Molecular Diagnostics & Cytogenetics Lecture 10 (2024) PDF

MLT552: MOLECULAR DIAGNOSTICS & CYTOGENETICS LECTURE 10: SEX CHROMOSOMES, X-CHROMOSOME INACTIVATION AND SEX CHROMOSOME ABNORMALITIES CHROMOSOME BREAKAGE AND INSTABILITY SYNDROMES. Ts. DR. MOHD FADLY MD AHID 17th DECEMBER 2024 LEA...

MLT552: MOLECULAR DIAGNOSTICS & CYTOGENETICS LECTURE 10: SEX CHROMOSOMES, X-CHROMOSOME INACTIVATION AND SEX CHROMOSOME ABNORMALITIES CHROMOSOME BREAKAGE AND INSTABILITY SYNDROMES. Ts. DR. MOHD FADLY MD AHID 17th DECEMBER 2024 LEARNING OUTCOMES At the end of this lesson, you should be able to: ▪ Describe the structure and functions of sex chromosomes. ▪ Explain the mechanisms of X-chromosome inactivation. ▪ Describe the types of sex chromosome abnormality. ▪ Define the term chromosome instability. ▪ Explain the mechanisms of chromosome instability. 2 SEX CHROMOSOMES ▪ In mammals, the gender of an individual is determined by the combination of sex chromosomes ▪ Female – 46,XX (homogametic) ▪ Male – 46,XY (heterogametic) 3 SEX CHROMOSOMES ▪ The distal region of the short arms of the X and Y chromosomes contains highly similar DNA sequences ▪ Pseudoautosomal region (PAR) – resembles crossing-over that occurs between autosomes ▪ X chromosome is larger than Y and has ~1400 genes compared to Y ~200 genes ▪ The X chromosome contains genetic information essential for both sexes; at least one copy of X is required ▪ A single Y, even in the presence of several X, still produces a male phenotype. The absence of Y results in a female phenotype 4 SEX CHROMOSOMES ▪ SRY (Sex determining Region of Y-chromosome) gene on the Y chromosome determines maleness ▪ encoded testis-determining factor (TDF) that triggers testes formation. ▪ testosterone produced by testes controls formation of male secondary traits. ▪ absence of SRY gene in females triggers development of ovaries, female characteristics. 5 HOMOGAMETIC vs HETEROGAMETIC ▪ Sex chromosomes are separated during meiosis into different gametes ▪ Human females produce all gametes with the same combination of chromosomes (X) = homogametic ▪ Human males produce gametes with two possible combinations of chromosomes (X/Y) = heterogametic ▪ Random fusion of gametes produces an F1 that is 1⁄2 female (XX) and 1⁄2 male (XY) 6 X-CHROMOSOME INACTIVATION ▪ Females have two copies of X while males have one. ▪ There are thousands of genes on the X chromosome but relatively few on the Y chromosome. ▪ The explanation for the fact that males survive with only one X chromosome while females have two involves a concept called dosage compensation. balances the dose of X chromosome gene expression in females and males. ▪ X-inactivation – one X-chromosome is inactivated to prevent cells from having twice (double-dose) of gene products 7 X-CHROMOSOME INACTIVATION ▪ X-inactivation occurs randomly in early embryonic development One cell may inactivate one X- chromosome from one parent & vice versa (50% chance) ▪ The inactivate X chromosome is visible as a Barr body (highly condensed chromatin) in the cell nucleus 8 Photomicrographs comparing cheek epithelial cell nuclei from a male that fails to reveal Barr bodies (right) with a female that demonstrates Barr bodies (indicated by an arrow in the left image). If one of the two X chromosomes is inactive in the cells of females, the dosage of genetic information that can be expressed in males and females will be equivalent. 9 X-CHROMOSOME INACTIVATION ▪ The inactive X has properties characteristic of heterochromatin, with late DNA replication in the S phase of the cell cycle and remaining condensed during interphase. ▪ Histone proteins associated with the inactive X are underacetylated, and the cytosines in the CpG islands are methylated. ▪ Techniques for detecting the inactive X have been based on the fact that it is late replicating. The most commonly used cytogenetic method involves the use of bromodeoxyuridine (BrdU). Newer methods for detecting the inactive X involve molecular techniques often using differential methylation analysis. 