WO2017124214A1 - 一种检测染色体罗氏易位的方法 - Google Patents

一种检测染色体罗氏易位的方法 Download PDF

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WO2017124214A1
WO2017124214A1 PCT/CN2016/070696 CN2016070696W WO2017124214A1 WO 2017124214 A1 WO2017124214 A1 WO 2017124214A1 CN 2016070696 W CN2016070696 W CN 2016070696W WO 2017124214 A1 WO2017124214 A1 WO 2017124214A1
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chr21
chr15
chr14
chr13
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冯涛
费嘉
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北京嘉宝仁和医疗科技有限公司
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Priority to US16/068,749 priority patent/US11345948B2/en
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • the invention relates to the field of molecular diagnostics, in particular to a method for detecting a Roche translocation of a chromosome, which is capable of detecting a normal and translocated carrier embryo and a fetus for a family whose parents are Roche translocation carriers.
  • the Robertsonian Translocation (ROB), named after the first American biologist William Rees Brebner Robertson who discovered this translocation, occurred mainly in five proximal centromere chromosomes (13, 14, 15 A chromosomal translocation of chromosomes 21 and 22 (Robertson WRB. Chromosome studies. I. Taxonomic aspects shown in the chromosomes of Tettigidae and Acrididae. V-shaped chromosomes and their significance in Acrididae, Locustidae and Gryllidae: chromosome and variation. J Morph 1916, 27: 179-331).
  • the short arm of the chromosome mainly includes random repetitive DNA and ribosomal RNA genes, and this gene is present in all five chromosomes, the Roche translocation does not cause a pathogenic phenotype despite the loss of the short arm of the chromosome.
  • the translocation chromosome and the corresponding two normal chromosome pairs will form a trivalent chromosome, which will lead to alternation, neighboring and uncommon 3 : 0 three ways of division, only alternating can produce normal or balanced gametes, the other two ways produce unbalanced gametes, the majority of non-equilibrium gametes are also difficult to carry to pregnancy, or repeated abortion during pregnancy.
  • 13-Patau syndrome which causes abnormal chromosome copy number.
  • More and more Roche translocation carriers are now seeking IVF technology for preimplantation diagnosis.
  • In vitro technology is to remove the egg and sperm and place it in a specific culture medium for cultivation and fertilization.
  • the fertilized egg develops into an embryo in a constant temperature incubator and is transplanted back to the mother uterus, eventually developing into a fetus. Selecting healthy embryo transfer is the key to successful prevention.
  • Preimplantation genetic screening refers to the detection of chromosome number and structural abnormalities before embryo implantation, and the selection of normal embryos into the uterus in order to obtain normal offspring.
  • Current pre-embryonic diagnostic methods for Roche translocation include SNP chips and fluorescence in situ hybridization (FISH).
  • FISH uses a fluorescently labeled specific nucleic acid probe to hybridize with a corresponding target DNA molecule in the cell, and the fluorescence signal is observed under a fluorescence microscope to determine the localization of the DNA molecule bound to the fluorescent probe in the chromosome.
  • FISH has certain defects in the method of examining single nuclei, with 7% of false negatives (Colls P, Escudero T, Cekleniak N, Sadowy S, Cohen J, Munné S. I ncreased efficiency of preimplantation genetic diagnosis for infertility using"no Result rescue".Fertil Steril.2007,88(1):53-61).
  • Spontaneous abortion is a common disease in obstetrics and gynecology. Its incidence is about 10% to 15% of all pregnancies. Its occurrence is affected by many factors, such as embryonic factors, placental factors, maternal factors and environmental factors. Chromosomal abnormalities are the main cause of miscarriage. Chromosome detection of abortion tissue analysis of embryonic cessation, abortion, not only provide a theoretical basis for clinical consultation, but also provide guidance for pregnant women to re-pregnancy. At present, FISH technology is used to detect the most common chromosomal abnormalities in the aborted villus and tissues of different parts of the fetus.
  • FISH technology As a targeted detection technology, FISH technology has limited commercial probes, which are largely limited by the type, quantity and cost of probes. At the same time, a large number of abnormal genes or The chromosome is tested. In addition, due to the limitations of FISH technology, FISH can not detect abnormal chromosome structure, which may lead to missed diagnosis of some patients with abnormal chromosome structure (Li Yangyang, Zhang Qin. Research status of karyotype analysis of spontaneous abortion villus chromosome. Journal of Practical Obstetrics and Gynecology, 2012 , 28:431-433).
  • the present invention contemplates a method for detecting the Roche translocation of a chromosome based on high-throughput sequencing technology to screen embryos, fetuses or abortion tissues of fully normal and translocated carriers.
  • a first aspect of the invention provides a method of detecting a Roche translocation of a chromosome comprising the steps of:
  • step (2) (4) determining the genotypes of the parental and progeny target sites for the SNP sites screened in step (2);
  • the chromosomes of the progeny were analyzed for Roche translocation.
  • the method is for detecting a translocation occurring between a human chromosome, in particular any two chromosomes of chromosomes 13, 14, 15, 21 and 22.
  • the parental DNA sample is parental genomic DNA extracted from a peripheral blood sample; the progeny DNA sample is selected from an embryonic or fetal trophoblast cell whole genome amplification product, or a genomic DNA extracted from aborted tissue, Whole-genome amplification products of blastocyst trophoblast cells of embryos cultured in vitro are preferred.
  • the screening criteria for the SNP locus in the above step (2) are:
  • the site is located in the 10 Mb region near the centromere;
  • the human genome database or the dbSNP database is included, and the SNP having the smallest allele frequency (MAF) greater than 0.2 in the target population is preferentially selected;
  • the SNP site comprises at least 1 SNP site, preferably at least 10 SNP sites, more preferably at least 50 SNP site SNP sites from each chromosome in which translocation occurs.
  • the SNP site is selected from the following SNP sites or any combination thereof: chr13: 19602195, chr13: 19625370, chr13: 19646283, chr13:19692745, chr13:19713634, chr13:19722801, chr13:19732341, chr13:19784913 , chr13:19843135,chr13:19877547,chr13:19883672,chr13:19896927,chr13:19917509,chr13:20494558,chr13:20709214,chr13:20747500,chr13:20784332,chr13:20825315,chr13:20864572,chr13:20881859,chr13 : 20908501, chr13: 20938952, chr13: 20951425, chr13: 20992333, chr13: 21012562, chr13: 21066
  • the methods of detecting chromosome copy number in steps (3) and (4) above are independently selected from the group consisting of sequencing methods and chip methods.
  • the sequencing method is a high-throughput sequencing method, preferably Ion Torrent PGM or Illumina Miseq;
  • the chip method is a human whole genome SNP chip, preferably an Illumina whole genome SNP chip.
  • one parent is a normal karyotype, and the other is a Roche translocation carrier, and the progeny includes an individual of a Roche translocation chromosome trisomy or a monomer.
  • the haplotype B derived from the parental Roche translocation carrier in chromosome n is a translocation chromosomal haplotype, correspondingly derived from the parental Roche translocation carrier chromosome
  • Another haplotype b of n is a normal chromosomal haplotype; further, by observing the haplotypes of other progeny, The chromosomal n haplotype B-linked chromosomal m haplotype A derived from the parental Roche translocation carrier is also a translocation chromosomal haplotype, and correspondingly, another monomer derived from the parental Roche translocation carrier chromosome m Type a is a normal chromosomal haplotype; thus, it can be judged that the progeny carrying chromosomal m haplotype A and chromosomal n haplotype B are translocation carriers carrying chromosomal m haplotype a and chromosomal
  • haplotype B derived from the parental Roche translocation carrier in chromosome n is a normal chromosomal haplotype, and correspondingly, derived from the parental Roche translocation carrier chromosome n
  • haplotype b is a translocation chromosomal haplotype; further, by observing the haplotypes of other progeny, a chromosomal m-single derived from a parental Roche translocation carrier linked to chromosomal n haplotype b
  • the body type a is also a translocation chromosomal haplotype.
  • haplotype A derived from the chromosome of the parental Roche translocation carrier is a normal chromosomal haplotype; thus, it can be judged that the chromosomal m haplotype is carried.
  • the progeny of A and chromosome n haplotype B are normal karyotypes, and the progeny carrying chromosomal m haplotype a and chromosomal n haplotype b are translocation carriers;
  • chromosome m and chromosome n are two chromosomes in the genome of the parental Roche translocation carrier, and A and a are haplotypes derived from the chromosome of the parental Roche translocation carrier, B and b are derived from The parental Roche translocation carrier haplotype of chromosome n.
  • the B-type of the chromosome 14 of the Roche translocation carrier is a translocation chromosome haplotype
  • the b-type is a normal chromosome haplotype.
  • Other embryos carrying type B or b can further speculate that type A is a translocation chromosome haplotype on chromosome 13, and type a is a normal chromosome haplotype;
  • chromosome 14 is a triploid or haploid
  • the type A of the Roche translocation carrier is a translocation chromosome haplotype
  • type a is a normal chromosome haplotype, which carries a type A or a by other
  • the type of embryo can be further speculated to carry chromosome B as the translocation chromosomal haplotype and b as the normal chromosomal haplotype.
  • carrying chromosome 13 a and 14 chromosome b is a normal embryo, while carrying chromosome 13 A and chromosome 14 B is a translocation carrier embryo.
  • the above method of the present invention is particularly suitable for preimplantation diagnosis of IVF infants.
  • a second aspect of the present invention provides a primer composition for detecting a Roche translocation of a chromosome, the primer composition capable of amplifying a sequence of a high frequency mutant SNP site in a region near a centromere of a proximal centromere chromosome .
  • the primer composition is used to detect a human chromosome Roche translocation, particularly a translocation occurring between any two chromosomes of chromosomes 13, 14, 15, 21 and 22.
  • the screening criteria for the SNP locus are:
  • the site is located in the 10 Mb region near the centromere;
  • the human genome database or the dbSNP database is included, and the SNP having the smallest allele frequency (MAF) greater than 0.2 in the target population is preferentially selected;
  • the SNP site comprises at least 1 SNP site, preferably at least 10 SNP sites, more preferably at least 50 SNP site SNP sites from each chromosome in which translocation occurs.
