Midterm - Cytogenetics.docx

**NON-MENDELIAN INHERITANCE PATTERNS** - Inheritance patterns that deviate from the fundamental laws of inheritance as stipulated by Gregor Mendel. - Non-mendelian may be influenced by factors such as the location of the gene, whether it is inside or outside the nucleus, location of...

**NON-MENDELIAN INHERITANCE PATTERNS** - Inheritance patterns that deviate from the fundamental laws of inheritance as stipulated by Gregor Mendel. - Non-mendelian may be influenced by factors such as the location of the gene, whether it is inside or outside the nucleus, location of the gene on the chromosomes, effects of the environment, and variation in the properties of the protein encoded by different alleles of the gene. NOT all genetic traits strictly follow the laws discovered by Gregor Mendel. Some variations can be observed in all animals including humans. Variations involving multiple genes/alleles: - Incomplete dominance - Codominance - Polygenic inheritance - Lethal Alleles - Pleiotropy A. **Incomplete/Partial Dominance** - A condition where during the heterozygous condition (Bb) the dominant allele DOES NOT completely overpower the recessive allele, therefore, there is a \"BLENDING\" of the traits. **Example \#1:** Incomplete Dominance in Four O\'clock Plants. **Example \#2:** In rabbits black fur is dominant over white fur. B. **Codominance** - A condition when during the heterozygous condition (Bb) the dominant allele DOES NOT completely overpower the recessive allele, so, both traits are seen at the same time. **Example \#1:** Rabbits **Example \#2:** Rhododendrons **Example \#3:** Humans C. **Polygenic Inheritance** - Inheritance pattern for traits that are controlled by more than one gene. Example is human height and skin complexion. Polygenic traits. The alleles of each gene have a minor additive effect on the phenotype. **Example \#1:** Skin color **Example \#2:** Height **Example \#3:** Eye Color D. **Lethal Genes** - Genes capable of causing death of organism carrying them. The gene responsible for pseudoachondroplasia (dwarfism) causes a dominant dwarfism but it is lethal when homozygous. **Example \#1:** Dwarfism - What is the probability of a dwarf child when a dwarf man marries a woman with normal stature? - Dwarf man genotype = Dd - Normal woman genotype = dd - Do a test cross **D** **d** ------- ------- ------- **d** Dd dd **d** Dd dd - If two dwarves marry? - Dwarf father genotype = Dd - Dwarf mother genotype = Dd - Do a test cross **D** **d** ------- ------- ------- **D** DD Dd **d** Dd dd **OTHER LETHAL GENES IN HUMANS** **Example:** - **Non-deadly lethal gene**: pattern baldness Male **androgenetic alopecia (AGA)** is the most common form of hair loss in men. It is characterized by a distinct pattern of progressive hair loss starting from the frontal area and the vertex of the scalp. E. **Pleiotropy** - When one gene affects multiple characteristics. **Pleiotropy** refers to the expression of multiple traits by a single gene. **Example \#1:** Sickle-cell disease **Example \#2:** Marfan Syndrome **OTHER NON-MENDELIAN INHERITANCE PATTERNS** - Extranuclear inheritance - Maternal ettect (also known as maternal influence) - Maternal inheritance (mitochondria/chloroplast) - Infectious heredity (cytoplasmic parasites) - Genomic imprinting - Mosaicism A specific phenotype is not controlled by genes on the chromosomes in the nucleus. **Maternal inheritance** (also called cytoplasmic inheritance or mitochondrial inheritance). - The transmission of traits through cytoplasmic genetic factors such as mitochondria or chloroplasts. - These cytoplasmic organelles are usually inherited with the egg\'s cytoplasm form the mother. **Maternal effects** - An individual\'s phenotype is controlled by gene products in the genome (e.g., proteins) of the mother (oocyte). - Non-nuclear DNA is often inherited uniparentally. - In humans, children get mitochondrial DNA from their mother (but not their father). Because of mutations in mitochondrial genes: There is **HETEROPLASMY** - A cell with mutant and normal mitochondria There is **HOMOPLASMY** - A cell has a uniform set of mitochondria: all completely normal mDNA or completely mutant mtDNA What is the characteristic of the cells/tissue that are frequently affected by mutations in the mtDNA? - Those that have high energy demand - CNS, the heart, and muscle (neuromuscular system) - Encephalopathy, myopathy, ataxia, retinal degeneration, loss of function of the external ocular muscles - LHON (Leber\'s Hereditary Optic Neuropathy) - MELAS (Mitochondrial Encephalomyopathy, Lactic Acidosis, and Stroke-like Episodes) **Mitochondria** have