysteroid dehydrogenase type 2 deficiency, 3 beta-HSD), congenital adrenal lipid increase (Lipoid CAH), Cytochrome P450 oxidoreductase deficiency (Cytochrome P450 oxidoreductase)Enzyme specificity, POR specificity, Cholesterol Side Chain lyase Deficiency (Cholesterol Side-Chain Cleavage Enzyme specificity, SCC specificity), respectivelyCYP21A2、CYP11B1、CYP17A1、HSD3B2、StAR、PORAndCYP11A1mutation of these 7 genes causes¹. Of these, 21-OHD is most common, about 90-95% of CAH, 11 β -OHD about 5-8%, 17 α -OHD and 3 β -HSD each about 1% or less than 1%, and the other three types are less common^{1, 2}。

From heavy to light clinical phenotype, 21-OHD can be classified into desalination (SW), Simple Virilization (SV), and atypical CAH (NCCAH), wherein desalination and simple virilization are collectively referred to as typical CAH. 21-OHD-provoking human sourceCYP21A2The gene is located in the HLAIII gene region of 6p21.3, is about 3.2kb in length, and has 10 exons and 9 introns.CYP21A2Genes and corresponding pseudogenesCYP21A1PThe homology of the exon and intron of (a) is 98% and 96%, respectively.CYP21A1PAndCYP21A2in contrast, mutations at 10 positions resulted in loss of gene function, including c.92C>T (p.P30L)、c.293-13C/A>G (In2G)、c.332_339del (p.G110Efs)、c.518T>A (p.I172N)、E6 Cluster（c.710T>A （p.I236N）、c.713T>A (p.V237E)、c.719T>A (p.M239K)）、c.845T>G (p.V281L)、c.923dup (p.L307fs)、c.955C>T (p.Q318X)、c.1069C>T (p.R356W)、c.1360C>T (p.453S)³. As shown in FIG. 1, the telomere side gene string RP1-C4A-CYP21A1P-TNXA and the centromere side homologous gene string RP2-C4B-CYP21A2-TNXB are connected in series to form a double RCCX pattern. The high homology of the pattern of RCCXs allows unequal interchanging or recombination to occur during mitosis or meiosis. About 70% ofCYP21A2The gene mutation is due to pseudogeneCYP21A1PGene conversion occurs to cause pathogenic point mutations. 20-25% ofCYP21A2Gene mutations are large deletions of 30kb due to unequal crossovers, includingCYP21A1PAndCYP21A29 kinds caused by gene recombinationCYP21A1P/CYP21A2Chimera CH1-CH9 large fragment deletion, andTNXAandTNXBcaused by gene recombination3 kinds ofTNXA/TNXBChimera CH1-CH3 large fragment deletion³。TNXA/TNXBThe chimera results in deletion of the entire CYP21a2 gene and partial exon of TNXB gene exchanged with TNXA to be nonfunctional, causing CAH-X. About 10% of the CAH population suffers from CAH-X, a connective tissue dysplasia consistent with the overactive Ehlers-Danlos syndrome (EDS) phenotype. The unequal crossovers resulting from homologous recombination also produce three or more patterns of RCCX, possibly related to p.Q318X point mutations⁴. At present, the most common method for detecting 21-OHD by molecules is to detect deletion or copy number increase of large fragments by MLPA and detect true genes by combining Sanger sequencing specificityCYP21A2Point mutation of (3)⁵. But instead of the other end of the tubeCYP21A2AndCYP21A1Pthere is a complex recombination relationship between them, i.e., true genesCYP21A2Will have pseudogeneCYP21A1PAbove characteristic base sequence, and pseudogeneCYP21A1PTrue genes will also be present inCYP21A2The characteristic sequences can interfere with the specificity of the MLPA probe and the specificity of PCR amplification primers and sequencing primers in Sanger sequencing, and can cause missed diagnosis and misdiagnosis in actual detection^{5, 6, 7}. The amplification primers are designed inCYP21A1PAndCYP21A2long fragment PCR at both ends of the homologous region is favorable for specific amplificationCYP21A2Fragment of, orCYP21A1P-CYP21A2Chimeras, combined with Next-generation sequencing (NGS), can specifically detect point mutations in true genes. However, each fragment of the method needs to construct an NGS sequencing library, cannot be compatible with other gene detection, has a more complicated process and cannot detect multiple RCCX patterns^{8, 9, 10}。

Pathogenic gene of 11 beta-OHDCYP11B1Located on chromosome 8q24.3 and consisting of 9 exons and 8 introns. Result inCYP11B1The loss-of-function mutations are mainly nonsense and missense mutations, and are mainly distributed in the coding region.CYP11B1From homologous genes that are about 40kb apartCYP11B2Will form a chimeric geneCYP11B2/CYP11B1Leading to glucocorticoid suppressive aldosteronism; is rareCYP11B2/CYP11B1Chimeric gene types may also lead to CAH^{1, 11}。17α-OHD、3β-HSD、Lipoid CAH, POR deficiency and SCC deficiency are relatively rare, and the pathogenic genes thereof are found through research at presentCYP17A1、HSD3B2、StAR、PORAndCYP11A1the type of mutation of (a) is mainly a point mutation¹。

Current methods based on qPCR, MLPA, Sanger sequencing or second generation sequencing can be implementedCYP21A2The detection of gene copy number and point mutation of most CAH related genes has the following limitations:

1. the detection of all mutation types of all CAH-related genes in the same system cannot be realized;

2. due to pseudogeneCYP21A1PHezhen geneCYP21A2The possibility of multiple recombination exists between the primers, the combination of MLPA probes and the design of Sanger sequencing primers are influenced, and the missed detection and the false detection are caused to a certain extent;

3. the inability to analyze multiple patterns of RCCX for recombination patterns and haplotypes in depth;

4. when two or more mutations exist on the same gene locus at the same time, the existing method cannot distinguish cis-mutation from trans-mutation.