  • the SNP site is selected from the following SNP sites or any combination thereof: chr13: 19602195, chr13: 19625370, chr13: 19646283, chr13:19692745, chr13:19713634, chr13:19722801, chr13:19732341, chr13:19784913 , chr13:19843135,chr13:19877547,chr13:19883672,chr13:19896927,chr13:19917509,chr13:20494558,chr13:20709214,chr13:20747500,chr13:20784332,chr13:20825315,chr13:20864572,chr13:20881859,chr13 : 20908501, chr13: 20938952, chr13: 20951425, chr13: 20992333, chr13: 21012562, chr13: 21066
  • the primer composition is selected from the group consisting of a primer set consisting of the following forward primer and reverse primer, or any combination thereof:
  • a primer for amplifying a SNP site of chromosome 13 is selected from the group consisting of a forward primer sequence of SEQ ID NO: 2n-1 and a reverse primer sequence of SEQ ID NO: 2n; wherein n is selected from 1 a natural number of -57;
  • the primer for amplifying the SNP site of chromosome 14 is selected from the following primer pairs: forward primer sequence SEQ ID NO: 2n-1, reverse primer sequence SEQ ID NO: 2n; wherein n is selected from 58 a natural number of -115;
  • the primer for amplifying the SNP site of chromosome 15 is selected from the following primer pairs: forward primer sequence SEQ ID NO: 2n-1, reverse primer sequence SEQ ID NO: 2n; wherein n is selected from 116 a natural number of -189;
  • the primer for amplifying the SNP site of chromosome 21 is selected from the following primer pairs: forward primer sequence SEQ ID NO: 2n-1, reverse primer sequence SEQ ID NO: 2n; wherein n is selected from 190 a natural number of -264;
  • a primer for amplifying a SNP site of chromosome 22 is selected from the group consisting of a forward primer sequence of SEQ ID NO: 2n-1 and a reverse primer sequence of SEQ ID NO: 2n; wherein n is selected from 265 The natural number of -344.
  • the primer composition comprises at least 1 pair of primers for amplifying a SNP site of each chromosome in which translocation occurs, preferably at least 10 pairs of primers, more preferably at least 50 pairs of primers, most preferably including amplification. All of the above primer pairs for the SNP site of each chromosome.
  • the invention also provides the use of the primer composition of the invention in the preparation of a diagnostic reagent or kit for detecting a Roche translocation of a chromosome.
  • the invention can solve the problem of detection of Roche translocation carriers of embryos, fetuses or abortion tissues which cannot be solved by conventional methods.
  • the present invention analyzes the chromosomal translocation of different cells based on the results of cell chromosome copy number analysis and haplotype, and does not need to observe the cells, and can quickly distinguish between normal and translocation embryos.
  • the present invention uses multiple SNPs for analysis, and can be used for pre-implantation diagnosis, fetal diagnosis or abortion tissue diagnosis of different spouses.
  • High-throughput Based on high-throughput sequencing technology, the present invention can analyze chromosome translocations, and by analyzing different sample sequences on each sample, a large number of samples can be analyzed at one time.
  • High sensitivity It can be used for the analysis of 3 ⁇ -5 cells. Therefore, in addition to abortion tissue, cervical-derived trophoblast cells, it is especially suitable for pre-embryo testing in IVF technology.
  • Figure 1 is a flow chart showing a method of detecting a Roche translocation of a chromosome of the present invention.
  • Fig. 2 is a diagram showing an embryo translocation chromosome analysis of the method for detecting a Roche translocation of a chromosome of the present invention.
  • Fig. 3 is a haplotype diagram of the chromosome 13 family in Example 1.
  • Fig. 4 is a haplotype diagram of the chromosome 14 family in Example 1.
  • Fig. 5 is a haplotype diagram of the chromosome 13 family in Example 2.
  • Fig. 6 is a haplotype diagram of the chromosome 14 family in Example 2.
  • a read refers to a sequence fragment obtained by sequencing.
  • single nucleotide polymorphism refers to DNA sequence polymorphisms caused by variations in single nucleotides at the genomic level.
  • a haplotype refers to a group of single nucleotide polymorphisms which are interrelated in a specific region of a chromosome and tend to be inherited to the progeny as a whole, also referred to as a haplotype or a haplotype. .
  • the embryonic genomic DNA is obtained by taking out 3 to 5 peripheral trophoblast cells when the embryo develops to the blastocyst stage, and enriching the genomic DNA in the cells by whole genome amplification method.
  • DNA molecule enrichment in a target region is carried out by a method of multiplex PCR amplification.
  • the instructions provided by the manufacturer please refer to the instructions provided by the manufacturer to enrich the DNA molecules into a relatively large number of fragments of a certain size.
  • the DNA fragment is sized from 125 to 275 bp.
  • 57, 58, 74, 75 and 80 pairs of sequence-specific primers are designed for high frequency mutant SNP sites in the vicinity of the centromere near the centromere of human chromosomes 13, 14, 15, 21 and 22. See Table 1-5 for primer sequences and corresponding SNP site information. These primers are characterized by: (1) unique sequence on the target chromosome; (2) identical annealing temperatures.
  • the SNP site is represented by the chromosome on which it is located and the position on the chromosome (based on the hg19 reference genome sequence), for example, chr13: 19602195 indicates that the SNP is located at chromosome 19602195 of chromosome 13 of the hg19 reference genome sequence. Nucleotide.
  • the sequencing method employed may be a high throughput sequencing method.
  • the length of the DNA fragments is between 125 and 275 bp.
  • the sequencing platform is Ion Torrent PGM, resulting in a DNA sequence molecule having a DNA length distribution between 125 and 275 bp.
  • the sequencing depth may be 300 to 3000 ⁇ , that is, each specific PCR amplification product is sequenced 300 to 3000 times, for example, in a specific embodiment of the present invention, the sequencing depth is 1000, that is, the specific PCR The amplified product was sequenced 1000 times.
  • each sample when the DNA molecule to be tested is derived from a plurality of test samples, each sample can be added with a different barcode for differentiation of the sample during the sequencing process (Micah Hamady, Jeffrey) J Walker, J Kirk Harris et al. Error-correcting barcoded primers for pyrosequencing hundreds of samples in multiplex. Nature Methods, 2008, 5(3)), thereby enabling simultaneous sequencing of multiple samples.
  • the genomic reference sequence can be from a public database.
  • the human genome sequence can be a human genome reference sequence in the NCBI or ucsc database.
  • sequence alignment can be compared by any sequence alignment program, such as the Torrent Mapping Alignment Program (TMAP) and BWA (Burrow-Wheeler-Aligner) available to those skilled in the art, and the reading and reference can be made.
  • TMAP Torrent Mapping Alignment Program
  • BWA Brown-Wheeler-Aligner
  • the raw data generated by the PGM sequencer is removed by the Torrent_Server_4.0_VM software, and the human hg19 reference genome is compared with the Tmap software, and finally the haplotype SNP coverage multiple and genotype are analyzed.
  • the detection method comprises the steps of:
  • DNA extraction and sequencing After extracting cellular DNA according to the MDA genome-wide amplification method (Qiagen kit), the library was constructed according to the Ion AmpliSeq TM Library Kits 2.0 standard library construction procedure. In this process, whole-genome amplification of embryonic MDA is amplified by target region multiplex PCR into DNA molecules concentrated at about 125-275 bp, with the ends used for sequencing, and each sample is labeled with a different barcode. Thus, the data of multiple samples can be distinguished in the data obtained by one sequencing.
  • Qiagen kit MDA genome-wide amplification method
  • the invention is used for pre-transplantation diagnosis of chromosome Roche translocation embryos in a suitable population, and is beneficial for providing genetic counseling and providing clinical decision-making basis.
  • the invention is particularly suitable for detecting normal embryos and chromosome Roche translocation carrier embryos in which the parental side is a carrier of the Roche translocation of the chromosome, and the other is a normal karyotype which cannot be distinguished according to conventional methods.
  • the sample source consisted of 8 embryonic blastocyst stage trophoblast cell whole genome amplification products and parental peripheral blood cell DNA samples.
  • the father was a Roche translocation carrier of chromosomes 13 and 14, the mother karyotype was normal, and the embryo chromosome copy number analysis results were as follows. Table 6.
  • the library was built according to the Ion AmpliSeq TM Library Kits 2.0 standard library building process. After the multiplex PCR reaction was carried out using the primers of Tables 1 and 2, the DNA molecule of the amplified product was ligated with the linker used for sequencing, and the nucleic acid molecule was clustered under certain conditions, and then sequenced on Ion Torrent PGM to obtain a fragment. A DNA fragment sequence having a length distributed in a target region of 125 bp to 275 bp.
  • Torrent_Server_4.0_VM software to remove the linker sequence from the original data obtained by PGM sequencing, compare the human hg19 reference genome with Tmap software, remove the low-quality sequence, count the sequencing depth N of the target site, and remove the N-less than 100 locus.
  • the haplotype of an individual is presumed by the genotype of the parent and the embryo, and the effective site is a site for providing effective SNP information for constructing the haplotype of the pedigree, and generally one of them is a heterozygous site.
  • Parental genotypes are ( ⁇ X1X1 ⁇ , ⁇ X2X2 ⁇ ), ( ⁇ X1X1 ⁇ , ⁇ X1X1 ⁇ ) are invalid sites, ( ⁇ X1X1 ⁇ , ⁇ X1X2 ⁇ ) are valid sites, ( ⁇ X1X2 ⁇ , ⁇ X1X2 ⁇ ) is a verifiable site.
  • the parental and embryonic haplotypes are inferred according to the effective site and the verification site.
  • the haplotypes of the male and female (LH) chromosomes 13 and 14 are Aa, Bb, and the female parent (YT).
  • the haplotypes of chromosomes 13 and 14 are Cc and Dd, respectively.
  • the chromosome 13 haplotype (type A) from LH carrying the chromosome 13 is translocated chromosome haplotype
  • type B is a translocation chromosome haplotype
  • the sample source is 6 embryonic blastocyst stage trophoblast cells whole genome amplification products and parental peripheral blood cell DNA samples, father is chromosome 13 and 14 Roche translocation carriers (JXD), mother karyotype normal (HCC), embryo
  • JXD chromosome 13 and 14 Roche translocation carriers
  • HCC mother karyotype normal
  • Table 8 The results of chromosome copy number analysis are shown in Table 8.
  • the library was built according to the Ion AmpliSeq TM Library Kits 2.0 standard library building process. After the multiplex PCR reaction was carried out using the primers of Tables 1 and 2, the DNA molecule of the amplified product was ligated with the linker used for sequencing, and the nucleic acid molecule was clustered under certain conditions, and then sequenced on Ion Torrent PGM to obtain a fragment. A DNA fragment sequence having a length distributed in a target region of 125 bp to 275 bp.
