Genetic L8: Origins of Genetic Variation Chromosome Aberrations PDF

Genetic L8: Origins of Genetic Variation Chromosome Aberrations ● Explain the origins of chromosomal aberrations. A. Aberrations result from errors in: 1. Somatic DNA repair mechanism 2. Meiosis 3. Mitosis B. DNA repair mechanisms use either the opposite strand, sister chromatid, or homologous chro...

Genetic L8: Origins of Genetic Variation Chromosome Aberrations ● Explain the origins of chromosomal aberrations. A. Aberrations result from errors in: 1. Somatic DNA repair mechanism 2. Meiosis 3. Mitosis B. DNA repair mechanisms use either the opposite strand, sister chromatid, or homologous chromosome as a template for base pairing/polymerization or end joining after trimming C. Error/mutations in the proteins involved in cell reductive division, DNA duplication, and DNA repair result in mutations ● Describe the mechanisms of DNA repair and how meiosis is a highly regulated form of DNA damage followed by homologous recombination as the repair mechanism. 1. Under intense study and new discoveries driven mostly by goal of CRISPer-based gene repair (a) Mechanism and proteins involved not well understood (i) New names and abbreviations galore! (b) CRISPer is not very efficient at gene repair at present because of our poor understanding of DNA repair mechanisms (c) Mechanisms involved are dependent upon where in the cell cycle the damage is repaired and cell type 2. Any error in the repair processes can result in mutations (a) Structural mutations via double strand break repair (dsbr) mechanisms (b) Copy number variations, insertions/deletions, and point mutations possibly via dsbr (c) Higher resolution via excision repair in a single strand 3. All repair pathways involve five main steps (a) Recognition of damage via specific proteins (i) High affinity interaction based upon DNA damage type (b) Stopping the cell cycle (if a mitotic cell) no-matter where in the process (checkpoints) (c) Recruitment of repair enzymes to damage site and removal of damaged DNA (i) From a single strand (ii)From both strands 4. Main types of DNA damage occurs within a single strand or double strand breaks (a) Base damage caused by deamination of cytosine to uracil or methylation of guanosine (i) C>U involved in demethylation of DNA (b) Base mismatch or short indels resulting from errors in DNA polymerization (c) UV-induced 6-4 thymine dimers along a strand (d) Double strand breaks 5. Based damage, mismatch/short indel, and base crosslinking remove bases from one strand and use the complement as the template for the DNA polymerase (a) Base excision repair (BER) removes the modified base, cleaves out the backbone, fills in and ligates (i) Used to (eventually) convert mC to C via thymidine deglycosylase (TDG) (ii)Can have excision of stretch of nucleotides followed by filling back in and ligating (long stretch BER) (b) Nucleotide excision repair (NER) occurs at cross-linked bases and excises a long stretch at the repair site (c) Mismatch/short indel repair (MMR) occurs at base mismatches and short insertions or deletions (i) Involves additional step of strand recognition for selection of which strand to excise 6. Double strand break repair (a) Breaks can occur during DNA replication and transcription (b) More frequent cell division, more likely to occur > cancer cells (c) Breaks can be induced by ionizing radiation and topoisomerase II inhibitors (i) Topoisomerases involved in untangling DNA during replication and transcription (d) Repair processes called classical non- homologous end joining (C-NHEJ), alternative non-homologous end joining (A-NHEJ), single- strand annealing (SSA) and homologous recombination (HR) (e) Repair pathway choice related to extent of resection of DNA at break point, chromatin context, and cell cycle phase (i) Greater resection favors A-NHEJ (aEJ), SSA (f) Classical non-homologous end joining (i) Process the breaks to allow ligation —>May remove some bases giving deletions (ii) Join blunted ends —-> Can join non-homologs if multiple chromosomes are broken to give structural aberrations (g) Alternative non-homologous end joining (micro-homology end joining) (i) One strand on each end trimmed (5 > 3 end resection) (ii)Annealing of micro-homologous regions Can join non-homologs if multiple chromosomes are broken (iii) Filling in of missing regions (iv) Ligation at end (h) Single stand