their own genome of about 16,500 bp that exists outside of the cell nucleus. Each contains 13 protein- coding genes, 22 tRNAs, and 2 rRNAs. **MATERNAL EFFECTS** - An individual\'s phenotype is controlled by gene products in the genome (e.g., proteins, mRNA) of the mother (oocyte). The phenotype of the individual is not determined by its own genotype but by the genotype of the mother. **Example:** Shell coiling in Lymnea peregra **INFECTIOUS INHERITANCE** - Refers to the inheritance of an agent that can be inherited with cytoplasm.Such agent may be intracellular bacteria or viruses. **GENOMIC IMPRINTING** - Imprints are formed due to the differential methylation of paternal and maternal alleles. This results in differing expression between alleles from the two parents. - **Methylated DNA** - low levels of gene expression - **Unmethylated DNA** - higher levels of gene expression **MOSAICISM** - Presence of two or more populations of cells with different genotypes in one individual who has developed from a single fertilized egg. - A result of mutations in the cells producing different populations of cells with different genes for a particular trait. **EXAMPLE 1:** Heterochromia iridum **EXAMPLE 2:** Cutaneous mosaicism **PEDIGREE ANALYSIS** - Determines if a trait is dominant, recessive, autosomal, or sex-linked. **PEDIGREE ANALYSIS/SYMBOLS** **Circle** = Female **Square** = Male **SYMBOL FOR TRAITS** **Clear Shapes** = Unaffected Individual **Shaded Symbol** = Affected Individual **Partial Shaded Symbol** = Heterozygous Individual for a particular trait. **SYMBOL FOR DECEASED** Diagonal line on top of the symbol. **SYMBOL FOR TWINS** **Horizontal Line** = Monozvaotic Twins - Identical twins **Slanted Line** = Dizyaotic Twins - Twins that do not look alike or they differ in sex **SYMBOL FOR ADOPTED** Bracket around the circle or square symbol. **SYMBOL FOR MISCARRIAGE** - **Triangle** - Sometimes information about miscarriage is placed on the symbol. **SYMBOL FOR UNKNOWN SEX** - **Diamond** - Happens when the mother is still pregnant and the baby\'s sex haven\'t been identified yet. These **symbols** are placed together to construct a pedigree. The **pedigree** describes what trait is present in the family and how is it passed down from one generation to another. **PEDIGREE ANALYSIS/SYMBOLS** **MARRIAGE LINE** - Horizontal Line that connects the parents. **SEPARATED** - If a diagonal line is present. **LINE OF DESCENT** - Vertical Line that connects the descent (offspring). **SIBLING LINE (SHIBSHIP LINE)** - Horizontal Line that connects the siblings. **GENERATION NUMBER** - Roman Numerals. - Each individual in each generation is label by numbers; \"1\", \"2\", \"3\", \... **When identifying individuals in the pedigree**, indicate the generation it came from (Roman numeral) and place a dash and add the individual\'s number in that generation. **PEDIGREE ANALYSIS/SYMBOLS** **Black Circle** = Living Affected Female **Black Square** Living Affected Male **Clear Circle** = Living Unaffected Female **Clear Square** = Living Unaffected Male **Symbols for miscarriage or abortion.** Notes about the pregnancy before the event of miscarriage or abortion are placed. **For pregnant women**, estimated delivery date is also placed. The interaction between alleles in heterozygotes may be: **DOMINANT** = One allele controls the phenotype. **RECESSIVE** = The recessive allele does not control the phenotype. **PEDIGREE: DOMINANT VS RECESSIVE** - **DOMINANT** - One of the parents must have the trait. - One, if not both parents have the trait. - Dominant traits will NOT skip a generation. - The trait is continuous in the affected family. - **RECESSIVE** - Neither parent is required to have the trait since they can be heterozygous. - The trait may escape/skip a generation in the affected family. - It is possible that the parents may not have the trait, but the trait reemerged in the children. **PEDIGREE: AUTOSOMAL VS SEX-LINKED** - **AUTOSOMAL** - Both males and females are equally likely to be affected. (Usually in equal proportions) - **SEX-LINKED** - Usually X-linked - Ex. In X-linked recessive traits, males are much more commonly affected than females. **X- LINKED RECESSIVE TRAITS** **Son** = Affected - In most cases, males are affected - the sons are affected. **Daughter** = Carrier - If the female will inherit the gene, usually they serve as carrier - especially if one copy only. - It is very rare to have homozygous recessive X chromosomes. - It is always possible that the male offspring will show- up the disease. **EXAMPLE 1** - **A Sex-Linked Trait** Mostly males are affected. - **A Sex-Linked