  • Torrent_Server_4.0_VM software to remove the linker sequence from the original data obtained by PGM sequencing, compare the human hg19 reference genome with Tmap software, remove the low-quality sequence, count the sequencing depth N of the target site, and remove the N-less than 100 locus.
  • the haplotype of an individual is presumed by the genotype of the parent and the embryo, and the effective site is a site for providing effective SNP information for constructing the haplotype of the pedigree, and generally one of them is a heterozygous site.
  • Parental genotypes are ( ⁇ X1X1 ⁇ , ⁇ X2X2 ⁇ ), ( ⁇ X1X1 ⁇ , ⁇ X1X1 ⁇ ) are invalid sites, ( ⁇ X1X1 ⁇ , ⁇ X1X2 ⁇ ) are valid sites, ( ⁇ X1X2 ⁇ , ⁇ X1X2 ⁇ ) is a verifiable site.
  • the parental and embryonic haplotypes are inferred based on the effective site and the verification site.
  • the haplotypes of the female and female (HCC) chromosomes 13 and 14 are Cc, Dd, and the male parent (JXD).
  • the haplotypes of chromosomes 13 and 14 are Aa and Bb, respectively.
  • the pedigrees were established according to 115 SNP sites in the target region (57 and 58 for chromosomes 13 and 14, respectively) (results shown in Fig. 1), and the haplotype analysis according to the family is shown in Table 9.
  • the chromosome 14 haplotype (type b) from JXD carried by embryo No. 2 is a normal chromosomal haplotype, ie No. 14 Chromosome B is a translocation chromosomal haplotype.
  • chromosome 13 is a translocation chromosomal haplotype by other embryos carrying B or b type. Therefore, it can be judged that the embryos No. 1 and No. 4 carry the chromosome 14 type B and the chromosome 13 type A, which are translocation carrier embryos; the embryos of No. 3 and No. 6 carry the chromosome 14 b type and the chromosome 13 a type, which are normal. Embryo; No. 5 embryo copy number analysis did not detect the results, this test did not get any valid sequence.
  • the invention is used for analyzing the chromosomal translocation of the embryo in the applicable population, is beneficial to providing genetic counseling and providing clinical decision-making basis, and pre-implantation diagnosis can effectively prevent the birth of the child.
  • the applicable population of the present invention may be a conventional karyotype analysis chromosome Roche translocation carrier.
  • the present invention is not limited to the above-described preferred embodiments, and any other form of product can be derived by anyone of the present invention, but without any change in its shape or structure, it is the same as or equivalent to the present application. Approximate technical solutions are all within the scope of the present invention. .

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Abstract

本发明提供了一种检测染色体罗氏易位的方法，以及该方法中使用的SNP位点及引物组合物。

Description

一种检测染色体罗氏易位的方法 技术领域
本发明涉及分子诊断学领域，具体涉及一种检测染色体罗氏易位的方法，其能够针对父母是罗氏易位携带者的家系，检测正常及易位携带者胚胎和胎儿。
背景技术
罗氏易位(Robertsonian translocation，ROB)，以第一次发现该种易位方式的美国生物学家William Rees Brebner Robertson命名，是主要发生于5条近端着丝粒染色体(13、14、15、21和22号染色体)的一种染色体易位(Robertson WRB.Chromosome studies.I.Taxonomic relationships shown in the chromosomes of Tettigidae and Acrididae.V-shaped chromosomes and their significance in Acrididae,Locustidae and Gryllidae:chromosome and variation.J Morph 1916,27:179-331)。当两个近端着丝粒染色体在着丝粒部位或在着丝粒附近部位发生断裂后，二者的长臂在着丝粒处接合在一起，形成一条由长臂构成的衍生染色体，也称丝端融合，是染色体平衡易位的一种。两个短臂则构成一个小染色体，小染色体往往在第二次分裂时丢失。罗氏易位是一种常见的染色体结构异常，在新生儿中的发生率为1.23/1000(Nielsen J,Wohlert M.Chromosome abnormalities found among 34910newborn children:results from 13-year incidence study in Arhus,Denmark.Hum Genet.1991,87:81–83)。由于染色体短臂主要包括随机重复DNA和核糖体RNA基因，而这种基因在五条染色体中都存在，所以罗氏易位虽然有染色体短臂的丢失，却不会造成致病表型。虽然表型正常，然而携带者生殖细胞在第一次减数分裂过程中，易位染色体和相应两个正常染色体配对会形成三价染色体，这种结构会导致交替、邻式和不常见的3:0三种分裂方式，只有交替可以产生正常或者平衡的配子，其他两种方式产生非平衡的配子，这种占大部分的非平衡配子也是易位携带者出现妊娠困难或者妊娠过程中反复流产的原因，甚至导致染色体拷贝数异常的13-Patau综合征等先天缺陷患儿的出生。目前越来越多的罗氏易位携带者寻求试管婴儿技术进行胚胎植入前诊断。
试管婴儿技术是将卵子与精子取出后置于特定的培养液内培养、受精，受精卵在恒温孵箱中发育为胚胎后移植回母体子宫，最终发育成胎儿。挑选健康胚胎移植是成功预防的关键。胚胎植入前遗传学筛查是指胚胎植入着床之前进行染色体数目和结构异常的检测，挑选染色体正常的胚胎植入子宫，以期获得正常的后代。目前罗氏易位的胚胎移植前诊断的方法包括SNP芯片和荧光原位杂交技术(fluorescence in situ hybridization，FISH)。SNP芯片是目前最常用的检测技术，但是这项技术只能检测胚胎染色体拷贝数的变化，不能区分罗氏易位中正常与携带易位的胚胎。FISH利用荧光标记的特异核酸探针与细胞内相应的靶DNA分子杂交，通过在荧光显微镜下观察荧光信号，来确定结合了荧光探针的DNA分子在染色体中的定位。但FISH作为检查单个细胞核的方法是存在一定缺陷的，有7％的假阴性(Colls P,Escudero T,Cekleniak N,Sadowy S,Cohen J,MunnéS.I ncreased efficiency of preimplantation genetic diagnosis for infertility using"no result rescue".Fertil Steril.2007,88(1):53-61)。
自然流产是妇产科的一种常见病，其发生率约占全部妊娠的10％～15％，其发生受很多因素的影响，如胚胎因素、胎盘因素、母体因素及环境因素等，其中胚胎染色体异常是导致流产发生的主要原因。对流产组织进行染色体检测分析胚胎停育、流产的原因，不仅为临床咨询提供理论依据，同时为孕妇再次妊娠提供指导。目前，利用FISH技术对稽留流产绒毛及胎儿不同部位组织进行最常见的染色体异常检测。FISH技术作为一种靶向性检测技术，商品化的探针有限，其在很大程度上是受到探针的类型、数量及成本较高等限制，在同一时间内也无法对大量的异常基因或染色体进行检测。此外由于FISH技术的局限性，FISH不能检测染色体结构异常，可能会导致一部分染色体结构异常的患者被漏诊(李阳洋，章勤.自然流产绒毛染色体核型分析的研究现状.实用妇产科杂志，2012，28：431-433)。
综上所述，急需开发一种能够检测试管婴儿技术中胚胎和流产组织罗氏易位的新方法。
发明内容
本发明设计了一种基于高通量测序技术检测染色体罗氏易位的方法，以筛查完全正常及易位携带者的胚胎、胎儿或流产组织。
本发明的第一方面提供了一种检测染色体罗氏易位的方法，包括以下步骤：
(1)获取亲代双方和子代的DNA样本；
(2)从染色体近端着丝粒附近区域筛选高频突变SNP位点作为标记；
(3)检测子代DNA样本的染色体拷贝数；
(4)针对步骤(2)筛选的SNP位点，确定亲代双方和子代目标位点的基因型；
(5)基于亲代双方和子代的基因型及家系关系，分析亲代双方和子代的单体型；
(6)根据子代的染色体拷贝数检测结果、亲代双方携带染色体罗氏易位的核型、
以及亲代双方和子代的单体型，分析子代的染色体罗氏易位情况。
优选地，所述方法用于检测人类染色体，尤其是第13、14、15、21和22号染色体中任意两条染色体之间发生的易位。
在一个特定的实施方案中，亲代DNA样本是从外周血样本中提取的父母基因组DNA；子代DNA样本选自胚胎或胎儿的滋养层细胞全基因组扩增产物，或流产组织提取的基因组DNA，优选体外培养的胚胎的囊胚期滋养层细胞全基因组扩增产物。
上述步骤(2)中SNP位点的筛选标准为：
a.位点位于着丝粒附近10Mb区域内；
b.千人基因组数据库或者dbSNP数据库中收录，优先选择目标人群中最小等位基因频率(MAF)大于0.2的SNP；
c.SNP位点附近序列在人类基因组中不具有同源性。
在一个特定的实施方案中，所述SNP位点包括来自发生易位的每条染色体的至少1个SNP位点，优选至少10个SNP位点，更优选至少50个SNP位点SNP位点。