annealing (i) Resect single strands to homologous overhang regions that can anneal (i) Homologous recombination (i) A diploid advantage (ii) Digest and leave 3’ overhang for template in broken chromosome (iii) Use homolog (in meiosis) or sister chromatid (interphase S/G2) as template for repair of damage strands →Homologs not necessarily in proximity for use as the template (iv) Does not result in cross-over (v) Can reduce heterozygosity when use the homolog →Region of repair is homozygous with template (j) RNA mediated repair suggested by some recent research (i) Various forms of RNA may be involved in regulating the overall process New name for RNAs involved (both long and short versions) Associated with transcription-mediated DNA breaks (ii) May be able to use mRNA transcript as template for RNA-directed DNA polymerase mediated repair →Suggested as a mechanism of repair for Go/G1 cells such as neurons and skeletal muscle cells (iii) mRNA transcript can be from other allele or made prior to damage 7. General features of double strand break repair (a) Repair pathways are turned off during mitosis (b) NHEJ (and derivatives – non-HR) is most common pathway throughout interphase (c) (NHEJ (and derivatives) more likely to result in structural variants and other small scale variants (d) HR (at least using DNA as the template) is favored during late S and G2 (i) Have sister nearby to serve as template (ii) Least error prone repair mechanism E. Cell Replication/division errors 1. (a) (b) (c) Most invaolve non-disjunction errors that occur during metaphase > anaphase Kinetochore attachment errors Cohesin errors Gives daughter cells with extra or missing chromosomes 2. Can involve errors in centromere replication (failure of or an additional) 3. Mitotic error either in somatic cells or gametogenic (germ line) cells (oogonia or spermatogonia) (a) Somatic cells: (i) Mosaics > earlier in development potential for greater phenome effects (ii)Cancer (b) Germ cell mitotic > gamete errors monosomic triosomic aneuploids 4. Meiosis error in gametes F. Meiosis review 1. Meiosis is a form of evolved DNA damage followed by repair to shuffle the genes between homologous chromosomes 2. Reductive cell division with one 2n (diploid) cell dividing two times without DNA synthesis between cell divisions to give four 1n (haploid) cells (nuclei) 3. Shuffles the genes between the maternal and paternal chromosomes via homologous recombination/crossing over 4. Shuffles combination of chromosomes via random G. A bit more molecular about meiosis 1. Evolved from homologous recombination (HR) repair (a) Designed to allow crossing over because of proximity and co-linear alignment of homologs at synaptonemal complex (b) Can result in non-cross over repair and thus loss of heterozygosity (i) Use other homolog sequence to repair 2. Sister chromatids linked on the same chromosome scaffold instead of individually as in mitosis 3. Makes double strand breaks and uses HR for repair to give crossover (a) Breaks thought to be restricted to specific regions instead of random (i) Synapsis hot-spots 4. Protein complexes link homologs together during synapsis (a) Non-sister homolog linkage favored (b) Positions homologs for processes 5. Homologous recombination can result in crossover and non-crossover between homologs (a) Usually only 1 sister of each homolog involved in cross-over (i) Maximum crossover frequency is 0.5 since only involves one sister of each homolog (b) Crossover results in swapping segment between break points or break point and end (i) Forms recombinant gamete from non-homologous sister chromatids with crossover (c)Non-crossover of homologous sisters do not swap regions (i) Forms non-recombinant gamete (d) Non-crossover may result in a small segment within one homolog having the sequence of the other (i) Results in gene conversion (converts one parental allele to the other parent) 6. After HR, linked region remains linked (chiasmata) but eventually 7. Kinetochore of sister fuse to form one giving separation of homologs instead of sisters as in mitosis H. Endometriosis results in polyploidies not directly associated with disease 1. A form of aneuploidy but not directly lethal and may be advantageous under some circumstances (a) Involved in evolution of most advanced animal and plants (b) Gives a double (or higher) dose of genes (c) Extras develop as variants with beneficial