Recessive Trait** Parents are not affected, most likely that the parents are carriers. It skips generations. **EXAMPLE 2** - **An Autosomal Trait** There is equal distribution of the trait to both sexes. It is not necessarily exactly 50:50 distribution but at least close to equal. - **An Autosomal Dominant Trait** Within the affected families, the trait does not skip a generation. **AUTOSOMAL DOMINANT** - One mutated copy of the gene in each cell is sufficient for a person to be affected. **AUTOSOMAL RECESSIVE** - Two mutated copies (Homozygous Recessive) of the gene are present in each cell. - Usually, 25% of the offspring will show the disease. - **25% of the Offspring** = Unaffected - **50% of the Offspring** = Could be heterozygous. (Carriers) **X-LINKED DOMINANT INHERITANCE** **Example:** Congenital Generalized Hypertrichosis **X-LINKED RECESSIVE INHERITANCE** - Hemophilia A & B - Bruton\'s agammaglobulinemia - Wiskott-Aldrich syndrome - G6PD deficiency - Ocular albinism - Lesch Nyhan syndrome - Dystrophy (Duchenne\'s and Becker\'s) - Hunter\'s Syndrome **For both X-linked Dominant and Recessive:** If mother is HOMOZYGOUS, ALL children will inherit one or two copies of the affected genes. **2^nd^ topic** **CHROMOSOMAL ABNORMALITIES** - Changes resulting in a visible alteration of the chromosome - Produced by: - Misrepair of broken chromosomes, - Improper recombination, or - Erroneous segregation due to (nondisjunction/anaphase lag) of chromosomes during mitosis or meiosis. **MEIOTIC NONDISJUNCTION** - Occurs when a homologous pair of chromosome fails to disjoin or separate during cell division. - The failure of the chromosomes to separate and migrate to opposite poles during meiosis / or meiosis Il, is the major cause of chromosomal abnormalities. **ANAPHASE LAG** - One homologous chromosome in meiosis or one chromatid in mitosis lags behind and is left out of the cell nucleus 0 this chromosome is lost (when the gamete is fertilized, you will produce) I one normal cell + one with monosomy. **Monosomy or trisomy** involving sex chromosomes or even more bizarre abnormalities, are compatible with life but are usually associated with variable degrees of phenotypic abnormalities. **Monosomy** involving an autosome generally represents loss of too much genetic materials to allow live birth or even embryogenesis, but a number of autosomal trisomies do allow survival. **NOMENCLATURE OF CHROMOSOMES** - Described according to the International System for Human Cytogenetic Nomenclature. - Somatic cells contain 46 chromosomes (22 homologous pairs of autosomes and 2 sex chromosomes, XX or XY). - Karyotype describe the chromosomal complement of an individual. - The total chromosome number is listed first, followed by the sex chromosomes, and numerical and structural abnormalities and band location in ascending order. - **46,2X**- Normal Female Karyotype - **46,XY** - Normal Male Karyotype **KARYOTYPE** - Standard arrangement of a photographed or imaged stained metaphase spread in which the chromosome pairs are arranged in order of decreasing length. **Karyotype** = total number of chromosomes + sex chromosome complement + description of abnormality - **p** = petit; short arm; **q** = long arm Each arm is divided into regions, regions into bands into sub-bands. **NOMENCLATURE OF NUMERICAL CHROMOSOMAL ABNORMALITY** - Triploidy: 69,XXX, 69,XXY, 69,XYY - Trisomy: 47,XX, +21 - Monosomy: 45,X - Mosaicism: 47, XXX / 46,XX **CODES IN STANDARD NOMENCLATURE** **ABBREVIATION** **MEANING** ---------------------- ---------------------------- ***cen*** Centromere ***del*** Deletion ***dic*** Dicentric ***dup*** Duplication ***ins*** Insertion ***inv*** Inversion ***i or iso*** Isochromosome ***mar*** Market chromosome ***Minus sign (-)*** Loss ***p*** Short arm of chromosome ***Plus sign (+)*** Gain ***q*** Long arm of chromosome ***r*** Ring chromosome ***rob*** Robertsonian translocation ***t*** Translocation ***tel*** Telomere ***ter*** Terminal end of chromosome **Interstitial deletion** - There are two breaks, and then the segment between the break gets detached and lost but the distal segment reattaches the chromosome fragment that was detached is lost during cell division. **Terminal deletion** - If you have a break, one break in the chromosome and the chromosome segment that is distal to the break get detached and do not reattach but is lost during cell division, that is terminal deletion. **Inversion** - 46,XY,inv(9)(p11q21.1) - There are 2 types: - **Pericentric inversion** - involves the centromere, involves both arms. - **Paracentric inversion** - occurs only in the same arm, involves only one arm (centromere is not involved in the centromere). **E.g.