Disclosure of Invention

In view of the above, the present invention is based on multiplex PCR amplification and third generation sequencing to detect multiple mutations of 9 CAH-associated genes simultaneously. The multiple PCR amplification can realize the simultaneous amplification of 9 CAH related genes in a single reaction tube (CYP21A2、CYP21A1P、CYP11B1、CYP11B2、CYP17A1、HSD3B2、StAR、PORAndCYP11A1) The point mutation, the structural variation and the copy number mutation are combined with the characteristics of reading length and measuring length of a third generation sequencing platform for reading length and measuring length and the like, so that the CAH related gene mutation can be accurately, quickly and high-flux detected. The method provided by the invention is simple and convenient to operate, the multiple PCR and third-generation library are reliable in quality and strong in repeatability, and the application of the third-generation sequencing technology to clinical detection is facilitated.

The invention aims to solve the problems of missed detection and false detection in clinic caused by incomplete CAH pathogenic gene coverage, difficult differentiation of CYP21A2 and CYP21A1P true and false genes, difficult copy number detection of RCCX modules, incapability of determining whether mutations are linked, and the like at the present stage. The aim of comprehensively, accurately and quickly detecting multiple mutations of CAH genes of multiple samples is fulfilled by simultaneously amplifying 12 segments of 9 pathogenic genes related to CAH through multiple PCR and preparing a third-generation sequencing library.

First, according to a first aspect of the present invention, the present invention relates to a primer set for simultaneously amplifying a plurality of mutations of 9 genes associated with adrenocortical hyperplasia, the primer set comprising one or more pairs of primers as follows: CYP21A2-F and CYP21A2-R, CYP21A1P-F and CYP21A1P-R, CYP11B1-F and CYP11B1-R, CYP11B1-F and CYP11B2-R, CYP17A1-F and CYP17A1-R, HSD3B2-F and HSD3B2-R, StAR-F and StAR-R, POR-F1 and POR-R1, POR-F2 and POR-R2, POR-F3 and POR-R3, CYP11A1-F1 and CYP11A1-R1, and CYP11A1-F2 and CYP11A1-R2 (primer positions are shown in FIG. 1).

Wherein, the CYP21A2-F and CYP21A2-R are respectively positioned at the upstream and downstream of a genome hg38 chr6:32,038,415-32,046, 127; CYP21A1P-F and CYP21A1P-R are respectively positioned at the upstream and downstream of the genome hg38 chr6:32,005,630-32,013, 273; CYP11B1-F and CYP11B1-R are respectively located at the upstream and downstream of genomes hg38 chr8:142,872,357 and 142,879 and 825, CYP11B1-F and CYP11B2-R are respectively located at the upstream and downstream of genomes hg38 chr8:142,872,357 and 142,917 and 843; CYP17A1-F and CYP17A1-R are located at the upstream and downstream of the genome hg38 chr10:102,830,531-102,837,472, respectively; HSD3B2-F and HSD3B2-R are respectively positioned at the upstream and downstream of the genome hg38 chr1:119,414,931 and 119,422 and 620; POR-F1 and POR-R1 are located at the upstream and downstream of the hg38 chr7:75,953,989-75,954,180 genome respectively; POR-F2 and POR-R2 are located at the upstream and downstream of the hg38 chr7:75,972,413-75,972,461, respectively; POR-F3 and POR-R3 are respectively located at the upstream and downstream of the genome hg38 chr7:75,979,451-75,986, 481; CYP11A1-F1 and CYP11A1-R1 are respectively located at the upstream and downstream of the genomes hg38chr15:74,337,972-74,348,055, and CYP11A1-F2 and CYP11A1-R2 are respectively located at the upstream and downstream of the genomes hg38chr15:74, 367,317-74,365, 752.

The primers can amplify the entire sequence on the genome within the primer range, including any type of mutated sequence within the primer range. Preferably, the amplification product of each primer is less than 15 Kb. Preferably, degenerate base primers are used if the primers have SNPs placed therein.

In a specific embodiment, wherein the primer sequences are shown as SEQ ID NO 1-23 in Table 1.

In a preferred embodiment, the primer set capable of simultaneously amplifying 9 gene mutations can simultaneously detect at least:CYP21A1PandCYP21A2large fragment deletion caused by gene recombination,TNXAandTNXBlarge fragment deletions resulting from gene recombination, and gene copy number increases corresponding to these deletions;CYP11B1large fragment deletion caused by recombination with CYP11B2 gene; as shown in Table 2CYP21A2951 point mutations in the gene; as shown in Table 3CYP11B1138 point mutations in the gene; as shown in Table 4HSD3B2114 point mutations on the gene; as shown in Table 5CYP17A1128 point mutations on the gene; as shown in Table 6StAR136 point mutations on the gene; as shown in Table 7POR248 point mutations on the gene; as shown in Table 8CYP11A162 point mutations on the gene.

