Chapter X - Medical Biology - Genetics 2023-2024 PDF

Exercise 10. Topic: Chromosomes and sex-linked traits. Cytogenetic techniques. Glossary: Constitutive heterochromatin - it is heterochromatin with very tightly packed (condensed) DNA, which mostly does not participate in the transcription process and occurs in this form in all cells of the body, reg...

Exercise 10. Topic: Chromosomes and sex-linked traits. Cytogenetic techniques. Glossary: Constitutive heterochromatin - it is heterochromatin with very tightly packed (condensed) DNA, which mostly does not participate in the transcription process and occurs in this form in all cells of the body, regardless of the degree of specialization; in mammalian cells, it occurs mainly around the centromere and at the ends of the chromosomes. It remains in a condensed state throughout the cell cycle and its further development; it contains highly repetitive sequences which are not transcribed and plays a role in the chromosome structure, so it is present during all stages of the cell. Comparative genomic hybridization (CGH) - a molecular cytogenetic method involving the detection of loss or amplification of chromosomal regions at the level of the chromosomal band without the need for culturing cells. Crossing over – a process of the exchange of genetic material between two homologous chromosomes non-sister chromatids;it consists in breaking and replacing corresponding positions of DNA segments between homologous chromosomes, leads to the creation of new gene systems, or genetic recombination. It occurs during the meiotic division when the gametes are formed. Euchromatin - is a form of chromatin that is a lightly packed (“relaxed”) form of chromatin that is transcriptionally active (contains mainly transcriptionally active genes). As a result of the condensation of euchromatin, a dense chromatin heterochromatin - is formed which again, in some periods of increased transcriptional activity, can transform (decondens) into loose chromatin. Facultative heterochromatin - an euchromatic material whose genetic activity at a given cell specialization has been suppressed, but in which there are coding genes that may be active in another cell specialization; the condensation is temporary. Fluorescent in situ hybridization (FISH) - a cytogenetic method for detecting and localizing specific nucleic acid sequences in cells by means of fluorescent DNA probes. Giant (polytene) chromosomes - chromosomes that formed as a result of repeated DNA endoreplication during the S phase of the cell cycle, bypassing mitosis. The chromatids produced in this way do not divide (chromatid beams - up to 2000 depending on the species), are 100 to 200 times larger than the normal chromosomes of a given organism; they occur in salivary glands and in various secretory tissues of Diptera larvae (including Malpighian tubules). Characteristic banding pattern and swellings (puffs, Balbiani rings) are visible in chromosomes, where transcription occurs with high activity (sites of mRNA synthesis).Their analysis allows to determine the activity of individual chromosomal fragments (cytogenetic studies). Holandric inheritance - inheritance of genes on the y chromosome (Y-linked); genes can only be transmitted from father to son. Karyogram - a graphical set of chromosomes of one arbitrarily selected cell, arranged according to the contractual rules; Agraphical representation of a karyotype. Karyotype - a graphical set of chromosomes typical for a given individual or species, ranked according to contractual rules (including the arm length, centromere position). Molecular probe - a labelled DNA or RNA fragment, used to locate complementary DNA or RNA sequences and to assess the level of RNA expression. Sex chromatin - suitably stained chromatin structures in the interphase nucleus corresponding to the condensed X and Y chromosome. The milestone in the research on the nature of inheritance was the achievement of Thomas Hunt Morgan, who, thanks to experiments at the fruit fly, Drosophila melanogaster, proved that genes are located on chromosomes and formulated the chromosomal theory of heredity. D. melanogaster is a convenient experimental organism for genetic research: it is characterized by a short development time (lasting for 10-14 days), high fertility (during its lifetime a female can lay up to 3 000 eggs), and the costs of maintaining these insects in laboratory conditions are low and do not require complicated treatments. In the genetic studies, very useful features of fruit flies are as follows: 1) the presence of so-called polytene (giant) chromosomes in the cells of the salivary glands of the insect larval forms, as well as 2) the occurrence of numerous gene mutations, the effects of which relate to many morphological features. Proper human karyotype 23 pairs of chromosomes: 22 pairs of autosomes and 1 pair of heterosomes. 