effects 2. Failure of karyokinesis and cytokinesis (mitosis) giving whole genome duplication 3. Can form multiple nuclei or one large nucleus and a large to very large cell 4. Can be repeated multiple times to give numerous nuclei/genomes per cell 5. Found in some terminally differentiated cells (a) Hepatocytes and cardiac myocytes can be bi- nucleate (b) Megakaryocytes can be up to 64N (as one BIG nucleus) (c) Trophoblast of the developing placenta 6. Endomitosis (polyploidation) can result in aneuploidies that do cause disease (a) May initiate cancer genomic instability (chromosomal and structural aberrations) (b) Can eb mediated by excess centrosomes resulting in mis-segregation of chromosomes ● Compare and contrast different methods used to analyze human chromosomes. A. Uses cytogenetics/molecular cytogenetics and sequencing B. Traditional staining cytogenetics has 2-10 Mb resolution so can only detect large changes in chromosome structure C. Molecular cytogenetics can give higher resolution but not at SNP level 1. Good for smaller scale repeats (copy number variants), insertions, and deletions beyond the resolution of traditional cytogenetics D. Whole genome sequencing (WGS) or targeted sequencing and comparison to reference genome 1. Alignment based method 2. What is the “reference” or non-diseased genome (a) Big problem with whole genome comparison (b) Less of an issue when you can target the likely region or use other information to restrict the region of interest 3. 4. 5. 6. Average sequence of multiple nuclei so may not detect mosaics Does have SNV/SNP level resolution Difficult to detect duplications and sequence repeats when using short read seq. May miss insertions E. Traditional approach is phenome > karyotyping > molecular cytogenetics (chromosomal microarray and others) > sequencing or WGS for congenital dysmorphias 1. Moving towards phenome > chromosomal microarray > WGS or single molecule sequencing and imaging F. Generalized chromosome structure from metaphase spread 1. Centromere (a) Acrocentric (b) Submetacentric (c)Metacentric 2. Telomeres 3. Arms (a) P (petite) short (b) Q long G. Cytogenetic analysis 1. Relatively simple unbiased method to detect large changes in either chromosome number, size or intra-chromosomal insertions or deletions 2. Involves preparation of a metaphase spread (a) Isolate cells, induce proliferation, and arrested in mitosis for cell population enriched in metaphase chromosomes (b) Lyse cells, prepare spread/smear, fix (c)Further processing or directly stain and image 3. Use different DNA stains the give a banding pattern related to AT or GC rich regions (a) G (Giemsa) staining for G-banding (i) Treat spread with trypsin to enhance staining (ii)AT rich (gene pore) stains more densely (iii) GC rich (gene rich) stains less densely (b) Q staining for Q-banding (i) Quinarcrine fluorescent dye (ii)Banding pattern similar to G ( c) R staining (i) Reverse of G-staining in terms of banding pattern (d) Pattern of bands reproducible (akin to a bar code) with the standard having 500 band resolution ● Compare and contrast the structural variation of human chromosomes and be able to differentiate between autosomes and sex chromosomes. 1. Autosomes named based upon length with 1 being the longest, 22 being the shortest, and the sex chromosomes 2. Complete patient karyotype description includes: (a) Chromosome number (i) Aneuploids note specific chromosomes additions (+) or deletions (-) (ii)Chromosome number can be haploid (23), diploid (46), triploid (69) or tetraploid (92) (b) Sex chromosomes (c) Chromosomal aberrations (d) Clones (cells) analyzed in cancer diagnostics because of potential diversity in population or for mosaics ● Describe the different types of numerical and structural aberrations and how they are coded for in a karyotype. 