Among these, point mutations and structural variations at the genetic loci described herein can be queried in LOVD, ClinVar, and references; see tables 2-8 for detailed mutation information.

In a preferred embodiment, the primer set can simultaneously detect and detect the copy numbers of the pseudogene CYP21A1P-TNXA, the eugene CYP21A2-TNXB and the recombinant products CYP21A1P-CYP21A2-TNXB, CYP21A2-CYP21A1P-TNXA and CYP21A 2-TNXA; can also detectCYP11B1AndCYP11B2mutations resulting from recombination between genes.

In one embodiment, 5 to 50nt of DNA with different sequences, i.e., DNA Barcode (Barcode), can be added to the 5' end of the primer for distinguishing different samples; preferably, the 5' end Barcode of the F and R primers may be the same or different, and may be selected by those skilled in the art as desired.

In a preferred embodiment, the primer set is used for multiplex PCR amplification of 12 fragments of 9 genes; it can also be used to detect whether different mutations within a single amplified gene fragment are linked.

The primer group can be used for multiplex primer PCR amplification of CAH related pathogenic gene fragments including mutation types in all primer ranges. In combination with subsequent PacBio or Nanopore sequencing platforms, the mutation patterns of all gene fragments within the primer range can be detected.

According to a second aspect of the present invention, there is provided a kit for simultaneously detecting multiple mutations of 9 CAH-associated genes, comprising the following reagents:

(1) reagents for multiplex PCR amplification;

(2) reagents for constructing a third generation sequencing library.

In one embodiment, wherein the reagents for multiplex PCR amplification comprise a DNA polymerase, a reaction buffer, and a primer set.

In a preferred embodiment, the primer set in the kit is selected from one or more primer pairs of the following 23 primers:

CYP21A2-F and CYP21A2-R, CYP21A1P-F and CYP21A1P-R, CYP11B1-F and CYP11B1-R, CYP11B1-F and CYP11B2-R, CYP17A1-F and CYP17A1-R, HSD3B2-F and HSD3B2-R, StAR-F and StAR-R, POR-F1 and POR-R1, POR-F2 and POR-R2, POR-F3 and POR-R3, CYP11A1-F1 and CYP11A1-R1, and CYP11A1-F2 and CYP11A1-R2 (primer positions are shown in FIG. 1).

Wherein, the CYP21A2-F and CYP21A2-R are respectively positioned at the upstream and downstream of a genome hg38 chr6:32,038,415-32,046, 127; CYP21A1P-F and CYP21A1P-R are respectively positioned at the upstream and downstream of the genome hg38 chr6:32,005,630-32,013, 273; CYP11B1-F and CYP11B1-R are respectively located at the upstream and downstream of genomes hg38 chr8:142,872,357 and 142,879 and 825, CYP11B1-F and CYP11B2-R are respectively located at the upstream and downstream of genomes hg38 chr8:142,872,357 and 142,917 and 843; CYP17A1-F and CYP17A1-R are located at the upstream and downstream of the genome hg38 chr10:102,830,531-102,837,472, respectively; HSD3B2-F and HSD3B2-R are respectively positioned at the upstream and downstream of the genome hg38 chr1:119,414,931 and 119,422 and 620; POR-F1 and POR-R1 are located at the upstream and downstream of the hg38 chr7:75,953,989-75,954,180 genome respectively; POR-F2 and POR-R2 are located at the upstream and downstream of the hg38 chr7:75,972,413-75,972,461, respectively; POR-F3 and POR-R3 are respectively located at the upstream and downstream of the genome hg38 chr7:75,979,451-75,986, 481; CYP11A1-F1 and CYP11A1-R1 are respectively located at the upstream and downstream of the genomes hg38chr15:74,337,972-74,348,055, and CYP11A1-F2 and CYP11A1-R2 are respectively located at the upstream and downstream of the genomes hg38chr15:74, 367,317-74,365, 752. The primers can amplify the entire sequence within the primer range, including any type of mutated sequence within the primer range.

Preferably, the amplification product of each primer is less than 15 Kb. Preferably, degenerate base primers are used if the primers have SNPs placed therein.

In a preferred embodiment, the primer sequences are shown as SEQ ID NO. 1-23 in Table 1.

In a preferred embodiment, 5 to 50nt of DNA (Barcode) of different sequences may be added to the 5' end of the primers in the kit for distinguishing between different samples; preferably, the 5' end Barcode of the F and R primers may be the same or different, and may be selected by those skilled in the art as desired.

In one embodiment, for the kit, the PCR amplification product may or may not be purified before proceeding to the next reaction, and may be selected as desired by one skilled in the art.

In another embodiment, wherein in the kit, the reagents for constructing the third generation sequencing library include a terminal repair enzyme, a linker, a ligase, a DNA purification magnetic bead, a reaction buffer, and an exonuclease.

