The basics of human chromosomes
In 1956, Tjio and Levan correctly determined the number of chromosomes in human cells to be 46, and only then did the scientific and systematic study of human chromosomes begin, leading to the formation of human cytogenetics. In 1959, Lejune discovered that congenital dysmorphism was due to trisomy 21, followed by Turner syndrome and Klinefelter syndrome, and a new subdiscipline, clinical cytogenetics, was formed. -Since the 1970s, the development of multiple chromosome banding techniques has improved the accuracy of chromosome analysis. In the mid-1980s, new techniques such as fluorescence in situ hybridization, microdissection techniques and chromosome staining allowed direct detection of changes in chromosomes and DNA fragments in the nucleus at the interrogation stage, which allowed cellular level research to be linked with molecular level exploration and combined to form a new field –Molecular cytogenetics.
Section I. Human chromosomes
I. Characteristics and types of human chromosomes
The morphological and structural characteristics of chromosomes are most typical in the cell cycle at mid-cell division and are called intermediate chromosomes. Each intermediate chromosome is composed of two chromosomes, each helixed by a DNA molecule, and the two chromosomes are called sister chromatids to each other. The two chromosomes are connected to each other by a single chromosome, which is deconvoluted, lightly stained and internally constricted, called the primary constriction. A special structure in the mitotic region is the spindle filament attachment site, which is associated with chromosome movement during cell division. The mitophagus divides the chromosome laterally into two arms, the longer one called the long arm (q) and the shorter one called the short arm (p). Each arm ends with a specialized part called a telomere, a highly repetitive DNA sequence, which is necessary for chromosome stability. Each chromosome needs a mitriole and two telomeres to exist stably. If a telomere is missing, the chromosome ends will lose their stability and the chromosomes will be connected abnormally, resulting in aberrant chromosomes; if a mitriole is missing, the chromosome cannot be connected to the spindle filament during cell division, resulting in chromosome deletion. In some chromosome arms, lightly stained inner constricted segments called paraconstrictions can also be seen. In human, there is a filament-like structure on the distal side of the short arm of the proximal chromosome called the follower, and the filament-like structure between the follower and the short arm is called the follower stalk, which is actually also a paracontractile region, where the ribosomal RNA (rRNA) gene exists. Its expression products are related to the composition of the nucleolus and maintenance of its structure and morphology, also known as nucleolus organizer (NOR). There are generally two types of follower, where the diameter of the follower is less than 1/2 the diameter of the chromosome arm is called small follower, while the diameter greater than 1/2 is called large follower. In the population, polymorphism in the length of paracontracts, follower size and number exists and is inherited in a Mendelian manner.
The position of the mitoses on the chromosomes is constant. By dividing chromosomes into eight equal parts along the longitudinal axis, human chromosomes can be divided into three categories according to the position of the trophoblast: (i) submedial trophoblast chromosomes, where the trophoblast is located at or near the center of the chromosome (1/2 to 5/8) and the long and short arms of the chromosome are similar; (ii) submedial trophoblast chromosomes, where the trophoblast is slightly biased towards one end (5/8 to 7/8) and divides the chromosome into two arms of significantly different lengths; (iii) proximal trophoblast chromosomes (iii) proximal mitotic chromosomes, with the mitotic granules near one end (7/8 to the end) (Figure 6-1, 6-2).
Second, the normal karyotype of human
(I) Denver regime Since 1956, when Tjio and Leven confirmed that the normal number of chromosomes in human somatic cells is 46, and 1959, when it was first discovered that congenital stupidity (Down syndrome) is due to an extra chromosome 21 than normal, human chromosome research has been gradually applied to clinical practice all over the world, and the number of chromosomal disorders discovered and reported has been increasing. In order to facilitate the description of aberrant chromosomes and international communication, the First International Cytogenetics Conference was held in Denver, USA, in 1960, which discussed and established the Denver system for the description of intracellular chromosome composition as a basis for identifying and analyzing human chromosomes. The Denver system is used as a basis for identifying and analyzing human chromosomes. According to the Denver system, the 46 chromosomes in human somatic cells are divided into 23 pairs, of which l-22 pairs are common to both sexes, called autosomes, and are numbered 1 to 22 in order; the other pair is related to sex, called sex chromosomes. The two sex chromosomes are XX chromosomes in women and X chromosomes and Y chromosomes in men.