46, XY - male (Figure 1); 46, XX - female. Figure 1. Human chromosomes (46,XY) stained with Giemsa reagent (G-bands). The rule of karyotyping is to arrange 22 autosomes following the size and sex chromosomes, X and Y, at the end. Chromosomes are classified into seven groups, A to G, by the length and centromere position (Table 1). Table 1. Classical division of human chromosomes. Group Chromosomes Size and shape A 1-3 Large metacentric B 4-5 Large submetacentric C 6-12 and X D 13-15 Medium acrocentric E 16-18 Short submetacentric F 19-20 Short metacentric G 21-22 and Y Short acrocentric Medium submetacentric The rules for recording the results of cytogenetic tests are described in ISCN (The International System for Human Cytogenetic Nomenclature), and it is necessary to follow these rules when describing an aberration. These rules contain lists of abbreviations describing chromosomes, chromosome segments, and aberrations (Table 2). The human karyotype is composed of a number that specifies the total number of chromosomes in the diploid cell, a comma and after the decimal of the letters denoting the sex chromosomes (e.g. 46,XX or 46,XY). Table 2. List of major symbols used to describe results of cytogenetic tests. Abbreviation Meaning cen centromere p shortarm of chromosome q longarm of chromosome del deletion h heterochromatin ins insertion inv inversion t translocation i isochromosome r ring chromosome dic dicentric chromosome + gain ; to separate altered chromosomes and breakpoints Methods of chromosome visualization used in cytogenetics For the karyotype testing, cells are collected, the number, size and structure of the chromosomes are estimated; in the case of adults, these are mostly peripheral blood lymphocytes or bone marrow cells. In order to perform prenatal tests trophoblast cells are collected for cell culture from a layer of external cells of one of the membranes of the foetus for example from chorion or amniotic fluid cells. Chromosomes are stained using one of the classical methods listed below, lighter and darker bands appear, which differ in the degree of chromatin condensation: - darker bands, staining intensely – called "positive" bands. - brighter bands that stain less – called "negative" bands. The band pattern is characteristic of the chromosome,allows its identification and detection of changes in the chromosome structure.  Methods of classical cytogenetics(banding) –chromosome analysis in the metaphase stage, usually after their previous digestion with trypsin and staining. As a result of staining, an image of darker and brighter bands, characteristic of a given chromosome, is obtained. The most commonlyusedstainingmethodsinclude:  G-banding (GTG) – which stains regions rich in AT pairs;  R- banding (RBA) – reverse Giemsa staining, which stains regions GC-rich;  Q- banding (OFQ) – there is a strong fluorescence of the regions rich in AT pairs;  C- banding (CBG) – visualization of constitutive chromatin (regions around the centromeres);  T-banding(HRBT) – exposes telomeres.  Silver-stained nucleolar organizer regions (Ag-NOR) – allows toanalyse nucleolus organizer regions- regions of satellites; Detection of chromosome aberrations - methods of molecular cytogenetics These methods are based on techniques: Comparative Genomic Hybridization (CGH), fluorescent in situ hybridization (FISH)and the use of microarrays.  FISH (fluorescent in situ hybridization) Fluorescent in situ hybridization– the use of molecular probes labelled with fluorescent dye (rhodamine, fluorescein, coumarin) or isotope (radioactive phosphorus or sulphur). The application and practical use of the FISH method are presented in Tables 3 and 4. In order to study polymorphism/mutation and DNA rearrangement, methods based on DNA multiplication/amplification in a PCR reaction or variants thereof, and methods based on DNA hybridization for molecular probes are used. The study in which the FISH technique is applied involves the use of probes or short fragments of DNA with a known sequence and location on the chromosome. The probe is labelled with fluorescent (using rhodamine or fluorescein), it can also be a radioactive label, e.g. phosphorus or sulphur isotope. FISH is a cytogenetic technique to detect a specific DNA sequence in genetic material using fluorescent DNA probes. Fluorescence microscopy is needed to analyse the material being tested. Table 3. Application of the FISH method. Application Limitations - identification of additional genetic material (marker chromosomes) - evaluation of structural aberrations - detection of submicroscopic aberrations (sensitivity 50-150 kb) - rapid detection of aneuploidy/polyploidy in pre- and postnatal