优选地，所述SNP位点选自以下SNP位点或其任意组合：chr13:19602195，chr13:19625370，chr13:19646283，chr13:19692745，chr13:19713634，chr13:19722801，chr13:19732341，chr13:19784913，chr13:19843135，chr13:19877547，chr13:19883672，chr13:19896927，chr13:19917509，chr13:20494558，chr13:20709214，chr13:20747500，chr13:20784332，chr13:20825315，chr13:20864572，chr13:20881859，chr13:20908501，chr13:20938952，chr13:20951425，chr13:20992333，chr13:21012562，chr13:21066214，chr13:21092894，chr13:21112935，chr13:21527671， chr13:21553971，chr13:21575458，chr13:21607760，chr13:21636136，chr13:21655335，chr13:21683681，chr13:21801501，chr13:22197910，chr13:22249975，chr13:22270788，chr13:22340074，chr13:22396664，chr13:22417688，chr13:22473749，chr13:22504691，chr13:22515844，chr13:22523279，chr13:22538311，chr13:22561684，chr13:22640820，chr13:22751654，chr13:22768476，chr13:22780737，chr13:22799808，chr13:22819627，chr13:22835073，chr13:22892864，chr13:22980146，chr14:19472367，chr14:20326905，chr14:20349694，chr14:20383719，chr14:20420387，chr14:20517369，chr14:20527044，chr14:20565487，chr14:20643775，chr14:20670979，chr14:20731036，chr14:20820537，chr14:20905141，chr14:20918598，chr14:21008574，chr14:21048774，chr14:21091127，chr14:21156472，chr14:21195967，chr14:21237137，chr14:21263166，chr14:21313819，chr14:21325985，chr14:21388266，chr14:21452591，chr14:21519915，chr14:21580671，chr14:21625262，chr14:21789092，chr14:21981923，chr14:22053817，chr14:22090208，chr14:22138437，chr14:22210067，chr14:22244363，chr14:22263821，chr14:22312275，chr14:22324460，chr14:22351977，chr14:22361282，chr14:22375208，chr14:22393124，chr14:22428984，chr14:22452496，chr14:22489611，chr14:22518909，chr14:22526966，chr14:22563283，chr14:22611651，chr14:22626948，chr14:22657897，chr14:22702039，chr14:22731529，chr14:22734948，chr14:22741160，chr14:22772663，chr14:22861948，chr14:22972955，chr15:20022190，chr15:20049922，chr15:20189367，chr15:20191854，chr15:20192138，chr15:20454253，chr15:20470948，chr15:20564500，chr15:20569241，chr15:20586537，chr15:20586966，chr15:20590656，chr15:20616721，chr15:20626721，chr15:20646773，chr15:20848359，chr15:20849625，chr15:20852181，chr15:20862156，chr15:20863958，chr15:21905797，chr15:21928397，chr15:21938184，chr15:21940649，chr15:22013203，chr15:22299435，chr15:22302182，chr15:22376163，chr15:22427155，chr15:22441959，chr15:22476195，chr15:22520388，chr15:22539952，chr15:22780065， chr15:22798800，chr15:22833272，chr15:22854479，chr15:22869870，chr15:22893990，chr15:22905599，chr15:22916637，chr15:22923869，chr15:22940961，chr15:22971662，chr15:23000363，chr15:23010627，chr15:23020535，chr15:23044004，chr15:23053813，chr15:23064687，chr15:23721906，chr15:23730971，chr15:23746510，chr15:23767135，chr15:23781336，chr15:23793989，chr15:23816231，chr15:23893430，chr15:23929209，chr15:24006330，chr15:24055535，chr15:24129894，chr15:24166516，chr15:24196094，chr15:24236177，chr15:24278723，chr15:24319610，chr15:24350583，chr15:24471640，chr15:24824114，chr15:24870875，chr15:24913763，chr15:24943341，chr15:24974072，chr21:14640496，chr21:14643423，chr21:14695894，chr21:14756515，chr21:14818516，chr21:14890028，chr21:15012159，chr21:15026905，chr21:15170049，chr21:15280687，chr21:15300595，chr21:15445542，chr21:15479041，chr21:15501432，chr21:15548748，chr21:15566056，chr21:15588845，chr21:15625530，chr21:15654618，chr21:15681452，chr21:15720558，chr21:15763305，chr21:15842397，chr21:15884516，chr21:15916232，chr21:15942816，chr21:15984234，chr21:16027704，chr21:16063848，chr21:16122140，chr21:16160041，chr21:16193235，chr21:16238898，chr21:16315932，chr21:16353639，chr21:16384555，chr21:16448591，chr21:16578538，chr21:16645724，chr21:16717265，chr21:16833315，chr21:16881542，chr21:17000938，chr21:17054831，chr21:17140290，chr21:17203891，chr21:17295288，chr21:17340912，chr21:17536247，chr21:17640426，chr21:17673255，chr21:17713264，chr21:17748703，chr21:17781159，chr21:17823782，chr21:18003355，chr21:18046650，chr21:18114449，chr21:18151760，chr21:18182817，chr21:18250262，chr21:18337678，chr21:18385770，chr21:18420873，chr21:18457287，chr21:18586049，chr21:18650837，chr21:18740886，chr21:18858018，chr21:18936951，chr21:19002939，chr21:19064396，chr21:19139888，chr21:19254923，chr21:19316600，chr22:16415691，chr22:16554800，chr22:16848067，chr22:16850056，chr22:16850858， chr22:16851557，chr22:16852464，chr22:16852792，chr22:16853453，chr22:16854397，chr22:16855647，chr22:16856500，chr22:16857432，chr22:16858287，chr22:16860547，chr22:16861742，chr22:16867995，chr22:17031697，chr22:17271213，chr22:17293257，chr22:17409957，chr22:17442318，chr22:17469090，chr22:17525677，chr22:17563259，chr22:17587785，chr22:17619511，chr22:17656792，chr22:17682387，chr22:17715164，chr22:17739757，chr22:17761489，chr22:17785311，chr22:17805528，chr22:17841251，chr22:17883346，chr22:17916190，chr22:17939998，chr22:17981144，chr22:18012297，chr22:18054406，chr22:18212058，chr22:18276198，chr22:18354015，chr22:18395978，chr22:18466951，chr22:18531582，chr22:18581715，chr22:18628321，chr22:18894617，chr22:18966077，chr22:19004846，chr22:19025459，chr22:19098323，chr22:19142652，chr22:19236590，chr22:19362714，chr22:19431588，chr22:19536367，chr22:19643965，chr22:19716983，chr22:19759532，chr22:19808057，chr22:19859087，chr22:19909387，chr22:19972494，chr22:20016622，chr22:20051573，chr22:20081918，chr22:20135421，chr22:20175034，chr22:20216874，chr22:20278637，chr22:20745699，chr22:20787955，chr22:20855585，chr22:20881195，chr22:20924097，chr22:20983921，chr22:21028224。最优选地，所述SNP位点包括来自发生易位的每条染色体的上述全部SNP位点。
在一个特定的实施方案中，上述步骤(3)和(4)中检测染色体拷贝数的方法独立地选自测序方法和芯片方法。其中，所述测序方法为高通量测序方法，优选Ion Torrent PGM或Illumina Miseq；所述的芯片方法为人全基因组SNP芯片，优选Illumina全基因组SNP芯片。
优选地，在本发明的检测方法中，亲代双方一方为正常核型，另一方为罗氏易位携带者，子代中包括罗氏易位染色体三体或者单体的个体。
特别地，上述步骤(6)中的判断标准如下：
a.如果某一子代为染色体m三体，则其染色体n中来源于亲代罗氏易位携带者的单体型B为易位染色体单体型，相应地，来源于亲代罗氏易位携带者染色体n的另一种单体型b为正常染色体单体型；进一步地，通过观察其他子代的单体型，与 染色体n单体型B连锁的来源于亲代罗氏易位携带者的染色体m单体型A也是易位染色体单体型，相应地，来源于亲代罗氏易位携带者染色体m的另一种单体型a为正常染色体单体型；由此，可以判断携带染色体m单体型A和染色体n单体型B的子代为易位携带者，携带染色体m单体型a和染色体n单体型b的子代为正常核型；
b.如果某一子代为染色体m单体，则其染色体n中来源于亲代罗氏易位携带者的单体型B为正常染色体单体型，相应地，来源于亲代罗氏易位携带者染色体n的另一种单体型b为易位染色体单体型；进一步地，通过观察其他子代的单体型，与染色体n单体型b连锁的来源于亲代罗氏易位携带者的染色体m单体型a也是易位染色体单体型，相应地，来源于亲代罗氏易位携带者染色体m的另一种单体型A为正常染色体单体型；由此，可以判断携带染色体m单体型A和染色体n单体型B的子代为正常核型，携带染色体m单体型a和染色体n单体型b的子代为易位携带者；
其中，染色体m和染色体n为亲代罗氏易位携带者基因组中发生罗氏易位的两条染色体，A和a为来源于亲代罗氏易位携带者染色体m的单体型，B和b为来源于亲代罗氏易位携带者染色体n的单体型。
以13、14号染色体易位为例：
  携带者 +13胚胎 +14胚胎 -13胚胎 -14胚胎 易位胚胎 正常胚胎
Chr13 Aa Aa A - a A a
Chr14 Bb B Bb b - B b
根据上表所示，若13号染色体为三倍体或者单倍体，可以推测罗氏易位携带者14号染色体中B型为易位染色体单体型，b型为正常染色体单体型，通过其他携带B型或者b型的胚胎可以进而推测13号染色体中A型为易位染色体单体型，a型为正常染色体单体型；
若14号染色体为三倍体或者单倍体，可以推测罗氏易位携带者13号染色体中A型为易位染色体单体型，a型为正常染色体单体型，通过其他携带A型或者a型的胚胎可以进而推测携带14号染色体B为易位染色体单体型，b为正常染色体单体型。
所以携带13号染色体a型及14号染色体b型为正常胚胎，而携带13号染色体A型及14号染色体B型为易位携带者胚胎。
本发明的上述方法尤其适用于试管婴儿的胚胎植入前诊断。
本发明的第二方面提供了一种用于检测染色体罗氏易位的引物组合物，所述引物组合物能够扩增近端着丝粒染色体的着丝粒附近区域高频突变SNP位点的序列。优选地，所述引物组合物用于检测人类染色体罗氏易位，尤其是第13、14、15、21和22号染色体中任意两条染色体之间发生的易位。
所述SNP位点的筛选标准为：
a.位点位于着丝粒附近10Mb区域内；
b.千人基因组数据库或者dbSNP数据库中收录，优先选择目标人群中最小等位基因频率(MAF)大于0.2的SNP；
c.SNP位点附近序列在人类基因组中不具有同源性。
在一个特定的实施方案中，所述SNP位点包括来自发生易位的每条染色体的至少1个SNP位点，优选至少10个SNP位点，更优选至少50个SNP位点SNP位点。