The image of all the chromosomes of a body cell is called karyotype. The process of identifying and determining the karyotype by pairing and arranging all chromosomes of the cell to be tested according to the Denver system is called karyotype analysis.
(b) Identification of karyotype of non-dominant chromosomes According to the Denver system, 23 pairs of chromosomes of a somatic cell are divided into 7 groups (A to G) according to their size and morphological characteristics. The chromosome groupings and their morphological characteristics of each group are shown in Table (6-1).
Karyotype analysis is usually a process in which chromosome photographs obtained by microphotography are identified, analyzed and cut and pasted according to grouping requirements. According to the international system, the description of the normal karyotype includes the total number of chromosomes and the composition of the sex chromosomes, which are written as follows
Normal male karyotype 46, XY
Normal female karyotype 46, XX
(III) Naming of chromosomal dominant bands and dominant chromosomes
In 1968, Swedish cytochemist Caspersson treated the specimens with the fluorescent dye quinacrine (QM). After treating the specimens with the fluorescent dye quinacrine (QM), he found that each chromosome showed a band with different widths and shades along its long axis under a fluorescence microscope, and that each of the 24 human chromosomes showed a distinctive band pattern, so that each chromosome could be accurately identified and characterized, and even minor chromosomal structural abnormalities could be detected. Therefore, the chromosome Q-banding technique was created, and the banding pattern displayed by this technique is called Q-banding. The reason why chromosomes show banding is generally believed to be due to the different base composition and degree of helix winding in different parts of the DNA molecules constituting chromosomes. Since the establishment of the Q-banding technique, there have been several other types of bands such as G-banding, R-banding, C-banding and T-banding.
C-banding, and T-banding.
Commonly used banding techniques.
Q-band chromosome specimens show bands after treatment with fluorescent dyes such as quinacrine (QM). Q-banding is the most widely used banding technique, but the fluorescence duration is short and the specimens cannot be stored for a long time and must be observed immediately.
G band is the most widely used band type at present. The operation is simple, the band pattern is clear, the specimen can be preserved for a long time, and the reproducibility is good. The method is: the chromosome is treated with reagents such as trypsin, NaOH, citrate or urea, and then stained with Jimsa dye, showing alternating dark and light banding patterns, called G-banding. The most commonly used of these is the trypsin treatment method. The band pattern shown by each chromosome number is basically similar to the Q-band band pattern. The dark band in the G-band is equivalent to the light band in the Q-band, while the light band is equivalent to the dark band.
The alternating dark and light band patterns shown by the R-band chromosome specimens treated with hot phosphate buffer and stained with Jimsa are called R-bands. It is also called the reverse band because it is exactly the opposite shade of the G band. Chromosomes with G- and Q-banding are lightly banded at the ends of both arms, so it is difficult to detect structural abnormalities such as end deletion and rearrangement, while R-banding has deep bands at the ends of chromosomes, so it is easy to identify if there are abnormalities at this part, so R-banding is mainly used to study chromosome end deletion and structural rearrangement.
C-band chromosome specimens are treated with alkaline solutions such as NaOH or Ba(OH)2, and then put into sodium citrate and sodium chloride solutions, and then stained with Gimsa dye, and it can be seen that the mitotic region of each chromosome is specifically colored, so it is called the mitotic band, also called C-band.
Strictly speaking, the C band shows the structurally heterochromatin region immediately adjacent to the mitoses, i.e., the paracontracts near the mitoses of human chromosomes 1, 9, and 16. In contrast, the long arm of the Y chromosome ends in a heterochromatin region, which also shows significant deep staining. Therefore, the C-banding technique was used to study the structural changes in the region of the mitotic region, the Y chromosome and the paracontractile region.
N-banding Treatment of chromosomal specimens with AgNO3 results in deep staining of the paraconstriction, or nucleolar organizer region (NOR), of the five proximal pairs of mitotic chromosomes (chromosomes 13, 14, 15, 2l, and 22) in human cells, called N-banding. Strictly speaking, AgNO3 can only stain transcriptionally active NORs black, and such silver-stain-positive NORs are called Ag-NOR. inactive NORs are not stained. Therefore, the N-banding technique can be used to study rRNA activity and its dynamics, and also as a way to observe whether the follower association of proximal chromosomes occurs, since follower association is one of the causes of chromosome non-separation.