In a preferred embodiment, said kit is capable of detecting at least simultaneously:CYP21A1PandCYP21A2large fragment deletion caused by gene recombination,TNXAandTNXBweight of geneLarge fragment deletions caused by the group, and gene copy number increases corresponding to these deletions;CYP11B1large fragment deletion caused by recombination with CYP11B2 gene; as shown in Table 2CYP21A2951 point mutations in the gene; as shown in Table 3CYP11B1138 point mutations in the gene; as shown in Table 4HSD3B2114 point mutations on the gene; as shown in Table 5CYP17A1128 point mutations on the gene; as shown in Table 6StAR136 point mutations on the gene; as shown in Table 7POR248 point mutations on the gene; as shown in Table 8CYP11A162 point mutations on the gene.

Among these, point mutations and structural variations at the genetic loci described herein can be queried in LOVD, ClinVar, and references; see tables 2-8 for detailed mutation information.

In a preferred embodiment, the kit can simultaneously detect the copy numbers of the pseudogene CYP21A1P-TNXA, the eugene CYP21A2-TNXB and the recombinant products CYP21A1P-CYP21A2-TNXB, CYP21A2-CYP21A1P-TNXA and CYP21A 2-TNXA; can also detectCYP11B1AndCYP11B2mutations resulting from recombination between genes.

In a preferred embodiment, the primer set is used for multiplex PCR amplification of 12 fragments of 9 genes; it can also be used to detect whether different mutations within a single amplified gene fragment are linked.

In a specific embodiment, wherein for said kit, multiplex PCR amplification is done in a single reaction tube.

In a preferred embodiment, the third generation sequencing is selected from PacBio sequencing by Pacific Biosciences or Nanopore sequencing by ONT.

In a specific embodiment, PacBio library adaptor ligation may use blunt end ligation or TA ligation.

In a specific embodiment, the PacBio universal blunt-ended linker sequence is 5 '-pATCTCTCTCTTCCTCCCTCCCTCCGTTGTTGTTGAGAGAGAT-3' (SEQ ID NO: 24), and a blunt-ended stem-loop structure linker aptamer is formed by annealing. Different linker aptamers with Barcode can be formed by adding DNA (Barcode) with 5-50nt different sequences to the stem. The PacBio libraries with different barcodes can be sequenced mixed together.

In a specific embodiment, the PacBio universal TA linker sequence is 5 '-pATCTCTCTCTTTTCCTCCCTCCCTCTGTTGTTGTTGAGAGAGATT-3' (SEQ ID NO: 25), and a blunt-ended stem-loop structure linker aptamer is formed by annealing. Different linker aptamers with Barcode can be formed by adding DNA (Barcode) with 5-50nt different sequences to the stem. The PacBio libraries with different barcodes can be sequenced mixed together.

In one embodiment, the PacBio linker may or may not be Barcode. Preferably, the PacBio linker is a Barcode designed by PacBio corporation or a Barcode designed by itself, which can be selected by those skilled in the art as desired.

In a preferred embodiment, the PacBio library is matched to the Pacific Biosciences sequencing platform.

In a preferred embodiment, wherein the reagents for constructing the three-generation Nanopore library include a terminal repair enzyme, a linker, a ligase, a DNA purification magnetic bead, 80% ethanol, and a reaction buffer.

In one embodiment, Nanopore library adaptor ligation may be by blunt end ligation or TA ligation.

In one embodiment, the Nanopore linker may or may not be Barcode. Preferably, the Nanopore linker is either Barcode, available from ONT corporation, or Barcode, available from self-designed sources, and can be selected by those skilled in the art as desired.

In a preferred embodiment, the Nanopore library is matched to the ONT corporation sequencing platform.

The third aspect of the invention provides a system for simultaneously detecting multiple mutations of 9 genes related to congenital adrenal cortical hyperplasia, which comprises the following parts:

(1) an acquisition module: a subject sample can be obtained;

(2) an amplification module: multiplex PCR amplification of 9 genes in the sample, said genes beingCYP21A2、CYP21A1P、CYP11B1、CYP11B2、CYP17A1、HSD3B2、StAR、PORAndCYP11A1；

(3) a library construction module: constructing a third generation sequencing library;

(4) a sequencing module: sequencing and analyzing the gene mutation type;

wherein the primer set for multiplex PCR amplification is the primer set described above in the present invention.

In some embodiments, in the amplification module in the system, the multiplex PCR amplification is done in a single reaction tube.

In some embodiments, the third generation sequencing is selected from PacBio sequencing by Pacific Biosciences, Inc., or Nanopore sequencing by Oxford Nanopore Technologies (ONT).

In another aspect, the present invention provides a method for simultaneously detecting multiple mutations of 9 genes associated with congenital adrenal cortical hyperplasia, comprising the following steps:

(1) preparing a sample of a subject;

(2) multiplex PCR amplification of 9 genes in the sample, said genes beingCYP21A2、CYP21A1P、CYP11B1、CYP11B2、CYP17A1、HSD3B2、StAR、PORAndCYP11A1；

(3) constructing a third generation sequencing library;

(4) sequencing and analyzing the gene mutation types.

In one embodiment, the multiplex PCR primer set used in the method of the invention is selected from the primer sets described above. Preferably, 5 to 50nt of DNA (Barcode) of different sequences may be added to the 5' end of the primers described above for distinguishing between different samples.