10 MECHANISM OF X-INACTIVATION ▪ A gene that controls X inactivation is XIST (X- inactive-specific transcript), located at the X- inactivation center (XIC) at band Xq13 ▪ Only inactive X expresses XIST gene. ▪ X-inactivation involves 3 steps: 1) Chromosome counting (determining the number of Xs in the cell) 2) Selection of an X for inactivation 3) Inactivation itself 11 MECHANISM OF X-INACTIVATION 1) Chromosome counting ▪ To ensure that inactivation only occurs in cells with two X chromosomes ▪ Involves the X-inactivation center (XIC) ▪ Requires the presence of at least two XIC sequences, one on each X chromosome 12 MECHANISM OF X-INACTIVATION 2) Selection of X ▪ Selection of an X for inactivation is made by the X-controlling element (XCE) in the XIC region ▪ There are different alleles of XCE, and each allele has a different probability that the X chromosome carrying it will be inactivated 13 MECHANISM OF X-INACTIVATION 3) Inactivation ▪ Mediated by XIST gene. ▪ XIST codes for Xist RNA (17-kb) – coats the chromosome to be inactivated and silences most of its genes. ▪ In active X, a blocking factor is produced in the cell to block the transcription of Xist. ▪ About 15% of genes on the X-chromosome escape inactivation and remain active to some degree on both X chromosomes in females. An additional 10% of genes show variable patterns of inactivation and are expressed to different extents from some inactive X chromosomes. Many more genes on Xp escape inactivation as compared to Xq. 14 NUMERICAL ABNORMALITIES OF SEX CHROMOSOME ▪ Common numerical abnormalities of sex chromosome: 1) 45,X (Turner syndrome) Sex chromosome aneuploidy 2) 47,XXY (Klinefelter syndrome) Caused by meiotic nondisjunction 15 SEX CHROMOSOME DISORDERS 1) 45,X (Turner syndrome) ) ▪ One of the X chromosomes is missing (monosomy X) or partially missing ▪ 1 in 2,500 live-born females ▪ One of the most common chromosome abnormalities in spontaneous abortions (~1–2% of all conceptuses) 16 SEX CHROMOSOME DISORDERS 1) 45,X (Turner syndrome) ▪ Clinical features of Turner syndrome may vary among girls and women with the disorder 17 SEX CHROMOSOME DISORDERS 2) 47,XXY (Klinefelter syndrome) Klinefelter syndrome karyotype (47,XXY) ▪ Presence of an extra chromosome X in male ▪ 1 in 575–1,000 newborn males ▪ The most common cause of hypogonadism and infertility in males 18 SEX CHROMOSOME DISORDERS 2) 47,XXY (Klinefelter syndrome) ▪ The extra X chromosome can affect physical, developmental, behavioural & cognitive functioning ▪ The clinical features of Klinefelter syndrome vary among boys and men with the disorder. In some cases, the features are so mild that the condition is not diagnosed until puberty or adulthood. 19 SEX LINKAGE ▪ The Y-chromosome lacks many genes found on its homologous X-chromosome. ▪ This leads to a pattern of inheritance called sex linkage ▪ In XX females, a recessive allele on one X can be masked by a dominant allele on the other X ▪ In XY males, a recessive allele on the X has no second copy to mask its effects ▪ Examples of X-linked disorder: Red-green colour blindness, Haemophilia, Duchenne muscular dystrophy, Vitamin D resistant rickets 20 WHAT IS CHROMOSOME INSTABILITY? ▪ Chromosome instability (CIN) – a type of genomic instability that refers to a higher-than- normal rate of missegregation of chromosomes or parts of chromosomes during cell divisions, leads to changes in both chromosome number and structure (numerical & structural chromosome abnormalities) ▪ Normal cells make errors in chromosome segregation in about 1% of cell divisions, whereas cells with CIN increase the error rate to 20% of cell divisions 21 CHROMOSOME SEGREGATION ▪ Mitosis is carefully choreographed to ensure that all sister chromatids segregate to opposite daughter cells ▪ Spindle assembly checkpoint (SAC) acts to maintain genome stability by delaying cell division to ensure that anaphase is triggered only after all kinetochores are bound to spindle microtubules ▪ Prior to the start of anaphase, the kinetochores must capture the microtubules of the spindle and connect the sister chromatids of each chromosome to the poles of the opposite spindle (amphitelic attachment) ▪ Once all chromosomes achieve proper bi-oriented attachments to spindle microtubules (amphitelic attachment), the SAC is inactivated, and chromosome segregation and cell division to proceed ▪ If the chromosomes are not correctly attached to the spindle (erroneous attachments), kinetochores activate the SAC network, which inhibits the initiation