T-banding is a technique that heats chromosome specimens and then stains them with Jimsa dye, which can make some chromosome terminal segments stained specifically and deeply, called T-banding or telobanding, which can exclusively show chromosome telomeres, and this technique can be applied to identify small aberrations in chromosome terminals.
The above Q, G and R banding methods can show the specific banding pattern of 24 human chromosomes in a constant manner, which indicates the objective existence and authenticity of the banding pattern and provides an analytical basis for identifying chromosomal alterations.
2.Naming of chromosomal dominant karyotype The application of banding technology further requires a unified standard for identification and description of dominant chromosomes to facilitate mutual communication. According to the recommendations of the Fourth International Conference on Human Cytogenetics held in Paris in 1971 and the decision of the Edinburgh Conference in 1972, a standard system for distinguishing each chromosomal region and band was proposed, called the International System of Human Cytogenetic Nomenclature (ISCN, 1971).
1and mark: It is a structural region on the chromosome that has significant morphological features and exists stably, and is an important indicator for identifying chromosomes with dominant bands. It includes the ends of the two arms of the chromosome, the mitoses and certain bands that are constant under different banding conditions.
Region: the area located between two boundary markers.
Band: Each chromosome should be considered as a series of bands, i.e. there are no non-banded regions. Each band can be distinguished from neighboring bands by differences in color intensity that are brighter (darker) or darker (lighter).
Each chromosome is distinguished into short and long arms using the trophectoderm as a boundary marker. The bands on both short and long arms are numbered starting from the mitophagus and numbered in the direction from near to far along the mitophagus. The two zones closest to the mitophagus are recorded as the “l” zone of the long arm or short arm, and the “2” zone and “3” zone from the near side to the distal side. The numbering of the bands in each zone follows the same principle, i.e., the band closest to the mitophagus in the zone is numbered as band 1 of the zone, followed by bands 2 and 3. In two cases, the band that is the boundary marker is counted as band 1 of the zone far from the boundary marker; the band that is divided into two by the filopodia
The band that is divided into two by the trophectoderm is the two bands belonging to the long and short arms, and they are recorded as band 1 of zone 1 of the long arm and band 1 of zone 1 of the short arm, respectively.
When describing a specific band, four elements should be written: ① chromosome number; ② arm symbol; ③ zone number; ④ band number. These elements are written in order without spacing or adding any punctuation. For example, the arrow in Figure 6-4 shows chromosome 1, short arm, zone 3, band 1, written as lp31.
The application of chromosomal banding techniques can identify a wide range of chromosomal microstructural abnormalities, such as breaks, translocations, and inversions of chromosomes, etc. In order to have a uniform format for describing these changes, the International System of Human Cytogenetic Nomenclature (1978, 1981, 1985), agreed at an international conference in Stockholm in 1997, proposed a nomenclature symbol and abbreviations for banded chromosomes terminology system (Table 6-2) for uniform application.
3, High-resolution chromosome banding and naming application of common G banding analysis is for mid-cell division chromosomes, when chromosome helicalization is the highest, so chromosomes contract to the shortest, banding tends to fuse, and the number of banding shown is less. Usually mid-phase haplochromosomes show only 320 bands. Since 1975, Yunis et al. established the high resolution banding technique, in which cells are synchronized with methotrexate and treated with colchicine for a short period of time to obtain many chromosomes in the prophase and late Then, by banding treatment, the human early and mid-stage haplochromosomes show 550 to 850 bands, while 850 to 1,250 bands can be observed in the late stage. These bands are due to the relatively low helicity of the elongated chromosomes, and bands fused in 320 bands are revealed, thus greatly improving the level of chromosome study. The chromosomal abnormalities that were previously thought to be “monogenic diseases” and the syndromes they manifest because they were not observed are actually due to minor chromosomal changes. For example, WAGR syndrome, which was originally thought to be an AD genetic disorder, is due to a deletion of the llpl4.1-p13 fragment; Prader-Willi syndrome, also thought to be an AD genetic disorder, is actually due to a deletion and rearrangement of 15q11.2. For example, the etiology of congenital dysgenesis was first identified by Weune (1959) as the result of an extra chromosome 2l, and the application of high-resolution banding techniques further determined that the main symptoms of congenital dysgenesis were only associated with the duplication of a tiny fragment of 21q22.3. This application of high-resolution banding techniques to study the microstructure of chromosomes and the genetic effects of structural alterations is also known as microcytogenetics. It is now possible to show 3,000 to 10,000 bands on chromosomes in the Gz or early prophase, which is close to the number of structural genes present in a cell (about 30,000), thus reducing the distance between cytogenetics and molecular genetics.