优选地，所述SNP位点选自以下SNP位点或其任意组合：chr13:19602195，chr13:19625370，chr13:19646283，chr13:19692745，chr13:19713634，chr13:19722801，chr13:19732341，chr13:19784913，chr13:19843135，chr13:19877547，chr13:19883672，chr13:19896927，chr13:19917509，chr13:20494558，chr13:20709214，chr13:20747500，chr13:20784332，chr13:20825315，chr13:20864572，chr13:20881859，chr13:20908501，chr13:20938952，chr13:20951425，chr13:20992333，chr13:21012562，chr13:21066214，chr13:21092894，chr13:21112935，chr13:21527671，chr13:21553971，chr13:21575458，chr13:21607760，chr13:21636136，chr13:21655335，chr13:21683681，chr13:21801501，chr13:22197910，chr13:22249975，chr13:22270788，chr13:22340074，chr13:22396664，chr13:22417688，chr13:22473749，chr13:22504691，chr13:22515844，chr13:22523279，chr13:22538311，chr13:22561684，chr13:22640820，chr13:22751654，chr13:22768476，chr13:22780737，chr13:22799808，chr13:22819627，chr13:22835073，chr13:22892864，chr13:22980146，chr14:19472367，chr14:20326905，chr14:20349694，chr14:20383719，chr14:20420387，chr14:20517369，chr14:20527044，chr14:20565487，chr14:20643775，chr14:20670979，chr14:20731036，chr14:20820537， chr14:20905141，chr14:20918598，chr14:21008574，chr14:21048774，chr14:21091127，chr14:21156472，chr14:21195967，chr14:21237137，chr14:21263166，chr14:21313819，chr14:21325985，chr14:21388266，chr14:21452591，chr14:21519915，chr14:21580671，chr14:21625262，chr14:21789092，chr14:21981923，chr14:22053817，chr14:22090208，chr14:22138437，chr14:22210067，chr14:22244363，chr14:22263821，chr14:22312275，chr14:22324460，chr14:22351977，chr14:22361282，chr14:22375208，chr14:22393124，chr14:22428984，chr14:22452496，chr14:22489611，chr14:22518909，chr14:22526966，chr14:22563283，chr14:22611651，chr14:22626948，chr14:22657897，chr14:22702039，chr14:22731529，chr14:22734948，chr14:22741160，chr14:22772663，chr14:22861948，chr14:22972955，chr15:20022190，chr15:20049922，chr15:20189367，chr15:20191854，chr15:20192138，chr15:20454253，chr15:20470948，chr15:20564500，chr15:20569241，chr15:20586537，chr15:20586966，chr15:20590656，chr15:20616721，chr15:20626721，chr15:20646773，chr15:20848359，chr15:20849625，chr15:20852181，chr15:20862156，chr15:20863958，chr15:21905797，chr15:21928397，chr15:21938184，chr15:21940649，chr15:22013203，chr15:22299435，chr15:22302182，chr15:22376163，chr15:22427155，chr15:22441959，chr15:22476195，chr15:22520388，chr15:22539952，chr15:22780065，chr15:22798800，chr15:22833272，chr15:22854479，chr15:22869870，chr15:22893990，chr15:22905599，chr15:22916637，chr15:22923869，chr15:22940961，chr15:22971662，chr15:23000363，chr15:23010627，chr15:23020535，chr15:23044004，chr15:23053813，chr15:23064687，chr15:23721906，chr15:23730971，chr15:23746510，chr15:23767135，chr15:23781336，chr15:23793989，chr15:23816231，chr15:23893430，chr15:23929209，chr15:24006330，chr15:24055535，chr15:24129894，chr15:24166516，chr15:24196094，chr15:24236177，chr15:24278723，chr15:24319610，chr15:24350583，chr15:24471640，chr15:24824114，chr15:24870875，chr15:24913763，chr15:24943341，chr15:24974072， chr21:14640496，chr21:14643423，chr21:14695894，chr21:14756515，chr21:14818516，chr21:14890028，chr21:15012159，chr21:15026905，chr21:15170049，chr21:15280687，chr21:15300595，chr21:15445542，chr21:15479041，chr21:15501432，chr21:15548748，chr21:15566056，chr21:15588845，chr21:15625530，chr21:15654618，chr21:15681452，chr21:15720558，chr21:15763305，chr21:15842397，chr21:15884516，chr21:15916232，chr21:15942816，chr21:15984234，chr21:16027704，chr21:16063848，chr21:16122140，chr21:16160041，chr21:16193235，chr21:16238898，chr21:16315932，chr21:16353639，chr21:16384555，chr21:16448591，chr21:16578538，chr21:16645724，chr21:16717265，chr21:16833315，chr21:16881542，chr21:17000938，chr21:17054831，chr21:17140290，chr21:17203891，chr21:17295288，chr21:17340912，chr21:17536247，chr21:17640426，chr21:17673255，chr21:17713264，chr21:17748703，chr21:17781159，chr21:17823782，chr21:18003355，chr21:18046650，chr21:18114449，chr21:18151760，chr21:18182817，chr21:18250262，chr21:18337678，chr21:18385770，chr21:18420873，chr21:18457287，chr21:18586049，chr21:18650837，chr21:18740886，chr21:18858018，chr21:18936951，chr21:19002939，chr21:19064396，chr21:19139888，chr21:19254923，chr21:19316600，chr22:16415691，chr22:16554800，chr22:16848067，chr22:16850056，chr22:16850858，chr22:16851557，chr22:16852464，chr22:16852792，chr22:16853453，chr22:16854397，chr22:16855647，chr22:16856500，chr22:16857432，chr22:16858287，chr22:16860547，chr22:16861742，chr22:16867995，chr22:17031697，chr22:17271213，chr22:17293257，chr22:17409957，chr22:17442318，chr22:17469090，chr22:17525677，chr22:17563259，chr22:17587785，chr22:17619511，chr22:17656792，chr22:17682387，chr22:17715164，chr22:17739757，chr22:17761489，chr22:17785311，chr22:17805528，chr22:17841251，chr22:17883346，chr22:17916190，chr22:17939998，chr22:17981144，chr22:18012297，chr22:18054406，chr22:18212058，chr22:18276198，chr22:18354015，chr22:18395978， chr22:18466951，chr22:18531582，chr22:18581715，chr22:18628321，chr22:18894617，chr22:18966077，chr22:19004846，chr22:19025459，chr22:19098323，chr22:19142652，chr22:19236590，chr22:19362714，chr22:19431588，chr22:19536367，chr22:19643965，chr22:19716983，chr22:19759532，chr22:19808057，chr22:19859087，chr22:19909387，chr22:19972494，chr22:20016622，chr22:20051573，chr22:20081918，chr22:20135421，chr22:20175034，chr22:20216874，chr22:20278637，chr22:20745699，chr22:20787955，chr22:20855585，chr22:20881195，chr22:20924097，chr22:20983921，chr22:21028224。最优选地，所述SNP位点包括来自发生易位的每条染色体的上述全部SNP位点。
在一个特别优选的实施方案中，所述引物组合物选自以下正向引物和反向引物组成的引物对，或其任意组合：
(1)扩增第13号染色体的SNP位点的引物选自以下引物对：正向引物序列SEQ ID NO:2n-1，反向引物序列SEQ ID NO:2n；其中，n为选自1-57的自然数；
(2)扩增第14号染色体的SNP位点的引物选自以下引物对：正向引物序列SEQ ID NO:2n-1，反向引物序列SEQ ID NO:2n；其中，n为选自58-115的自然数；
(3)扩增第15号染色体的SNP位点的引物选自以下引物对：正向引物序列SEQ ID NO:2n-1，反向引物序列SEQ ID NO:2n；其中，n为选自116-189的自然数；
(4)扩增第21号染色体的SNP位点的引物选自以下引物对：正向引物序列SEQ ID NO:2n-1，反向引物序列SEQ ID NO:2n；其中，n为选自190-264的自然数；
(5)扩增第22号染色体的SNP位点的引物选自以下引物对：正向引物序列SEQ ID NO:2n-1，反向引物序列SEQ ID NO:2n；其中，n为选自265-344的自然数。
优选地，所述引物组合物包括用于扩增发生易位的每条染色体的SNP位点的至少1对引物，优选至少10对引物，更优选至少50对引物，最优选包括扩增发生易位的每条染色体的SNP位点的上述全部引物对。
本发明还提供了本发明的引物组合物在制备检测染色体罗氏易位的诊断试剂或试剂盒中的应用。
本发明的优越性主要有以下几点：
实用性：本发明可以解决目前常规方法无法解决的胚胎、胎儿或流产组织罗氏易位携带者检测问题。
简便性：本发明基于细胞染色体拷贝数分析结果和单体型分析不同细胞的染色体易位情况，不需要对细胞进行观察，可以快速地区分正常和易位携带胚胎。
通用性：本发明采用了多SNP进行分析，可用于不同配偶的胚胎移植前诊断、胎儿诊断或流产组织诊断。
高通量：基于高通量测序技术，本发明可以分析染色体的易位，通过在每个样品上加上不同的标签序列，可以一次地对大量样品进行分析。
成本低：随着测序技术的不断发展和测序成本的不断降低，本发明对染色体易位检测的成本也在不断下降。
高灵敏度：可用于3～-5个细胞的分析。因此除了流产组织、宫颈来源的滋养层细胞外，尤其适合于试管婴儿技术中胚胎移植前的检测。
特异性：选取千人基因组计划数据中染色体近着丝粒区域10Mb范围内CHB(北方汉人)和CHS(南方汉人)最小等位基因频率均大于0.2的高频突变位点，去除多聚核苷酸(polyN)和位点上下游50bp序列中GC含量>70％的多态性位点，并选择唯一比对到人类基因组的SNP突变位点作为目标区域。登陆https://www.ampliseq.com/网站提交目标位点和区域设计引物。这些引物具有很高的特异性。
准确性：根据易位染色体拷贝数的变化，及携带对应染色体的样本，可以推测易位的染色体中，各自的衍生染色体单体型，进而作相互验证，准确性更高。
附图说明
图1是本发明的检测染色体罗氏易位的方法的流程图。
图2是本发明的检测染色体罗氏易位的方法的胚胎易位染色体分析图。
图3是实施例1中的13号染色体家系单体型图。
图4是实施例1中的14号染色体家系单体型图。
图5是实施例2中的13号染色体家系单体型图。
图6是实施例2中的14号染色体家系单体型图。
具体实施方式
在本发明中，读段(reads)是指测序获得的序列片段。