diagnosis -requires prior knowledge in the DNA sequence of the test - testing a few sequences at the same time does not provide information about non-tested sequences Human sex chromosomes (heterosomes, X and Y chromosomes) Chromosome X In terms of the size and position of the centromere (submetacentric), the X chromosome is similar to the chromosomes from group C. Numerous genes are located on the X chromosome, but most of them are not genes responsible for determination and sex differentiation. So far, 1094 features coupled to the X chromosome have been identified. The lenght of the DNA on the X chromosome is 1.8x108 base pairs (bp). Genes important for determination and sex differentiation are located on the long arms of the chromosome. These are the genes encoding nuclear and cytoplasmic protein receptors that mediate the response of target cells to testosterone and dihydrotestosterone (Figure 2). PAR1 and PAR2 – pseudoautosomal regions (also present on the Y chromosome, homologous regions) – thanks to them during meiosis in spermatogenesis, conjugation is possible and crossing over between genes can occur.Pseudoautosomal regions contain genes encoding traits inherited as autosomal traits (e.g., a gene encoding a structural tooth enamel protein). Figure 2.The X chromosome. The Y chromosome The Y chromosome is small, acrocentric, similar to chromosomes from group G (the size may vary in a wide range). It constitutes about 1% of total genomic DNA, the size of the Y chromosome is 58 Mbp. On the Y chromosome, 78 genes have been identified that include: - holandric genes (genes inherited only from father to son), ­ initiating the differentiation of the embryo towards the male sex, ­ encoding factors involved in the control of spermatogenesis, ­ tissue histocompatibility complex antigens (HY), ­ genes of basic metabolism. In terms of cytogenetics, two regions can be distinguished: heterochromatic and euchromatin. Constitutive heterochromatin contains repetitive satellite DNA; the euchromatin region includes the entire short arm, the pericentromeric region and a part of the proximal long arm.This region contains all previously identified chromosome genes Y (Figure 4). Very close to the PAR1 region is the SRY gene (Yp11.3, Sex determining Region of Y) determining the male sex (involved in the formation of testicles). This is the one gene known so far specific only for the Y chromosome. It is also a weak transplant antigen that can cause a male to reject a male transplant even within one inbred strain. The pericentromeric region contains a group of genes responsible for spermatogenesis. At the ends of both arms, there are pseudoautosomal regions PAR, which allow during meiosis to connect to the X chromosome and recombine, covering about 5% of the base pair of the Y chromosome. The remaining part, 95% of the base pairs, creates the region NRY – non-recombining region of Y. NRY is not recombined, therefore it is transmitted in a normally unchanged state from father to son - then both have the same profile of Y chromosome-related polymorphism, i.e. haplotype.The haplotype ofY chromosome differs between geographic regions following a different location of de novomutations. Therefore, its analysis is important in the process of determining the origin of humans (searching for Ychromosomal Adam) and migration routes. Figure 3. The Y chromosome. Chromatin X (Barr body) and Y chromatin (Y-body) In 1949, Barr and Bertram observed a darker papule of basophilic chromatin stained adjacent to the nuclear membrane stains in the interphase nuclei in females. This part of nuclear chromatin is called the Barr body. Barr bodies are present in the cells of almost all tissues. They do not occur in normal male cells (exception Klinefelter syndrome) and female germline cells.They are examined in the smears of the buccal mucosal cells (oral epithelium) and in the cells of the amniotic fluid. The standard is the presence of Barr bodies in 20-60% of the tested cell nuclei.In women, X chromatin is visible in 20 to 70% of cells, and the positive result (female chromatin sex) indicates the presence of Barr bodies in at least 20% of the testes. In the correct female cell 46,XX, one Barr body is present.Numerical aberrations of the X chromosome lead to a lack or increase in the number of Barr bodies, in these cases their number will always be one less than the number of X chromosomes.In neutrophilic granulates, after appropriate coloration, chromatin X is visualized in the form of thrown out of the nucleus – but still stay in touch with it - chromatin clot, so-called drummer's sticks. Chromatin Y (Y-body) -a distal, condensed, heterochromatic part of the long arms of Yq12 – visualized by the fluorescent method in the interphase cell nucleus (highly fluorescent