In a preferred embodiment, the 5' end of the F and R primers, Barcode, may be the same or different and may be selected by one skilled in the art as desired.

In a preferred embodiment, wherein said method is capable of detecting at least simultaneously:CYP21A1PandCYP21A2large fragment deletion caused by gene recombination,TNXAandTNXBlarge fragment deletions resulting from gene recombination, and gene copy number increases corresponding to these deletions;CYP11B1large fragment deletion caused by recombination with CYP11B2 gene; as shown in Table 2CYP21A2951 point mutations in the gene; as shown in Table 3CYP11B1138 point mutations in the gene; as shown in Table 4HSD3B2114 point mutations on the gene; as shown in Table 5CYP17A1128 point mutations on the gene; as shown in Table 6StAR136 point mutations on the gene; as shown in Table 7POR248 point mutations on the gene; as shown in Table 8CYP11A162 point mutations on the gene.

Among these, point mutations and structural variations at the genetic loci described herein can be queried in LOVD, ClinVar, and references; see tables 2-8 for detailed mutation information.

In a preferred embodiment, the method can simultaneously detect the copy numbers of the pseudogene CYP21A1P-TNXA, the eugene CYP21A2-TNXB and the recombinant products CYP21A1P-CYP21A2-TNXB, CYP21A2-CYP21A1P-TNXA and CYP21A 2-TNXA; can also detectCYP11B1AndCYP11B2mutations resulting from recombination between genes.

In a preferred embodiment, the primer set is used for multiplex PCR amplification of 12 fragments of 9 genes; it can also be used to detect whether different mutations within a single amplified gene fragment are linked.

In a preferred embodiment, wherein the method, multiplex PCR amplification is performed in a single reaction tube.

In one embodiment, wherein the sample is selected from a biological sample or gDNA extracted from a sample. Wherein the biological sample is selected from cultured cell lines, blood, amniotic fluid, villi, gametes, blastocytes, synovial fluid, urine, sweat, saliva, stool, cerebrospinal fluid, ascites, pleural fluid, bile or pancreatic fluid.

In a specific embodiment, wherein the third generation sequencing of the method is selected from PacBio sequencing by Pacific Biosciences or Nanopore sequencing by ONT.

In one embodiment, PacBio library adaptor ligation may use blunt-end ligation or TA ligation.

In one embodiment, the PacBio universal blunt-ended linker sequence is 5 '-pATCTCTCTCTTCCTCCCTCCCTCTGTTGTTGTTGAGAGAGAGAT-3' (SEQ ID NO: 24), and a blunt-ended stem-loop structure linker aptamer is formed by annealing. Different linker aptamers with Barcode can be formed by adding DNA (Barcode) with 5-50nt different sequences to the stem. The PacBio libraries with different barcodes can be sequenced mixed together.

In one embodiment, the PacBio universal TA linker sequence is 5 '-pATCTCTCTCTTTTCCTCCCTCCCTCTGTTGTTGTTGAGAGAGATT-3' (SEQ ID NO: 25), and a blunt-ended stem-loop structure linker aptamer is formed by annealing. Different Barcode-bearing linker aptamers can be formed by adding 5-50nt of DNA (Barcode) with different sequences to the stems, and PacBio libraries with different Barcode can be mixed together for sequencing.

In one embodiment, the PacBio linker may or may not be Barcode. In a preferred embodiment, the PacBio linker is a Barcode designed by PacBio Inc. or a Barcode designed by itself. One skilled in the art can select as desired.

In a preferred embodiment, the PacBio library is matched to the Pacific Biosciences sequencing platform.

In a preferred embodiment, wherein the reagents for constructing the three-generation Nanopore library comprise a terminal repair enzyme, a linker, a ligase, a DNA purification magnetic bead, 80% ethanol and a reaction buffer.

In one embodiment, Nanopore library adaptor ligation may be by blunt end ligation or TA ligation.

In one embodiment, the Nanopore linker may or may not be a Barcode, and may be selected by one skilled in the art as desired. Preferably, the Nanopore linker is either Barcode, available from ONT corporation, or Barcode, available from self-designed sources, and can be selected by those skilled in the art as desired.

In a preferred embodiment, the Nanopore library is matched to the ONT corporation sequencing platform.

The method based on the specific combination of long-fragment multiplex PCR amplification and third-generation high-throughput sequencing can be used for detecting multiple mutations of 9 pathogenic genes related to CAH of multiple samples at the same time with high specificity, accuracy and rapidness.

The excellent technical effects of the method and the kit mainly lie in the following aspects:

(1) the detection range is wide. The invention can simultaneously detect the mutation of all CAH related genes which are researched and found at present. Comprises thatCYP21A1PAndCYP21A2large fragment deletion caused by gene recombination,TNXAandTNXBlarge fragment deletions resulting from gene recombination, and gene copy number increases corresponding to these deletions;CYP11B1large fragment deletion caused by recombination with CYP11B2 gene; 951 speciesCYP21A2Point mutations in the gene; 138 kinds ofCYP11B1Point mutations in the gene; 114 kinds ofHSD3B2Point mutations in the gene; 128 kinds ofCYP17A1Point mutations in the gene; 136 kinds ofStARPoint mutations in the gene; 248 kinds ofPORPoint mutations in the gene; 62 kinds ofCYP11A1Point mutations in the gene. Meanwhile, due to the characteristics of third-generation sequencing, length reading and length measuring, the invention can also detect whether different mutations are linked.