在本发明中，单核苷酸多态性(single nucleotide polymorphism，SNP)是指 在基因组水平上由单个核苷酸的变异所引起的DNA序列多态性。
在本发明中，单体型(Haplotype)是指位于一条染色体特定区域的一组相互关联，并倾向于以整体遗传给后代的单核苷酸多态的组合，又称单倍体型或单元型。
本发明中，胚胎基因组DNA的获取是当胚胎发育至囊胚期时取出外围滋养层细胞3～5个，运用全基因组扩增方法对细胞中基因组DNA进行富集。
在本发明中，目标区域DNA分子富集采用的是多重PCR扩增的方法。具体原理和方法请参见生厂商提供的说明书，将DNA分子富集为比较集中的一定大小的片段。在本发明的一个特定实施方案中，DNA片段大小在125～275bp的大小。
在本发明中，针对人类第13、14、15、21和22号染色体近端着丝粒附近区域高频突变SNP位点，分别设计57、58、74、75和80对序列特异性引物(引物序列及相应的SNP位点信息参见表1-5)。这些引物的特点为：(1)在目标染色体上序列唯一；(2)具有相同的退火温度。
本发明中，SNP位点由其所在染色体以及在该染色体上的位置(以hg19参考基因组序列为标准)来表示，例如，chr13:19602195表示该SNP位于hg19参考基因组序列第13号染色体第19602195位核苷酸。
表1第13号染色体近端着丝粒附近区域高频突变SNP位点及检测引物
Figure PCTCN2016070696-appb-000001
Figure PCTCN2016070696-appb-000002
Figure PCTCN2016070696-appb-000003
Figure PCTCN2016070696-appb-000004
表2第14号染色体近端着丝粒附近区域高频突变SNP位点及检测引物
Figure PCTCN2016070696-appb-000005
Figure PCTCN2016070696-appb-000006
Figure PCTCN2016070696-appb-000007
Figure PCTCN2016070696-appb-000008
表3第15号染色体近端着丝粒附近区域高频突变SNP位点及检测引物
Figure PCTCN2016070696-appb-000009
Figure PCTCN2016070696-appb-000010
Figure PCTCN2016070696-appb-000011
Figure PCTCN2016070696-appb-000012
表4第21号染色体近端着丝粒附近区域高频突变SNP位点及检测引物
Figure PCTCN2016070696-appb-000013
Figure PCTCN2016070696-appb-000014
Figure PCTCN2016070696-appb-000015
Figure PCTCN2016070696-appb-000016
Figure PCTCN2016070696-appb-000017
表5第22号染色体近端着丝粒附近区域高频突变SNP位点及检测引物
Figure PCTCN2016070696-appb-000018
Figure PCTCN2016070696-appb-000019
Figure PCTCN2016070696-appb-000020
Figure PCTCN2016070696-appb-000021
Figure PCTCN2016070696-appb-000022
在本发明中，所采用的测序方法可以为高通量测序方法。DNA片段长度分布在125～275bp之间。在本发明的一个特定实施方案中，测序平台为Ion Torrent PGM，得到DNA长度分布在125～275bp之间的DNA序列分子。
在本发明中，测序深度可以是300～3000×，即每条特异PCR扩增产物被测序300～3000次，例如在本发明的一个特定实施方案中，测序深度为1000，即该条特异PCR扩增产物被测序1000次。
本发明中，当待测的DNA分子来自多个受试样品时，每个样品可以被加上不同的标签序列(barcode)，以用于在测序过程中进行样品的区分(Micah Hamady,Jeffrey J Walker,J Kirk Harris et al.Error-correcting barcoded primers for pyrosequencing hundreds of samples in multiplex.Nature Methods,2008,5(3))，从而实现同时对多个样品进行测序。
本发明中，基因组参考序列可以来自公共数据库。例如，人类基因组序列可以是NCBI或者ucsc数据库中的人类基因组参考序列。
本发明中，序列比对可以通过任何一种序列比对程序，例如本领域技术人员可获得的Torrent Mapping Alignment Program(TMAP)和BWA(Burrow-Wheeler-Aligner)进行比对，将读段与参考基因组序列比对，得到读段在参考基因组上的位置。
本发明中，利用Torrent_Server_4.0_VM软件对PGM测序仪产生的原始数据去除接头序列，用Tmap软件比对到人类hg19参考基因组，最后分析单体型SNP覆盖倍数和基因型。
在本发明的一个特定实施方案中，检测方法包括以下步骤：
DNA提取及测序：按照MDA全基因组扩增法(Qiagen试剂盒)提取细胞 DNA后，按照Ion AmpliSeqTMLibrary Kits 2.0标准建库流程进行建库。在这个过程中，胚胎MDA全基因组扩增通过目标区域多重PCR扩增为集中在125～275bp左右的DNA分子，两端加上测序所用接头，每个样品被加上不同的标签序列(barcode)，从而在一次测序得到的数据中可以使多个样品的数据区分开。
比对及统计：利用Torrent_Server_4.0_VM软件对PGM测序仪产生的原始数据去除接头序列，用Tmap软件比对到人类hg19参考基因组，最后分析单体型SNP覆盖倍数和基因型。
本发明用于对适用人群进行染色体罗氏易位胚胎移植前诊断，有利于提供遗传咨询和提供临床决策依据。本发明尤其适用于检测亲代双方一方为染色体罗氏易位携带者，另一方为正常核型，按照常规方法无法区分的正常胚胎和染色体罗氏易位携带者胚胎。
下面将对本发明实施例中的方案进行清楚完整的描述，但本发明并不受其限制，所描述的实施例仅仅是本发明的一部分实施例，基于本发明的实施例，本领域技术人员所获得的所有其他实施例，都属于本发明的保护范围；同样地，实施例的附图仅仅是本发明一部分实施例的附图，本领域技术人员根据这些附图所获得的其他附图，也都属于本发明的保护范围。
下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。
实施例1胚胎染色体罗氏易位的检测
一、材料
样本来源为8例胚胎囊胚期滋养层细胞全基因组扩增产物及父母外周血细胞DNA样本，父亲为13、14号染色体罗氏易位携带者，母亲染色体核型正常，胚胎染色体拷贝数分析结果如表6。
表6父母及胚胎染色体核型信息
Figure PCTCN2016070696-appb-000023
Figure PCTCN2016070696-appb-000024
二、方法
1、建库测序
按照Ion AmpliSeqTM Library Kits 2.0标准建库流程进行建库。使用表1和表2的引物进行多重PCR反应后，扩增产物的DNA分子两端加上测序所用的接头，在一定条件下使核酸分子成簇生长，然后在Ion Torrent PGM上测序，得到片段长度分布在125bp～275bp目标区域的DNA片段序列。
本实施例中，对于获自上述8例胚胎细胞的DNA样品及父母双方的全血DNA样本按照Ion Torrent官方公布的测序说明书进行操作。
2、数据分析
利用Torrent_Server_4.0_VM软件对PGM测序得到的原始数据去除接头序列，用Tmap软件比对到人类hg19参考基因组，去除低质量序列，统计目标位点的测序深度N，去除N小于100的位点，分析有效位点的所有序列方向和各碱基比例，将正反向序列数大于20％并且碱基比例大于10％的碱基类型定义为该位点的一种基因型(X，X＝{A,T,C,G})。
利用父母和胚胎的基因型推测个体的单体型，有效位点即对于构建家系单体型提供有效SNP信息的位点，一般为其中一方为杂合的位点。父母双方基因型为({X1X1}、{X2X2})、({X1X1}、{X1X1})的为无效位点，({X1X1}、{X1X2})为有效位点，({X1X2}、{X1X2})为可验证位点，根据有效位点和验证位点推断父母及各胚胎单体型，父本(LH)13、14号染色体单体型分别为Aa、Bb，母本(YT)13、14号染色体单体型分别为Cc、Dd。
三、结果分析
根据目标区域115个(13、14号染色体分别为57和58个)SNP位点建立家系单体型(结果如图3和图4)，根据家系单体型分析如表7所示。
表7家系单体型表
Figure PCTCN2016070696-appb-000025
Figure PCTCN2016070696-appb-000026
根据LH为易位携带者及3号胚胎为14号染色体重复的信息，可以推断3号胚胎带有的来自LH的13号染色体单体型(A型)为易位染色体单体型，进一步地，通过其他携带A型或a型的胚胎可以推测14号染色体中B型为易位染色体单体型。由此，可以判断1、4、5号胚胎携带13号染色体a型及14号染色体b型，为正常胚胎；2、6、7、8号胚胎携带13号染色体A型及14号染色体B型，为易位携带者胚胎。
实施例2胚胎染色体罗氏易位的检测
一、材料
样本来源为6例胚胎囊胚期滋养层细胞全基因组扩增产物及父母外周血细胞DNA样本，父亲为13、14号染色体罗氏易位携带者(JXD)，母亲染色体核型正常(HCC)，胚胎染色体拷贝数分析结果如表8。
表8父母及胚胎染色体核型信息
Figure PCTCN2016070696-appb-000027
二、方法
1、建库测序
按照Ion AmpliSeqTM Library Kits 2.0标准建库流程进行建库。使用表1和表2的引物进行多重PCR反应后，扩增产物的DNA分子两端加上测序所用的接头，在一定条件下使核酸分子成簇生长，然后在Ion Torrent PGM上测序，得到片段长度分布在125bp～275bp目标区域的DNA片段序列。
本实施例中，对于获自上述6例胚胎细胞的DNA样品及父母双方的全血DNA按照Ion Torrent官方公布的测序说明书进行操作。
2、数据分析
利用Torrent_Server_4.0_VM软件对PGM测序得到的原始数据去除接头序列，用Tmap软件比对到人类hg19参考基因组，去除低质量序列，统计目标位点的测序深度N，去除N小于100的位点，分析有效位点的所有序列方向和各碱基比例，将正反向序列数大于20％并且碱基比例大于10％的碱基类型定义为该位点的一种基因型(X，X＝{A,T,C,G})。
利用父母和胚胎的基因型推测个体的单体型，有效位点即对于构建家系单体型提供有效SNP信息的位点，一般为其中一方为杂合的位点。父母双方基因型为({X1X1}、{X2X2})、({X1X1}、{X1X1})的为无效位点，({X1X1}、{X1X2})为有效位点，({X1X2}、{X1X2})为可验证位点，根据有效位点和验证位点推断父母及各胚胎单体型，母本(HCC)13、14号染色体单体型分别为Cc、Dd，父本(JXD)13、14号染色体单体型分别为Aa、Bb。
三、结果分析
根据目标区域115个(13、14号染色体分别为57和58个)SNP位点建立家系单体型(结果如图1)，根据家系单体型分析如表9所示。
表9家系单体型表
  HCC JXD HCC-1 HCC-2 HCC-3 HCC-4 HCC-5 HCC-6
chr13 Cc Aa AC C aC AC - ac
chr14 Dd Bb BD bD bD BD - bD
根据JXD为易位携带者及2号胚胎为13号染色体缺失的信息，可以推断2号胚胎带有的来自JXD的14号染色体单体型(b型)为正常染色体单体型，即14号染色体B型为易位染色体单体型，进一步地，通过其他携带B型或b型的胚胎可以推测13号染色体A型为易位染色体单体型。由此，可以判断1、4号胚胎携带14号染色体B型及13号染色体A型，为易位携带者胚胎；3、6号胚胎携带14号染色体b型及13号染色体a型，为正常胚胎；5号胚胎染色体拷贝数分析未检出结果，本次检测也没有得到任何有效序列。
工业实用性
本发明用于对适用人群进行胚胎染色体易位分析，有利于提供遗传咨询和提供临床决策依据，进行植入前诊断可有效防止患儿出生。
本发明适用人群可以是常规染色体核型分析染色体罗氏易位携带者。
本发明不局限于上述最佳实施方式，任何人在本发明的启示下都可得出其它各种形式的产品，但不论在其形状或结构上作任何变化，凡是具有与本申请相同或相近似的技术方案，均落在本发明的保护范围之内。。
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Claims (23)

  1. 一种检测染色体罗氏易位的方法，其特征在于，包括以下步骤：
    (1)获取亲代双方和子代的DNA样本；
    (2)从近端着丝粒染色体的着丝粒附近区域筛选高频突变SNP位点作为标记；
    (3)检测子代DNA样本的染色体拷贝数；
    (4)针对步骤(2)筛选的SNP位点，确定亲代双方和子代目标位点的基因型；
    (5)基于亲代双方和子代的基因型及家系关系，分析亲代双方和子代的单体型；
    (6)根据子代的染色体拷贝数检测结果、亲代双方携带染色体罗氏易位的核型、以及亲代双方和子代的单体型，分析子代的染色体罗氏易位情况。
  2. 根据权利要求1的方法，其中，所述亲代DNA样本是从外周血样本中提取的父母基因组DNA。
  3. 根据权利要求1或2的方法，其中，所述子代DNA样本选自胚胎或胎儿的滋养层细胞全基因组扩增产物，或流产组织提取的基因组DNA，优选体外培养的胚胎的囊胚期滋养层细胞全基因组扩增产物。
  4. 根据权利要求1-3中任一项的方法，其用于检测人类染色体罗氏易位，优选地，所述人类染色体罗氏易位是第13、14、15、21和22号染色体中任意两条染色体之间发生的易位。
  5. 根据权利要求4的方法，其中，步骤(2)中SNP位点的筛选标准为：
    a.位点位于着丝粒附近10Mb区域内；
    b.千人基因组数据库或者dbSNP数据库中收录，优先选择目标人群中最小等位基因频率(MAF)大于0.2的SNP；
    c.SNP位点附近序列在人类基因组中不具有同源性。