body). The number of Y bodies is equal to the number of Y chromosomes. The Y-body is found in 30-50% of the studied male cells (including: sperm, lymphocytes, oral epithelial cells). Chromatin Y - like X - can be seen in neutrophils in the form of Y-drummer's sticks (fluorescent staining). Lyonisation The hypothesis of inactivation of the X chromosome was first formulated in 1961 by the English geneticist Mary Lyon.Currently known as the "Lyon law", the process of inactivation (condensation) of one of the X chromosomes is referred to as "lyonisation". The inactivationprocessis:  independent,  random,  indivertible. It is a compensatory mechanism which balances the level of expression of genes located on the sexchromosomes (the individual XX have the same number of active copies of genes linked to the X chromosome as the individual XY). The genes of the pseudoautosomal region and some genes occupying the proximal part of the long and short arms are not inactivated. Both X chromosomes are active at the early embryonic stage. Next, in female embryonic cells, about 16 days of embryonic life, there is random inactivation of one X chromosome. These changes are transmitted during mitotic divisions. Both X chromosomes can be inactivated with equal probability. Inactivation is a permanent process, affects the whole generation of a given cell, i.e. the same X chromosome remains inactive in all daughter cells.Thus, the woman's body in terms of the X chromosome is a mosaic consisting of cells containing an inactive X paternal chromosome (Xpat) or maternal (Xmat) chromosome. Lyonisationis the most spectacular example of epigenetic silencing.It is a process that includes the effect of different factors, including histone modification and methylation CpG sites (CpG islands)related to the gene promoter XIST. The inactivation centre (XIC), i.e. the place where the inactivation process is initiated, is located atXq13 band. The high level of gene expression XIST, substitution of histone H2A by macro-H2A and lysine methylation at position 9 and 27 of histone H3 leads to inactivation of the X chromosome. The X chromosome is reactivated during oogenesis. The study of chromatin X is the simplest indirect method of detection of numerical aberrations of chromosome X (Table 6). Until recently, it was commonly used in clinical diagnostics (determination of sex on the level of chromatin, bodily-sexual development disorders, diagnosis of numerical aberrations of the X chromosome) - currently replaced by other, more precise methods, e.g. FISH (similarly - for the Y chromosome). Table 4. The number of chromatin bodies in the diploid nucleus of the somatic cell. Sex chromosomes The maximum number of chromatin bodies in the diploid nucleus of the somatic cell X-body Y-body 45,X 0 0 46,XX 1 0 46,XY 0 1 47,XXX 2 0 47,XXY 1 1 47,XYY 0 2 48,XXXX 3 0 Inheritance of sex-linkedtraits Holandric inheritance - the transmission of the traits takes place only from father to son (Figure 5). Figure 5. Diagram of holandric inheritance. Inheritance of sex-linked recessive traits Table 5. Genotypes and phenotypes of recessive sex-linked traits. WOMEN MEN GENOTYPE PHENOTYPE XAXA Geneticallyhealthy XAXa Clinicallyhealthy, carrier XaXa ill XAY healthy Xa Y ill Table 6. Inheritance of recessive sex-linked traits. Healthyman x Healthywoman Genotype: XAY XAXa Gametes: XA Offspring: half of the sons are ill, half of the daughters are carriers Y Illman XA x Xa Healthywoman Genotype: Xa Y XAXA Gametes: Xa Y XA Offspring: all sons are healthy, all daughters are carriers  XA Hemizygous – man has only one X chromosome and only one copy of each gene linked to the X chromosome (Table 5).  Mainly hemizygous men get ill, heterozygous women with a mutant gene do not get ill (carriers).  The situation when both X chromosomes have a mutated gene occurs very rarely (homozygous) – only then do women get sick.  Female carriers (heterozygotes) of the mutated gene transmit it with the same probability (50%) to both daughters and sons (Table 6).  From the relationship of a female carrier (heterozygote) and a healthy man, 50% of sons will be sick and 50% healthy, among daughters, 50% will be carriers and 50% will be healthy.  An ill man (hemizygous) does not pass the disease to the sons, but all daughters will be carriers of the disease. Inheritance of sex-linked dominant traits  Hemizygous – man has only one X chromosome and only one copy of each gene linked to the X chromosome (Table 7).  Hemizygous men and heterozygous women both get ill.  Heterozygous women with a mutated gene usually have a mild form of disease, while hemizygous males severe.  Ill man (hemizygous) does not pass the disease on to his sons, but all daughters will be sick.  