Nomenclature representation of high-resolution chromosomes 320 bands level of a band usually in high-resolution chromosomes can be divided into several bands – sub-bands. Regarding the nomenclature and representation of subbands, ISCN (1981) stipulates that the subbands divided from any of the bands named in ISCN (1978) maintain the original zone and boundary marker band numbers, and each band is subdivided by adding a dot after the original band number and writing the number of each subband, and its numbering principle is still sequential numbering from the mitotic granule to the arm end. For example, the original lp3l band is divided into three subbands, which should be written as lp31.1, 1p31.2 and 1p31.3.
Among them, lp31.1 is close to the mitophagus and 1p31.3 is far from the mitophagus. If the subband is subdivided into subbands, the number can be added after the original subband number, but it is not necessary to add punctuation. For example, when subband 1p31.3 is subdivided, it is written as 1p31.3l, lp31.32 and lp31.33.
III. Progress in human cytogenetics research
In summary, chromosome banding technology has entered the level of high-resolution chromosome banding, which has greatly improved the ability to analyze chromosomes, but under the light microscope, it can only identify DNA fragments above 4,500 kb, while chromosomal abnormalities below 4,500 kb cannot be recognized. Nowadays, DNA molecules of tens of kilobases to 2,000 kilobases can be effectively detected and analyzed using molecular biology techniques. Therefore, combining molecular biology techniques with microcytogenetics is of great value for the analysis of the causes of genetic diseases and the identification of disease-causing genes, and molecular cytogenetics, which is the study of the structure of chromosomes and the genetic effects of aberrations at the molecular level based on microcytology using molecular biology methods and
Molecular cytogenetics is the study of chromosome structure and the genetic effects of aberrations and disease development at the molecular level, based on microcytology. This technique is the direction of development of human cytogenetics research.
The main research techniques of molecular cytogenetics are as follows.
1, fluorescence in situ hybridization (fluorescence in situ hybridization,: FISH) This technique was developed on the basis of the in situ hybridization technology established in the 1960s. The method is to use a known DNA sequence labeled with a special fluorescein as a probe to hybridize with the chromosomal DNA to be tested, and if there is a sequence homologous to the probe on the chromosome to be tested, complementary binding can occur, and then the hybridization signal is displayed in the interphase nucleus or chromosome in situ. FISH has the advantages of rapid, safe, economical, high sensitivity and high specificity compared with general in situ hybridization (ISH). The probes are detected by non-radioactive fluorescent staining in combination with antibodies or proteins, no special safety precautions are required, the specimens can be stored for a long time without inactivation, and the resolution can reach 100-200 kb, which is the same or higher than that of radioactive probes. Therefore, FISH technology has been widely used for the detection of exogenous genes such as gene localization, chromosomal aberrations, gene amplification and viruses integrated into human cells. For example, high-resolution banding techniques have shown that the cause of congenital stupidity is the duplication of a tiny fragment of 2lq22.3, which can be cut down using microdissection techniques, the DNA fragment contained in it can be cloned, and fluorescently labeled DNA can be used as a probe to perform fluorescence in situ hybridization on interphase cells of the fetus in the chorion or amniotic fluid of a pregnant woman at high risk for congenital The use of fluorescent in situ hybridization on interstitial fetal cells in the chorionic villi or amniotic fluid of pregnant women at risk of fetal development allows for a rapid prenatal diagnosis of congenital dimorphism. At present, multiple in situ hybridizations can be performed simultaneously with different fluorescent dyes showing different fluorescent shades. This multicolor FISH technique has developed rapidly in recent years and has become an important tool for gene localization and genetic disease diagnosis.