(2) Single tube detection of multiple mutation types. The traditional method needs to set a detection system for each mutation type, and the invention simultaneously detects a plurality of mutations in a reaction primer system, including SNV, Indel, gene recombination and copy number variation.

(3) The detection error detection rate and the detection omission rate are low. The current general method for detecting the most common pathogenic gene CYP21A2 of CAH is MLPA combined with Sanger sequencing. Since the homology between the true gene CYP21A2 and the false gene CYP21A1P exceeds 96%, multiple recombination can occur between the genes, and characteristic sites between the true and false genes are not specific, so that the Sanger sequencing result can be interfered. The invention directly amplifies full-length fragments of CYP21A1, CYP21A1P and recombinant genes, so that the risk of false detection and missing detection does not exist.

(4) The samples were diversified. The template for PCR may be peripheral blood, dried blood spots or extracted genomic DNA, or may be a human cell line or other specific tissue.

(5) High-throughput detection. The third generation sequencing can realize 384 Barcode linkers, and actually more Barcode linkers can be designed according to needs. Or more kinds of Barcode combinations are realized by using a primer-strip Barcode and a linker-strip Barcode double Barcode. The high throughput characteristics of the third generation sequencing platform determine that high throughput sample detection can be achieved.

(6) The accuracy is high. The dumbbell library of PacBio can be subjected to multiple rounds of reading during sequencing, and the base accuracy of the corrected sequencing result is more than 99%. And PacBio sequencing errors were random and corrected for base accuracy by sequencing depth to greater than 99.9%. Therefore, gene mutations within the detection range of the primers can be accurately read.

(7) The detection time is flexible. The Nanopore platform can generate data in minutes, and can start data analysis in minutes or hours according to actual data volume requirements. The Nanopore platform has time advantages when the requirement for detection time efficiency is high.

Drawings

FIG. 1: schematic diagram of multiplex PCR primer design.

FIG. 2: DNA gel electrophoresis images of different samples were amplified according to the multiplex PCR method in example 1.

Fig. 3A to 3E: a representative PacBio sequencing result graph of CAH-related gene mutation sample, wherein fig. 3A is a CYP21a2 point mutation sample; FIG. 3B is a sample of a large fragment deletion of CYP21A 2; fig. 3C is a CYP21a2 multicopy sample; FIG. 3D is a sample of HSB3D point mutations; FIG. 3E is a sample of CYP17A1 point mutations.

Detailed Description

Example 1: amplification of CAH-related Gene mutations Using the multiplex PCR method of the present invention

Reaction systems were prepared to amplify peripheral blood, dried blood spots and genomic DNA samples according to table 9 below.

On a PCR instrument, pre-amplification was performed under the conditions shown in Table 10 below:

after amplification, 5 ul of each sample was taken and tested on 1% DNA gel, and the results are shown in FIG. 2, and CAH-related genes were efficiently amplified using different samples as templates.

Example 2: construction of PacBio sequencing library Using the multiplex PCR method of the present invention

Step 1: multiplex PCR amplification

The reaction system was prepared to amplify peripheral blood samples of different types of CAH-related gene mutations according to table 11 below:

on a PCR instrument, pre-amplification was performed under the conditions shown in Table 12 below:

after amplification, the amplification product was put into a centrifuge at 10000rpm for 20 min. After the centrifugation, the mixture was left standing horizontally, and 4. mu.L of the supernatant was added to a new tube.

Step 2: construction of PacBio sequencing library

The reaction system was prepared as follows:

on a PCR instrument, the reaction is carried out according to the following conditions: c, 20min at 37 ℃; 25 ℃ for 15 min; and (5) performing temperature regulation at 65 ℃ for 10 min. After the reaction was completed, 0.5. mu.L of Exonase III (NEB, Cat # M0206L) and 0.5. mu.L of Exonase VII (NEB, Cat # M0379L) were added and the reaction was continued at 37 ℃ for 1 hour. The DNA was purified twice using 0.6X Ampure PB beads (PacBio, Cat # 100-. The resulting DNA eluate was the target DNACBio sequencing library. The DNA concentration was determined on a Qubit 3 Fluorometer (ThermoFisher, Cat # Q33216) using a Qubit dsDNA HS reagent (ThermoFisher, Cat # Q32851). When there are multiple samples of the PacBio sequencing library, equal amounts of the library can be mixed together to prepare a mixed library.

And step 3: sequencing and analysis on a PacBio machine

According to the total concentration and molar concentration of the library, the library with an appropriate volume is reacted with a binding reagent (PacBio, Cat #101-820-200) and a primer (PacBio, Cat # 100-970-100) to prepare the final operable library. Representative sequencing results are shown in fig. 3, a CYP21a2 point mutation sample in fig. 3A, a large deletion of CYP21a2 fragment in fig. 3B, a CYP21a2 multicopy sample in fig. 3C, a HSB3D point mutation sample in fig. 3D, and a CYP17a1 point mutation sample in fig. 3E.