  6. 根据权利要求5的方法，其中，所述SNP位点选自以下SNP位点或其任意组合：chr13:19602195，chr13:19625370，chr13:19646283，chr13:19692745，chr13:19713634，chr13:19722801，chr13:19732341，chr13:19784913，chr13:19843135，chr13:19877547，chr13:19883672，chr13:19896927，chr13:19917509，chr13:20494558，chr13:20709214，chr13:20747500， chr13:20784332，chr13:20825315，chr13:20864572，chr13:20881859，chr13:20908501，chr13:20938952，chr13:20951425，chr13:20992333，chr13:21012562，chr13:21066214，chr13:21092894，chr13:21112935，chr13:21527671，chr13:21553971，chr13:21575458，chr13:21607760，chr13:21636136，chr13:21655335，chr13:21683681，chr13:21801501，chr13:22197910，chr13:22249975，chr13:22270788，chr13:22340074，chr13:22396664，chr13:22417688，chr13:22473749，chr13:22504691，chr13:22515844，chr13:22523279，chr13:22538311，chr13:22561684，chr13:22640820，chr13:22751654，chr13:22768476，chr13:22780737，chr13:22799808，chr13:22819627，chr13:22835073，chr13:22892864，chr13:22980146，chr14:19472367，chr14:20326905，chr14:20349694，chr14:20383719，chr14:20420387，chr14:20517369，chr14:20527044，chr14:20565487，chr14:20643775，chr14:20670979，chr14:20731036，chr14:20820537，chr14:20905141，chr14:20918598，chr14:21008574，chr14:21048774，chr14:21091127，chr14:21156472，chr14:21195967，chr14:21237137，chr14:21263166，chr14:21313819，chr14:21325985，chr14:21388266，chr14:21452591，chr14:21519915，chr14:21580671，chr14:21625262，chr14:21789092，chr14:21981923，chr14:22053817，chr14:22090208，chr14:22138437，chr14:22210067，chr14:22244363，chr14:22263821，chr14:22312275，chr14:22324460，chr14:22351977，chr14:22361282，chr14:22375208，chr14:22393124，chr14:22428984，chr14:22452496，chr14:22489611，chr14:22518909，chr14:22526966，chr14:22563283，chr14:22611651，chr14:22626948，chr14:22657897，chr14:22702039，chr14:22731529，chr14:22734948，chr14:22741160，chr14:22772663，chr14:22861948，chr14:22972955，chr15:20022190，chr15:20049922，chr15:20189367，chr15:20191854，chr15:20192138，chr15:20454253，chr15:20470948，chr15:20564500，chr15:20569241，chr15:20586537，chr15:20586966，chr15:20590656，chr15:20616721，chr15:20626721，chr15:20646773，chr15:20848359，chr15:20849625，chr15:20852181，chr15:20862156，chr15:20863958，chr15:21905797， chr15:21928397，chr15:21938184，chr15:21940649，chr15:22013203，chr15:22299435，chr15:22302182，chr15:22376163，chr15:22427155，chr15:22441959，chr15:22476195，chr15:22520388，chr15:22539952，chr15:22780065，chr15:22798800，chr15:22833272，chr15:22854479，chr15:22869870，chr15:22893990，chr15:22905599，chr15:22916637，chr15:22923869，chr15:22940961，chr15:22971662，chr15:23000363，chr15:23010627，chr15:23020535，chr15:23044004，chr15:23053813，chr15:23064687，chr15:23721906，chr15:23730971，chr15:23746510，chr15:23767135，chr15:23781336，chr15:23793989，chr15:23816231，chr15:23893430，chr15:23929209，chr15:24006330，chr15:24055535，chr15:24129894，chr15:24166516，chr15:24196094，chr15:24236177，chr15:24278723，chr15:24319610，chr15:24350583，chr15:24471640，chr15:24824114，chr15:24870875，chr15:24913763，chr15:24943341，chr15:24974072，chr21:14640496，chr21:14643423，chr21:14695894，chr21:14756515，chr21:14818516，chr21:14890028，chr21:15012159，chr21:15026905，chr21:15170049，chr21:15280687，chr21:15300595，chr21:15445542，chr21:15479041，chr21:15501432，chr21:15548748，chr21:15566056，chr21:15588845，chr21:15625530，chr21:15654618，chr21:15681452，chr21:15720558，chr21:15763305，chr21:15842397，chr21:15884516，chr21:15916232，chr21:15942816，chr21:15984234，chr21:16027704，chr21:16063848，chr21:16122140，chr21:16160041，chr21:16193235，chr21:16238898，chr21:16315932，chr21:16353639，chr21:16384555，chr21:16448591，chr21:16578538，chr21:16645724，chr21:16717265，chr21:16833315，chr21:16881542，chr21:17000938，chr21:17054831，chr21:17140290，chr21:17203891，chr21:17295288，chr21:17340912，chr21:17536247，chr21:17640426，chr21:17673255，chr21:17713264，chr21:17748703，chr21:17781159，chr21:17823782，chr21:18003355，chr21:18046650，chr21:18114449，chr21:18151760，chr21:18182817，chr21:18250262，chr21:18337678，chr21:18385770，chr21:18420873，chr21:18457287，chr21:18586049，chr21:18650837， chr21:18740886，chr21:18858018，chr21:18936951，chr21:19002939，chr21:19064396，chr21:19139888，chr21:19254923，chr21:19316600，chr22:16415691，chr22:16554800，chr22:16848067，chr22:16850056，chr22:16850858，chr22:16851557，chr22:16852464，chr22:16852792，chr22:16853453，chr22:16854397，chr22:16855647，chr22:16856500，chr22:16857432，chr22:16858287，chr22:16860547，chr22:16861742，chr22:16867995，chr22:17031697，chr22:17271213，chr22:17293257，chr22:17409957，chr22:17442318，chr22:17469090，chr22:17525677，chr22:17563259，chr22:17587785，chr22:17619511，chr22:17656792，chr22:17682387，chr22:17715164，chr22:17739757，chr22:17761489，chr22:17785311，chr22:17805528，chr22:17841251，chr22:17883346，chr22:17916190，chr22:17939998，chr22:17981144，chr22:18012297，chr22:18054406，chr22:18212058，chr22:18276198，chr22:18354015，chr22:18395978，chr22:18466951，chr22:18531582，chr22:18581715，chr22:18628321，chr22:18894617，chr22:18966077，chr22:19004846，chr22:19025459，chr22:19098323，chr22:19142652，chr22:19236590，chr22:19362714，chr22:19431588，chr22:19536367，chr22:19643965，chr22:19716983，chr22:19759532，chr22:19808057，chr22:19859087，chr22:19909387，chr22:19972494，chr22:20016622，chr22:20051573，chr22:20081918，chr22:20135421，chr22:20175034，chr22:20216874，chr22:20278637，chr22:20745699，chr22:20787955，chr22:20855585，chr22:20881195，chr22:20924097，chr22:20983921，chr22:21028224。
  7. 根据权利要求1-6中任一项的方法，其中，所述SNP位点包括来自发生易位的每条染色体的至少1个SNP位点，优选至少10个SNP位点，更优选至少50个SNP位点SNP位点。
  8. 根据权利要求6的方法，其中，所述SNP位点包括来自发生易位的每条染色体的权利要求6中限定的全部SNP位点。
  9. 根据权利要求1-8中任一项的方法，其中，步骤(3)中检测染色体拷贝数的方法和步骤(4)中确定样本基因型的方法独立地选自测序方法和芯片方法。
  10. 根据权利要求9的方法，其中，所述测序方法为高通量测序方法，优选Ion  Torrent PGM或Illumina Miseq；所述的芯片方法为人全基因组SNP芯片，优选Illumina全基因组SNP芯片。
  11. 根据权利要求1-10中任一项的方法，其中，亲代双方一方为正常核型，另一方为罗氏易位携带者，子代中包括罗氏易位染色体三体或者单体的个体。
  12. 根据权利要求11的方法，其中，步骤(6)中的判断标准如下：
    a.如果某一子代为染色体m三体，则其染色体n中来源于亲代罗氏易位携带者的单体型B为易位染色体单体型，相应地，来源于亲代罗氏易位携带者染色体n的另一种单体型b为正常染色体单体型；进一步地，通过观察其他子代的单体型，与染色体n单体型B连锁的来源于亲代罗氏易位携带者的染色体m单体型A也是易位染色体单体型，相应地，来源于亲代罗氏易位携带者染色体m的另一种单体型a为正常染色体单体型；由此，可以判断携带染色体m单体型A和染色体n单体型B的子代为易位携带者，携带染色体m单体型a和染色体n单体型b的子代为正常核型；
    b.如果某一子代为染色体m单体，则其染色体n中来源于亲代罗氏易位携带者的单体型B为正常染色体单体型，相应地，来源于亲代罗氏易位携带者染色体n的另一种单体型b为易位染色体单体型；进一步地，通过观察其他子代的单体型，与染色体n单体型b连锁的来源于亲代罗氏易位携带者的染色体m单体型a也是易位染色体单体型，相应地，来源于亲代罗氏易位携带者染色体m的另一种单体型A为正常染色体单体型；由此，可以判断携带染色体m单体型A和染色体n单体型B的子代为正常核型，携带染色体m单体型a和染色体n单体型b的子代为易位携带者；
    其中，染色体m和染色体n为亲代罗氏易位携带者基因组中发生罗氏易位的两条染色体，A和a为来源于亲代罗氏易位携带者染色体m的单体型，B和b为来源于亲代罗氏易位携带者染色体n的单体型。
  13. 根据权利要求1-12任一项的方法在胚胎植入前诊断中的应用。
  14. 一种用于检测染色体罗氏易位的引物组合物，其特征在于所述引物组合物能够扩增近端着丝粒染色体的着丝粒附近区域高频突变SNP位点的序列。
  15. 根据权利要求14的引物组合物，其用于检测人类染色体罗氏易位，优选地， 所述人类染色体罗氏易位是第13、14、15、21和22号染色体中任意两条染色体之间发生的易位。