3. Structural aberrations note chromosome, chromosome position, and aberration and possibly other details 4. Chromosome position based upon: (a) Arm of segment (b) Band regions within an arm has name based upon distance from centromere (ii) 1, 2, … (c ) Higher resolution banding gives sub-bands within region increasing with increasing distance from centromere (i) Centromere is 1 0 (one zero – not ten) (ii) 11.22 is band 1 sub-band 22 (.22) (iii) pter and qter for telomere ends (d) Example locus at 7p14.1 (i) Chromosome 7 (ii) P arm (iii) Region 1 5. Diagram of chromosome called an ideogram 6. Other descriptors based upon ISCN (International System for Human Cytogenetic) nomenclature (a) Punctuation used for further details about karyotyping procedures (b) Abbreviations used to describe (some) cytogenetic aberrations (i) Derivative (structurally rearranged chromosome with intact centromere): der (ii) Deletions: del (iii) Dicentric: dic (iv) Duplication: dup (v) Insertion: ins (vi) Inversion: inv (vii) Ring: r (vii) Translocation: t I. Patient chromosomal karyotype determined by staining, imaging, and analyzing for autosome number, sex, and chromosome aberrations 1. Example description of patient karyotype (a)46,XX,t(1;22)(q25.1;q13.2) (i) 46 chromosomes (44 autosomes + sex chromosomes) (ii) Female (iii) Translocation (reciprocal) involving chromosome 1 and 22 - q25.1 of chromosome 1 - q13.1 of chromosome 22 J. Cell-lines (transformed) and cancer cells have many aberrations 1. Cell-lines can mutate during culture (and repeated culture) giving somewhat of a moving target (a) Enhanced mutation rate over primary culture (that’s why they are transformed! 2. Cells within cancerous tissue can have heterogenous karyotypes (a) Typically karyotype several clones from the tissue (b) Enhanced mutation rate and ability to evolve and develop drug resistance ● Summarize how cytogenetics can be used to identify different human diseases. A. Use DNA probes and hybridization to dramatically improve resolution potentially to single nucleotide level B. FISH methods can use either metaphase or interphase chromosomes 1. Use probes to sequence/segments of interest (a)Highly flexible diagnostic tool by designing probes to specific sequence 2. Generally directly labeled probe 3. Ability to do interphase cells speeds the process 4. Can detect differences in the level/number of segments for detection of repeats 5. 5) Example probes: (a)Centromere repeats (b)Telomeres (c) Unique regions for each chromosome (chromosome painting) (d) Specific chromosome (e) Gene of interest FISH methods can use either in metaphase or interphase chromosomes 6. Aberration type (structural or numerical) dictates whether interphase or metaphase chromosomes are used (a) Metaphase spread for: (i) Inversions (ii) Translocations (iii) Ring (b) Interphase for: (i) Insertions (ii) Deletions (iii) Chromosome number (iv) Dicentric C. Virtual karyotypes (aka chromosomal microarrays/comparative genomic hybridization) are a large microarrays spanning the whole genome, uses bait along the chromosome and known mutational hot spots 1. Array bait (sDNA) based upon OMIN (Online Mendelian Inheritance in Man) data using common disease variants (a) 1K to 1000K bait probes 2. Use total fragmented DNA (individual’s genome and a normal sample) for catch (a) Compare signal to normal on Y-axis of plot (i) O = same as (ii) +1 = extra copy (trisomy) (iii) -1 = deletion (loss of heterozygosity) (b) X-axis is chromosomes of genome starting at chromosome 1 and ending at sex chromosomes 3. Cell-to-cell genomic heterogeneity a challenge (a) Cancers (b) Mosaics 4. Kit-based assay for reproducibility 5. Disadvantage of what is on the array (get what you are looking for or what is known) D. Whole genome sequencing using second (next) generation (short read) sequencing 1. Detects chromosomal aberrations at single nucleotide level 2. Can detect aberrations that are too small for karyotyping and not detected by arrays such as balanced translocations 3. Allows further characterization of anomalous junctions/break points and information on the mechanism 4. A study of balanced chromosomal translocation found. (a) Could resolve known karyotype aberrations at single base resolution (b) Many different mechanisms involved in formation (c) Chromosome 5 is a hot-spot for translocations (d) Many are associated with mental disorders (e) Break points happen intragenic and in “positional” regions of the genome (i) Positional = cis or trans DNA elements involved in transcriptional regulation E. Single molecule optical mapping of dsDNA (BioNano Genomics) 1. High resolution form of cytogenetics 2. Isolate high molecular weight DNA (very long ds fragments at greater than 150 kbp) 3. Label with probes at known locations along the fragments (a) Essentially along known parts of a chromosome 4. Linearize labeled fragment in nanochannel as a long ssDNA strand 5. Image strand to find labeled fragments 6. Analyze distance between labels to determine location along strand (a) Distances unique to regions of the genome (b) Insertions or deletions detected by distance difference relative to control samples 7. Detects structural aberrations at > 500bp resolution (a) Depends on the type of structural aberration F. Indications for chromosome analysis (karyotyping and/or comparative genomic hybridization) 1. 