Example 3: detection and validation of CAH-associated Gene mutations

Heel blood of 14 subjects and peripheral blood genomic DNA of 11 subjects were collected from the Hospital of the Hunan Jiahui genetics specialty as 25 verification samples, and referring to example 2, the primer set (and kit) of the present invention was used to simultaneously detect multiple mutations at 9 CAH-associated gene sites. Meanwhile, the copy number of CYP21A2 gene is detected by using an MLPA method, and point mutation of related genes is detected by combining Sanger sequencing. The results obtained by the present invention were compared with the control results, and the results are shown in table 14, where the results of 25 samples are completely consistent.

Therefore, the detection result obtained by the method of the invention is compared with the MLPA combined PCR-Sanger sequencing method, and the specificity and the sensitivity reach 100 percent. In addition, 10 samples out of 25 samples have determined the range of large fragment deletion by the method of the invention, or have clarified cis-trans relationship between different mutation points.

It should be noted that although some features of the present invention have been illustrated by the above embodiments, they are not intended to limit the present invention, and various modifications and changes can be made by those skilled in the art. The reagents, reaction conditions, etc. involved in the multiplex PCR reaction and the third-generation sequencing library construction can be adjusted and changed according to specific needs. It will thus be appreciated by those skilled in the art that changes may be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims.

Reference to the literature

[1] Fady Hannah-Shmouni, Wuyan Chen, Deborah P Merke. Endocrinol Metab Clin North Am. Genetics of Congenital Adrenal Hyperplasia. 2017 Jun;46(2):435-458. doi: 10.1016/j.ecl.2017.01.008

[2] The national institutional and health commission clinical testing center neonatal hereditary metabolic disease screening laboratory interstitial evaluation committee. The screening of congenital adrenal cortical hyperplasia of newborn is commonly recognized by technical experts of diagnostic laboratory detection. China journal of laboratory medicine, vol 42, No. 12, 12 months 2019.

[3] Merke DP, Auchus RJ. Congenital Adrenal Hyperplasis Due to 21-Hydroxylase Deficiency. N Engl J Med. 2020 Sep 24;383(13):1248-1261. doi:10.1056/NEJMra1909786.

[4] Maria I New 1, Moolamannil Abraham, Brian Gonzalez, Miroslav Dumic, Maryam Razzaghy-Azar, David Chitayat, Li Sun, Mone Zaidi, Robert C Wilson, Tony Yuen. Genotype-phenotype correlation in 1,507 families with congenital adrenal hyperplasia owing to 21-hydroxylase deficiency. Proc Natl Acad Sci U S A. 2013 Feb 12;110(7):2611-6. doi: 10.1073/pnas.1300057110.

[5] Duarte Pignatelli , Berta L Carvalho, Aida Palmeiro, Alberto Barros, Susana G Guerreiro, Djuro Macut. The Complexities in Genotyping of Congenital Adrenal Hyperplasia: 21-Hydroxylase Deficiency. Front Endocrinol (Lausanne). 2019 Jul 4;10:432. doi: 10.3389/fendo.2019.00432.

[6] Paola Concolino. Issues with the Detection of Large Genomic Rearrangements in Molecular Diagnosis of 21-Hydroxylase Deficiency. Mol Diagn Ther. 2019 Oct;23(5):563-567. doi: 10.1007/s40291-019 -00415-z.

[7] Wuyan Chen, Zhi Xu, Annie Sullivan, Gabriela P Finkielstain, Carol Van Ryzin, Deborah P Merke, Nazli B McDonnell. Junction site analysis of chimeric CYP21A1P/CYP21A2 genes in 21-hydroxylase deficiency. Clin Chem. 2012 Feb;58(2):421-30. doi: 10.1373/clinchem. 2011.174037.

[8] Hsien-Hsiung Lee. Mutational analysis of CYP21A2 gene and CYP21A1P pseudogene: long-range PCR on genomic DNA. Methods Mol Biol. 2014;1167:275-87. doi: 10.1007/978-1-4939-0835-6_19.

[9] Christopher N Greene, Suzanne K Cordovado, Daniel P Turner, Lisa M Keong, Dorothy Shulman, Patricia W Mueller. Novel method to characterize CYP21A2 in Florida patients with congenital adrenal hyperplasia and commercially available cell lines. Mol Genet Metab Rep. 2014 Aug 8;1:312-323. doi: 10.1016/j.ymgmr.2014.07.002.

[10] Wencui Wang, Rulai Han, Zuwei Yang, Sichang Zheng, Haorong Li, Zhihan Wan, Yan Qi, Shouyue Sun, Lei Ye, Guang Ning. Targeted gene panel sequencing for molecular diagnosis of congenital adrenal hyperplasia. J Steroid Biochem Mol Biol. 2021 Apr 14;211:105899. doi: 10.1016/j.jsbmb.2021.105899.

[11] M Hampf, N T Dao, N T Hoan, R Bernhardt. Unequal crossing-over between aldosterone synthase and 11beta-hydroxylase genes causes congenital adrenal hyperplasia. J Clin Endocrinol Metab. 2001 Sep;86(9):4445-52.doi:10.1210/jcem.86.9.7820.