2, DNA fiber fluorescence in situ hybridization (DNA fiber-FISH) This technique is a newly established visual high-resolution genomic mapping technique, the main point of this technique is to first treat the cells to be tested with alkaline solution or formaldehyde solution to loosen the organization of interphase nuclear chromatin to release chromatin (filaments) from the nuclear skeleton in order to prepare DNA fibers on slides, and then the different color DNA fiber-FISH has the same procedure as FISH analysis for detecting chromosomal abnormalities, but the former has the advantage of higher resolution (1~500kb) and the length of probes can be in the range of 1~300kb. 300kb range. Therefore, DNA fiber-FISH technique is widely used in human genome mapping, chromosome structure analysis and analysis of chromosomal diseases, tumors and certain monogenic diseases.
3.Chromosome coating Chromosome coating is a combination of FISH and chromosome in situ suppression hybridization (CISS) technique, which is a new technique of using single-stranded DNA and Cot-1 to close the repetitive sequences of genome to reduce the non-specific hybridization signal and thus enhance the specific hybridization intensity, and using chromosome-specific DNA library as a probe pool to coat the whole chromosome or chromosome-specific region with different fluorescence.
The basic step of chromosome coating technique is to label chromosome-specific DNA (whole chromosome or a chromosome-specific region) with a non-isotopic substance, e.g., biotin, prepare a DNA probe and then hybridize it in situ with the target cell chromosome, and then detect and amplify it with an antigen-antibody reaction using streptavidin anti-biotin protein with a fluorescent substance. That is, the whole chromosome or chromosome segment to be tested can display the hybridization signal of departure fluorescence, so that the analysis and diagnosis can be made accordingly. For example, to analyze microscopic translocations involving two chromosomes, digoxigenin-1l-duTp and common biotin can be used to label the two chromosomes separately, making two different DNA probes, and then detected and amplified by antigen-antibody reaction after hybridization, which can make the two chromosomes in the same karyotype stained in different colors (e.g., one in red and the other in green), and the mutual translocation of the two derived chromosomes formed The two derived chromosomes, each with red and green colors, can be clearly identified. This suggests that this new technique will have a wide application prospect and important application value for analyzing complex translocations of human chromosomes, cytogenetics of tumors and gene localization.
4.Comparative genomic hybridization (CGH) Kallioniemi (1992) and Manoir (1993) established the CGH technique based on FISH technique, the main point of CGH technique is to label the tumor genomic DNA (DNA to be tested) and normal control DNA with different non-isotopic markers, and then mix them in a 1:1 ratio to make probes, and then mix them with normal human peripheral blood. The tumor genomic DNA (DNA to be tested) and normal control DNA are then mixed in a 1:1 ratio to make probes, which are simultaneously hybridized with normal human peripheral blood for in situ suppression of mid mitotic chromosomes, and detected with different fluorescent dye labeled probes after hybridization. Tumor DNA is generally labeled in green and normal genomic DNA is labeled in red. The relative amount of tumor DNA hybridized with a certain chromosomal region and normal control DNA depends on the content of this sequence in both samples, which can be determined by visual observation of the difference between the two colors in different chromosomal regions to roughly determine the relative copy number of the two different sources of DNA, and can also be quantified by a fluorescence microscope connected to a computerized color image analysis system for the red-green fluorescence ratio. This can be used to analyze the copy number variation of the examined DNA sequences and to make a determination of the genetic imbalance of the tumor, for example, tumor DNA amplification (green enhancement) or deletion (red but not green). This new technology can detect and localize DNA sequence copies between different genomes at the level of all chromosomes or chromosomal sub-bands, and also identify the origin of certain components of tumor chromosomes (e.g., double microsomes, marker chromosomes) that are difficult to determine by cytogenetic techniques, as well as provide information on chromosomal trisomies, monosomes, or changes in the copy number of larger segments of chromosomes by rapid detection, thus facilitating the diagnosis of Rapid diagnosis of diseases such as congenital dysmorphism and spontaneous abortion.