2. 3. 4. 5. 6. 7. Multiple congenital abnormalities Presence of dysmorphic features Unexplained disability and neurodevelopmental disorders Sexual ambiguity or disorders of sexual development Infertility Recurrent miscarriage Unexplained stillbirth 8) Malignancy and chromosome breakage syndromes Genetics L8: chromosome copy number and effects A. Chromosomal number aneuploidies (numerical aberrations) result from errors in chromosome separation 1. Sister chromatid non-disjunction in mitosis (a) Deviations from disomy of monosomy and trisomy or higher somy for individual chromosomes or multiple chromosomes 2. Homolog non-disjunction in meiosis I and sister non-disjunction in meiosis II (a) Deviations from gamete monosomy of nullisomy or disomy or up to tetrasomy 3. Can be constitutional or mosaic/acquired (a) Constitutional: all cells have aneuploidy (i) Meiotic origin or mitosis at zygote (1 cell > 2 cell) stage (b) Mosaic/acquired: some cells have aneuploidy (i) Mitosis during early development (> 2 cell stage) (ii) More difficult to detect because of dilution of normal and mosaic (i.e. cannot look at just one cell or clone) B. Chromosome abnormality percentages are 10% in spermatozoa and 25% in oocytes with most resulting zygotes being lost during early pregnancy with 50% of spontaneous abortions resulting from these numerical aberrations C. Human aneuploidies observed in infants mostly in sex chromosomes and chromosome 21 1. Other aneuploidies result in miscarriage/death of cells and for chromosome 13 and 18, death of the infant 2. Observed are likely the result of chr 21 being gene poor and/or genes where dose is not important or because of gene inactivation mechanisms as for the X-chromosome D. Types of female sex chromosome number aneuploidies 1. Generally less effect on phenotype than male sex aberrations because of X-chromosome inactivation (a) Increasing X generally increases phenotypic effects 2. XO (Turner syndrome): short stature, reduced sexual development and most infertile (a) Can be mosaics and thus less effected 3. XXX (triple X): generally taller, normal sexual development and reproductive capabilities 4. XXXX (tetrasomy X): dentition defects, mild cognitive impairment, developmental delays, generally taller E. Types of male sex chromosome aneuploidies 1. Generally greater effect on phenotype than in females especially with excess Y 2. XXY(Klinefelter syndrome): hypogonadism, low testosterone and delayed puberty, may have low sperm count 3. XXXY: hypogonadism, developmental delays, infertility 4. XXYY: hypogonadism, low testosterone, developmental delays, tall stature, infertility, learning disabilities 5. XYY: minimal to some effects on phenotype, taller, possible learning disabilities and behavioral abnormalities F. Types of autosomes aneuploidies 1. Polyploidy (greater than diploid) embryonic lethal so not found in human population (a) Can have in some populations of cells in differentiated cells 2. Possible with all autosomes but most are embryonic lethal 3. Can be constitutional or mosaic/acquired (a) Constitutional show the greatest disease/syndrome phenotype (b) Mosaics typically have reduced disease phenotype to no disease phenotype (i) Phenotypes of skin pigment abnormalities, developmental delays and asymmetrical anatomy 4. Non-disjunction more common in oocytes than sperm Types of autosomes aneuploidies 5. Trisomies observed for chromosome 13, 18, and 21 in live births (a) Trisomy 13 (Patau syndrome): many physical abnormalities and typically only survive less than one week (b) Trisomy 18 (Edwards syndrome): low birth weight, anatomical abnormalities, typically survive less than one month (c) Trisomy 21 (Down syndrome): survive into adulthood into 50s, intellectual disabilities, short stature and characteristic facial features (i) 95% trisomic, 4% via translocation of part of 21, 1% mosaics (trisomic/disomic 21) 6. Other autosomal aneuploidies are not observed as they are reproductively lethal (a) Failure of gamete development (b) Fertilization