Sequence listing

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Claims (13)

1. A primer set for simultaneously amplifying a plurality of mutations of 9 genes associated with adrenal cortical hyperplasia; wherein the primer set comprises the following primer pairs:

CYP21A2-F and CYP21A 2-R;

CYP21A1P-F and CYP21A 1P-R;

CYP11B1-F and CYP11B 1-R;

CYP11B1-F and CYP11B 2-R;

CYP17A1-F and CYP17A 1-R;

HSD3B2-F and HSD3B 2-R;

StAR-F and StAR-R;

POR-F1 and POR-R1;

POR-F2 and POR-R2;

POR-F3 and POR-R3;

CYP11A1-F1 and CYP11A 1-R1; and the number of the first and second groups,

CYP11A1-F2 and CYP11A 1-R2;

wherein, the primer group can simultaneously detect a plurality of mutations in the primer range, and the mutations at least comprise:

CYP21A1PandCYP21A2large fragment deletion caused by gene recombination,TNXAandTNXBlarge fragment deletions resulting from gene recombination, and gene copy number increases corresponding to these deletions;CYP11B1large fragment deletion caused by recombination with CYP11B2 gene; as shown in Table 2CYP21A2951 point mutations in the gene; as shown in Table 3CYP11B1138 point mutations in the gene; as shown in Table 4HSD3B2114 point mutations on the gene; as shown in Table 5CYP17A1128 point mutations on the gene; as shown in Table 6StAR136 point mutations on the gene; as shown in Table 7POR248 point mutations on the gene; as shown in Table 8CYP11A162 point mutations on the gene;

wherein the sequences of the primers CYP21A2-F, CYP21A2-R, CYP21A1P-F, CYP21A1P-R, CYP11B1-F, CYP11B1-R, CYP11B2-R, CYP17A1-F, CYP17A1-R, HSD3B2-F, HSD3B2-R, StAR-F, StAR-R, POR-F R, POR, POR-R R, POR, CYP11A R, POR-F R, POR, CYP11A R, POR-R R, POR, CYP11A R, POR-F R, POR and CYP11A R, POR-R R, POR are respectively shown in SEQ ID NO 1-23.

2. The primer set according to claim 1, wherein the primer is added with 5-50nt of DNA with different sequences at the 5' end, i.e., DNA barcode, for distinguishing different samples.

3. The primer set of claim 1, wherein the primer set is useful for detecting whether different mutations within an amplification product fragment are linked.

4. A kit capable of simultaneously detecting multiple mutations of 9 genes related to adrenal cortical hyperplasia comprises the following reagents:

(1) reagents for multiplex PCR amplification;

(2) reagents for constructing a third generation sequencing library;

wherein the 9 genes related to the adrenal cortical hyperplasia areCYP21A2、CYP21A1P、CYP11B1、CYP11B2、CYP17A1、HSD3B2、StAR、PORAndCYP11A1；

wherein the reagents for multiplex PCR amplification comprise the primer set of any one of claims 1-3.

5. The kit according to claim 4, wherein a plurality of mutations of 9 genes associated with adrenocortical hyperplasia are detected simultaneously, said mutations comprising at least:

CYP21A1PandCYP21A2large fragment deletion caused by gene recombination,TNXAandTNXBlarge fragment deletions resulting from gene recombination, and gene copy number increases corresponding to these deletions;CYP11B1large fragment deletion caused by recombination with CYP11B2 gene; as shown in Table 2CYP21A2951 point mutations in the gene; as shown in Table 3CYP11B1138 point mutations in the gene; as shown in Table 4HSD3B2114 point mutations on the gene; as shown in Table 5CYP17A1128 point mutations on the gene; as shown in Table 6StAR136 point mutations on the gene; as shown in Table 7POR248 point mutations on the gene; as shown in Table 8CYP11A162 point mutations on the gene.

6. The kit of claim 4, wherein the kit is used to detect whether different mutations within the same amplified fragment are linked.

7. The kit of claim 4, wherein the reagents for multiplex PCR amplification further comprise a DNA polymerase, a reaction buffer.

8. The kit of claim 7, wherein the reagents for constructing a third generation sequencing library comprise linkers, ligases, DNA purification magnetic beads, reaction buffers, and exonucleases.

9. The kit of claim 4, wherein the multiplex PCR amplification is accomplished in a single reaction tube.

10. The kit of claim 4, wherein the third generation sequencing is selected from PacBio sequencing by Pacific Biosciences or Nanopore sequencing by Oxford Nanopore Technologies (ONT).

11. A system for simultaneously detecting multiple mutations of 9 genes related to congenital adrenal cortical hyperplasia comprises the following parts:

(1) an acquisition module: a subject sample can be obtained;

(2) an amplification module: multiplex PCR amplification of fragments of 9 genes in the sample, said genes beingCYP21A2、CYP21A1P、CYP11B1、CYP11B2、CYP17A1、HSD3B2、StAR、PORAndCYP11A1；

(3) a library construction module: constructing a third generation sequencing library;

(4) a sequencing module: sequencing and analyzing the gene mutation type;

wherein the primer set for multiplex PCR amplification is the primer set of any one of claims 1-3.

12. The system of claim 11, wherein the multiplex PCR amplification is accomplished in a single reaction tube.

13. The system of claim 11, wherein the third generation sequencing is selected from PacBio sequencing by Pacific Biosciences, Inc., or Nanopore sequencing by Oxford Nanopore Technologies (ONT).

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