of anaphase and preserves the cohesion of the sister chromatid 22 MECHANISM OF CHROMOSOME INSTABILITY ▪ The mechanisms underlying CIN remain poorly understood but likely reflect dysfunctional chromosome duplication or segregation during mitosis ▪ Mechanisms include: 1. Kinetochore-microtubule attachment errors 2. Aberrant sister chromatid cohesion 3. Abnormal centrosome replication 4. Telomere attrition 5. Spindle assembly checkpoint (SAC) abnormalities 23 CHROMOSOME INSTABILITY SYNDROME ▪ The chromosome instability syndromes (formerly known as chromosome breakage syndromes) comprise a number of rare but distinct clinical entities ▪ Associated with an increased risk of development of malignancies ▪ Caused by defective DNA repair, cell cycle control or apoptosis ▪ Classic chromosome instability syndromes: 1) Fanconi Anaemia (FA) 2) Ataxia Telangiectasia (A-T) 3) Bloom syndrome 4) Nijmegen breakage syndrome 5) Immunodeficiency, centromeric instability and facial anomalies (ICF) syndrome 6) Robert syndrome 7) Werner syndrome 24 CHROMOSOME INSTABILITY SYNDROME 1) Fanconi Anaemia (FA) ▪ a rare genetic disorder characterised by diverse congenital anomalies and a predisposition to bone marrow failure and malignancy ▪ Half (50%) of FA patients have congenital anomalies include: short stature, skin hyperpigmentation (including café au lait spots), radial ray anomalies (ranging from bilateral absent thumbs and radii to unilateral hypoplastic thumb or bifid thumb), malformations of heart & kidney ▪ Bone marrow failure leading to progressive pancytopenia and predisposition to cancers (especially AML), is the major cause of death in FA patients 25 CHROMOSOME INSTABILITY SYNDROME 1) Fanconi Anaemia (FA) ▪ FA is caused by mutations in FANC gene. Genetic defects of FA proteins result in a failure of recognition of interstrand DNA cross-links and leave damaged DNA unrepaired ▪ FA is usually inherited in an autosomal recessive pattern ▪ 1 in 160,000 births, with a higher frequency in Ashkenazi Jews, the Roma population of Spain & Black South Africans. The carrier frequency in the Ashkenazi Jewish population is approximately 1 in 90 26 CHROMOSOME INSTABILITY SYNDROME 1) Fanconi Anaemia (FA) ▪ The definitive test for FA is a chromosome breakage test: Chromosome breakage is examined in short-term cultures of peripheral blood T-cell mitogen–stimulated lymphocytes in the presence of DNA cross-linkers, such as DEB (diepoxybutane) or MMC (mitomycin C) These agents lead to increased numbers of breaks, gaps, rearrangements & quadraradii in FA homozygote cells Normal cells are able to correct most of the damage and are not severely affected whereas FA cells show marked chromosome breakage 27 CHROMOSOME INSTABILITY SYNDROME 2) Ataxia Telangiectasia (A-T) ▪ Ataxia – poor coordination, Telangiectasia – small dilated blood vessels (“spider veins”) ▪ an autosomal recessive genetic disorder characterised by progressive cerebellar degeneration, oculocutaneous Ocular telangiectasia telangiectasias, immunodeficiency, chromosome instability, radiosensitivity & cancer predisposition ▪ 1 in 40,000 – 100,000 people worldwide ▪ A-T is caused by mutations in ATM gene. ATM gene encodes a protein that plays a role in regulating cell division after DNA damage Cutaneous telangiectasia 28 CHROMOSOME INSTABILITY SYNDROME 2) Ataxia Telangiectasia (A-T) ▪ A diagnosis of ataxia telangiectasia is made based upon a detailed patient history, a thorough clinical evaluation & a variety of specialized tests including blood tests, MRI & karyotyping ▪ Elevated spontaneous chromosome breakage has been observed in fibroblasts and peripheral lymphocytes from A-T patients ▪ For example, a high frequency of balanced rearrangements involving chromosomes 7 and 14 at loci for immunoglobulin and T-cell receptor genes is often seen in lymphocytes from A-T affected individuals R-banding karyotype of A-T patients 29  CHROMOSOME INSTABILITY SYNDROME Other types of chromosome instability: Chromosome 1 multiradial configuration G-banded and C-banded (insert) image Multiple siter chromatid from a patient with ICF syndrome of cells from a exchanges (SCEs) in a cell from a patient with Robert syndrome, patient with Bloom syndrome demonstrating premature centromere 30 separation (arrows) THANK YOU 31