incompetent (c) Lost prior to implantation into the uterine wall (d) Cause miscarriage early in pregnancy —----------------------------------------------------------------------------------------------------------------- A. Structural aberrations occur from errors in double strand break repair either in somatic cells or crossing over in meiosis one B. Types of structural aberrations are: 1. Deletions: loss of a segment of a chromosome 2. Duplications: serially duplicate segments of a chromosome 3. Inversions: invert segment within a chromosome 4. Rings: chromosome fuses at ends or at breaks near the ends, typically occurs after removal of telomeres by end breaks (a) Ends normally protected from fusion by telomere-associated protein complex called shelterin 5. Isochromosomes: two identical arms instead of unequal p and q resulting from ds break within centromere 6. Dicentric: fusion of two chromosome segments both containing centromeres, likely lose genetic material in centromere-lacking segments, typically unstable/breaks during anaphase/telophase 7. Insertions: segment removed from one chromosome and inserted into a different chromosome 8. Translocations: parts of chromosomes translocated between different chromosomes (a) Reciprocal translocation: segments swap between chromosomes (b) Robertsonian translocation: entire long arms of two chromosomes fuse and is found mostly in acrocentric chromosomes (i) Most common translocation (ii) P-arm fragment lost reducing karyotype to 45 (iii) Have fertility issues resulting from disomic or nullisomic gametes C. Structural aberrations can be balanced or unbalanced 1. Balanced: no extra or missing segments (a) Inversions, insertions, translocations, isochromosomes and rings (b) Generally milder phenotypic effects resulting from different 3D position of gene in relation to transcriptional regulation (“position effects”) (c) Meiosis can be problematic with inversions causing loops and translocations giving more than two homologs paring 2. Unbalanced: extra or missing segments (a) Duplications, deletions, and dicentric (b) Generally abnormal phenotypes because of missing genes or gene dosage effects for duplications (c) Exception are loss or duplication of gene-poor regions or highly duplicated genes D. Structural aberrations that cut or cut and paste can result in mutations resulting from formation of a new sequence variant at the cut/ligation or cut and paste sites E. Large number of possible aberrations and extent of effects 1. Frequency estimated at 1/400 for all different types of structural aberrations 2. Most do not cause disease but may impact reproduction (a) Meiotic errors that occur during gametogenesis via incorrect pairing of homologous regions (i) “Non-homologous” pairing involving homologous regions on different homologs F. Some examples 1. XX male syndrome: translocation of part of Y onto X chromosome, phenotype includes ambiguous/intersex gender phenotype but mostly male biased 2. Cri du chat: deletion of the p-arm of chromosome 5 (5p-), infant’s cry sounds like a cat, delayed development, small head, distinctive facial features, intellectual disability H. A combination of both a numerical and structural aberration of pigmentary mosaicism and ambiguous biological sex resulting from trisomy 14 and der(Y)t(Y;14) translocation (Romero et al. Human Genome Variation (2020) 7:28 ) 1. Young patient presented with slowed growth, hypotonia, pigmentary mosaicism, ambiguous genitalia, no visible testicles 2. Ultrasound used to clarify biological sex showed a vaginal channel, and a right testicle in the inguinal canal (failure to descend) 3. Initial karyotype of 45,X[80]/46,XX[20], subsequent karyotype gave 45,X[24]/ 46,X,der(Y)t(Y;14)(p11.32;q12) 4. Molecular cytogenetics via FISH showed the SRY and several other genes on the derivative chromosome 5. Whole genome sequencing could not resolve NHEJ junction of derivative chromosome likely because of low frequency of derivative chromosome (a) Sequencing depth not good enough 6. Mosaic for Turner syndrome, no imprinted genes in the derivative chromosome, low level of trisomy 14 region is compatible with life 7. Likely mechanism involved paternal meiotic translocation of most the Y chromosome onto the p region of chromosome 14 giving a disomic gamete followed by fusion with a monosomic egg and disomic rescue during early zygotic development to give mosaicism