Sick women (heterozygotes) pass the mutant gene to both daughters and sonswith 50% probability (Table 8).  The mutant dominant gene (on the X chromosome) in men is often lethal. Table 7. Genotypes and phenotypes of dominant sex-linked traits. WOMEN MEN GENOTYPE PHENOTYPE XAXA ill, most common lethal genotype XAXa ill XaXa healthy XAY ill, often a lethal feature Xa Y healthy Table 8 Inheritance of recessive sex-linked traits. Illman x Healthywoman Genotype: XAY XaXa Gametes: XA Y XaXa Offspring: all daughters are ill, all sons are healthy, Genotype: Healthyman Xa Y Gametes: Xa Y Offspring: x Illwoman XAXa XA Xa 50% of daughters are healthy, 50% of daughters are ill, 50%of sons are healthy, 50% of sons are ill Recessive diseases linked with X chromosome Recessive sex-linked diseases Haemophilia Congenital blood coagulation disorders are caused by the lack of blood coagulation factors involved in the conversion of fibrinogen to fibrin.Molecularbasesaremutations in the genes:  F8C, locus Xq28 (factor VIII) – haemophilia A (HA)  F9, locus Xq27.1-27.2 (factor IX) - haemophilia B (HB) Frequency of occurrence: HA – 1:10 thousand.- 1:20 thousandof births; HB – 1:30 thousand of births. Clinically, haemophilia A and B do not differ from each other, symptoms appear in early childhood (Table 11). Patient mortality is twice higher than in men in the general population. Haemophilia A Usually, the disease has a more severe clinical courseif the cause is mutation, which results in the loss of a larger fragment of the gene or premature introduction of the stop codon to the sequence, because these changes lead to a shortening of the protein chain. Almost half of the identified causative mutations in severe HA are inversion mutations. Haemophilia B Most often (74% of all mutations), substitutions of single nucleotides are detected - missense mutations (75%) and nonsense mutations (11%), less frequently splicing mutations (11%) and others (3%).The remaining identified changes (26% of all mutations) in F9 are small ( 50 bp) gene rearrangements (4%). Table 9. Clinical forms of haemophilia A and B (References: Drewa G, Ferenc T. Genetykamedyczna [Medical genetics]). Clinical form, level of factor VIII or IX in plasma Mild Symptoms 0.05-0.40 IU/ml (5-40% norm) do not occur by itself Moderate Severe Prolonged bleeding - after minor injuries, operations, tooth extractions; Intra-articular bleeding– rare, medium severity Severe bleeding – as a result of injuries, micro-trauma, after XP2 similarly, the X-norm allele dominates over the deuteranomaly allele Xd1 , and this dominates over the deuteranopia allele Xd2 : X > Xd1> Xd2 Women can be doubly loaded by genes of abnormal colour vision. It has been found that if there is an abnormality gene for a different colourin every X chromosome, the woman sees correctly. Examples of genotypes and phenotypes are presented in Table 10. Table 10. Genotypes and phenotypes of colour vision. Genotypes Phenotype XP1 XP2 protanomaly Xd1Xd2 deuteranomaly XP2XP2 protanopia Xd1Xd1 deuteranomaly Xd1Xp2 correctvision X Xd1 correctvision Xd2Xp2 correctvision Examples of genetic cross: ♀ XP1Xd1 XX P1 XP1Y daughter son seeing correctly with protanomaly (carrier of protanomaly) x XY ♂ X X d1 Xd1Y daughter son seeing correctly with deuteranomaly (carrier of deuteranomaly) In rare cases, red-green axis vision disturbances may be caused by point mutations within the deutan gene. In North European populations, red and green colour vision disturbances affect about 8% of men and 0.5% of women, with deuteranomaly being 4-5 times more common than protanopia, protanomaly and deuteranopia. The disturbances of red and green colour vision caused by the abnormal amount of visual dyes are erroneously referred to as daltonism. Ishihara pseudoisochromatic tables (1917) - tables used to check the ability to distinguish colours - red and green: Ishihara full test - 36 tables; Screening versions - 14 or 24 tables. The test is carried out showing boards in daylight, within 3 seconds from a distance of 75 cm. On the 21 consecutive tables (1-21), the person correctly distinguishes the colour, 17 reads, and the persons with the disorder - 13. The next tables with the numbers 22-25 allow to distinguish protanopia from deuteranopia; the next 26-36 tables allow to distinguish amblyopia of red and green. References: 1. Drewa G., Ferenc T. Genetyka medyczna, Wydawnictwo Elsevier Urban & Partner Sp. z o.o., Wrocław 2011 2. Gardner D.G., Shoback D. Endokrynologia ogólna i kliniczna Greenspana, Tom II, Wydawnictwo Czelej Sp. z o.o., Lublin 2011 3. Campbell, J.B. Reece: Biology. Pearson, Benjamin Cummings, Seventh Edition 2005 4. Meder S., Windelspecht M.: Human Biology. McGraw-Hill Science/Engineering/Math, Twelfth Edition 2011 5. Jorde L.B.; Carey J.C.; Bamshad M.J.; White R.L.: Medical Genetics, Third Edition