  16. 根据权利要求15的引物组合物，其中，所述SNP位点的筛选标准为：
    a.位点位于着丝粒附近10Mb区域内；
    b.千人基因组数据库或者dbSNP数据库中收录，优先选择目标人群中最小等位基因频率(MAF)大于0.2的SNP；
    c.SNP位点附近序列在人类基因组中不具有同源性。
  17. 根据权利要求16的引物组合物，其中，所述SNP位点选自以下SNP位点或其任意组合：chr13:19602195，chr13:19625370，chr13:19646283，chr13:19692745，chr13:19713634，chr13:19722801，chr13:19732341，chr13:19784913，chr13:19843135，chr13:19877547，chr13:19883672，chr13:19896927，chr13:19917509，chr13:20494558，chr13:20709214，chr13:20747500，chr13:20784332，chr13:20825315，chr13:20864572，chr13:20881859，chr13:20908501，chr13:20938952，chr13:20951425，chr13:20992333，chr13:21012562，chr13:21066214，chr13:21092894，chr13:21112935，chr13:21527671，chr13:21553971，chr13:21575458，chr13:21607760，chr13:21636136，chr13:21655335，chr13:21683681，chr13:21801501，chr13:22197910，chr13:22249975，chr13:22270788，chr13:22340074，chr13:22396664，chr13:22417688，chr13:22473749，chr13:22504691，chr13:22515844，chr13:22523279，chr13:22538311，chr13:22561684，chr13:22640820，chr13:22751654，chr13:22768476，chr13:22780737，chr13:22799808，chr13:22819627，chr13:22835073，chr13:22892864，chr13:22980146，chr14:19472367，chr14:20326905，chr14:20349694，chr14:20383719，chr14:20420387，chr14:20517369，chr14:20527044，chr14:20565487，chr14:20643775，chr14:20670979，chr14:20731036，chr14:20820537，chr14:20905141，chr14:20918598，chr14:21008574，chr14:21048774，chr14:21091127，chr14:21156472，chr14:21195967，chr14:21237137，chr14:21263166，chr14:21313819，chr14:21325985，chr14:21388266，chr14:21452591，chr14:21519915，chr14:21580671，chr14:21625262，chr14:21789092，chr14:21981923， chr14:22053817，chr14:22090208，chr14:22138437，chr14:22210067，chr14:22244363，chr14:22263821，chr14:22312275，chr14:22324460，chr14:22351977，chr14:22361282，chr14:22375208，chr14:22393124，chr14:22428984，chr14:22452496，chr14:22489611，chr14:22518909，chr14:22526966，chr14:22563283，chr14:22611651，chr14:22626948，chr14:22657897，chr14:22702039，chr14:22731529，chr14:22734948，chr14:22741160，chr14:22772663，chr14:22861948，chr14:22972955，chr15:20022190，chr15:20049922，chr15:20189367，chr15:20191854，chr15:20192138，chr15:20454253，chr15:20470948，chr15:20564500，chr15:20569241，chr15:20586537，chr15:20586966，chr15:20590656，chr15:20616721，chr15:20626721，chr15:20646773，chr15:20848359，chr15:20849625，chr15:20852181，chr15:20862156，chr15:20863958，chr15:21905797，chr15:21928397，chr15:21938184，chr15:21940649，chr15:22013203，chr15:22299435，chr15:22302182，chr15:22376163，chr15:22427155，chr15:22441959，chr15:22476195，chr15:22520388，chr15:22539952，chr15:22780065，chr15:22798800，chr15:22833272，chr15:22854479，chr15:22869870，chr15:22893990，chr15:22905599，chr15:22916637，chr15:22923869，chr15:22940961，chr15:22971662，chr15:23000363，chr15:23010627，chr15:23020535，chr15:23044004，chr15:23053813，chr15:23064687，chr15:23721906，chr15:23730971，chr15:23746510，chr15:23767135，chr15:23781336，chr15:23793989，chr15:23816231，chr15:23893430，chr15:23929209，chr15:24006330，chr15:24055535，chr15:24129894，chr15:24166516，chr15:24196094，chr15:24236177，chr15:24278723，chr15:24319610，chr15:24350583，chr15:24471640，chr15:24824114，chr15:24870875，chr15:24913763，chr15:24943341，chr15:24974072，chr21:14640496，chr21:14643423，chr21:14695894，chr21:14756515，chr21:14818516，chr21:14890028，chr21:15012159，chr21:15026905，chr21:15170049，chr21:15280687，chr21:15300595，chr21:15445542，chr21:15479041，chr21:15501432，chr21:15548748，chr21:15566056，chr21:15588845，chr21:15625530， chr21:15654618，chr21:15681452，chr21:15720558，chr21:15763305，chr21:15842397，chr21:15884516，chr21:15916232，chr21:15942816，chr21:15984234，chr21:16027704，chr21:16063848，chr21:16122140，chr21:16160041，chr21:16193235，chr21:16238898，chr21:16315932，chr21:16353639，chr21:16384555，chr21:16448591，chr21:16578538，chr21:16645724，chr21:16717265，chr21:16833315，chr21:16881542，chr21:17000938，chr21:17054831，chr21:17140290，chr21:17203891，chr21:17295288，chr21:17340912，chr21:17536247，chr21:17640426，chr21:17673255，chr21:17713264，chr21:17748703，chr21:17781159，chr21:17823782，chr21:18003355，chr21:18046650，chr21:18114449，chr21:18151760，chr21:18182817，chr21:18250262，chr21:18337678，chr21:18385770，chr21:18420873，chr21:18457287，chr21:18586049，chr21:18650837，chr21:18740886，chr21:18858018，chr21:18936951，chr21:19002939，chr21:19064396，chr21:19139888，chr21:19254923，chr21:19316600，chr22:16415691，chr22:16554800，chr22:16848067，chr22:16850056，chr22:16850858，chr22:16851557，chr22:16852464，chr22:16852792，chr22:16853453，chr22:16854397，chr22:16855647，chr22:16856500，chr22:16857432，chr22:16858287，chr22:16860547，chr22:16861742，chr22:16867995，chr22:17031697，chr22:17271213，chr22:17293257，chr22:17409957，chr22:17442318，chr22:17469090，chr22:17525677，chr22:17563259，chr22:17587785，chr22:17619511，chr22:17656792，chr22:17682387，chr22:17715164，chr22:17739757，chr22:17761489，chr22:17785311，chr22:17805528，chr22:17841251，chr22:17883346，chr22:17916190，chr22:17939998，chr22:17981144，chr22:18012297，chr22:18054406，chr22:18212058，chr22:18276198，chr22:18354015，chr22:18395978，chr22:18466951，chr22:18531582，chr22:18581715，chr22:18628321，chr22:18894617，chr22:18966077，chr22:19004846，chr22:19025459，chr22:19098323，chr22:19142652，chr22:19236590，chr22:19362714，chr22:19431588，chr22:19536367，chr22:19643965，chr22:19716983，chr22:19759532，chr22:19808057， chr22:19859087，chr22:19909387，chr22:19972494，chr22:20016622，chr22:20051573，chr22:20081918，chr22:20135421，chr22:20175034，chr22:20216874，chr22:20278637，chr22:20745699，chr22:20787955，chr22:20855585，chr22:20881195，chr22:20924097，chr22:20983921，chr22:21028224。
  18. 根据权利要求14-17中任一项的引物组合物，其中，所述SNP位点包括来自发生易位的每条染色体的至少1个SNP位点，优选至少10个SNP位点，更优选至少50个SNP位点SNP位点。
  19. 根据权利要求17的引物组合物，其中，所述SNP位点包括来自发生易位的每条染色体的权利要求17中限定的全部SNP位点。
  20. 根据权利要求17的引物组合物，其选自以下正向引物和反向引物组成的引物对，或其任意组合：
    (1)扩增第13号染色体的SNP位点的引物选自以下引物对：正向引物序列SEQ ID NO:2n-1，反向引物序列SEQ ID NO:2n；其中，n为选自1-57的自然数；
    (2)扩增第14号染色体的SNP位点的引物选自以下引物对：正向引物序列SEQ ID NO:2n-1，反向引物序列SEQ ID NO:2n；其中，n为选自58-115的自然数；
    (3)扩增第15号染色体的SNP位点的引物选自以下引物对：正向引物序列SEQ ID NO:2n-1，反向引物序列SEQ ID NO:2n；其中，n为选自116-189的自然数；
    (4)扩增第21号染色体的SNP位点的引物选自以下引物对：正向引物序列SEQ ID NO:2n-1，反向引物序列SEQ ID NO:2n；其中，n为选自190-264的自然数；
    (5)扩增第22号染色体的SNP位点的引物选自以下引物对：正向引物序列SEQ ID NO:2n-1，反向引物序列SEQ ID NO:2n；其中，n为选自265-344的自然数。
  21. 根据权利要求20的引物组合物，其包括用于扩增发生易位的每条染色体的SNP位点的至少1对引物，优选至少10对引物，更优选至少50对引物，最优选包括扩增发生易位的每条染色体的SNP位点的权利要求20中限定的全部 引物对。
  22. 根据权利要求14-21中任一项的引物组合物在制备检测染色体罗氏易位的诊断试剂中的应用。
  23. 根据权利要求14-21中任一项的引物组合物在制备检测染色体罗氏易位的试剂盒中的应用。
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