** 46,XY,inv(9)(p11q21.1) **Duplication** - 46,XX,dup (1)(q22q25) **Translocation** - 46,XY,t(4;9)(q21.2;22) **Ring chromosome** - 46,XY,(9)(p12q21) **TYPES OF CHROMOSOMAL ABNORMALITIES** Abnormalities based on cell chromosomal constituent: - Constitutional - Somatic or Acquired **Constitutional abnormality** - Found in all cells of the body - Occurs very early in development - Due to defect in sperm or egg, anomalous fertilization, or abnormal episode in early embryo **Somatic or acquired abnormality** - Present only in certain tissues or cells - The individual has cell types with different chromosome compositions - Mosaic - Chimera **Euploidy** - A state of an individuals with a normal set of chromosomes; no extra or missing; normal human somatic cells are diploid, with 2 sets of 23 chromosomes. - Numerical abnormalities results from gain or loss of whole chromosome. **TYPES OF NUMERICAL ABNORMALITIES** - **Polyploidy** with extra sets of chromosomes caused by fertilization of egg by \>1 or 2 sperm; not compatible with life. - **Mixoploidy** state of having 2 or more genetically different cell lines. - **Mosaic** occurs when you have/ with two cell populations originating from the same zygote. - **Chimera** these are those that arise from two different zygotes. **Aneuploidy** - with extra or missing chromosome **TYPES OF ANEUPLOIDY** - **Nullisomy** is a state of having a missing a pair of homologs occurring during the pre- implementation stage; lethal. For example, both of chromosome 6 are absent. - **Monosomy** is when one chromosome missing; occurs during embryonic stage lethal and abort in 99% of cases Exception, Monosomy X, Turner Syndrome (45,X). - **Trisomy** is a state where in three copies of a particular chromosome are present; gain of one chromosome; occurs during embryonic (1-2 weeks) or fetal development (9-36 weeks) stage; may be lethal; Exception, Trisomy 21, may survive or reach 40 y/o or more. **3^rd^ topic** **MOLECULAR TECHNIQUES** - Is a type of genetic test in which molecular genetic techniques are merged with the classical cytogenetics. - Ex. Fluorescence in Situ Hybridization (FISH) **Situ hybridization** - Indicates the localization of gene expression in their cellular environment. - A **labeled RNA or DNA** probe can be used to hybridize to a known target mRNA or DNA sequence within a sample. **CYTOGENETIC TECHNIQUES** **Fluorescent in situ hybridization (FISH)** - Allows much smaller changes to be seen. **Classical/Standard Cytogenetic Method** - Allows visualization of loss or gain of genetic material that contains at least 4 megabases of DNA. **CYTOGENETICS** - Study of the physical size and structure of chromosomes. **KARYOTYPE** - Complete set of chromosomes in the cells of an organism. - 23 Pairs of chromosomes - 22 Somatic Pairs - 1 Pair of Sex Chromosomes - Changes in the karyotype correlate with changes in the phenotype. **CLINICAL CYTOGENETICS** - Shows that Cytogenetic abnormalities may be found in: - Apparently normal individuals. - 2 Patients with phenotypic anomalies. - Patients with diagnosed genetic disorders. **Testing may be done during:** - Prenatal period - Postnatal period - Childhood and Adulthood **FORMS OF NEURAL TUBE DEFECTS** **CRANIORACHISCHISIS** - Completely open brain and spinal cord. **ANENCEPHALY** - Open brain and lack of skull vault. **ENCEPHALOCELE** - Herniation of the meninges and brain. **INIENCEPHALY** - Occipital skull and spine defects with extreme retroflexion of the head. **SPINA BIFIDA OCCULTA** - Closed asymptomatic neural tube defect (NTD) in which some of the vertebrae are not completely closed. **CLOSED SPINAL DYSRAPHISM** - Deficiency of at least two vertebral arches. **MENINGOCELE** - Protrusion of the meninges filled with CSF, through a defect in the skull or spine. **MYELOMENINGOCELE** - One spinal cord with a meninges cyst. **INDICATIONS FOR POST-NATAL GENETICS** - Presence if multiple congenital anomalies. - Suspected aneuploidy (e.g., Features of Down Syndrome). **INDICATIONS FOR ADULT AND CHILDHOOD GENETICS** - Unexplained mental retardation or developmental delay. - Suspected unbalanced autosome (e.g., Prader-Willi Syndrome). - Suspected sex chromosomal abnormality. - Suspected fragile - X syndrome. - Infertility - To rule out sex chromosomal abnormality as a cause of it. - Multiple spontaneous abortions - To rule out the parents as carriers of balanced translocations where both parents should be evaluated. **CHROMOSOME BANDING** - This will usually tell the number of chromosomes, the length, the location of centromeres, and banding patterns. **Chromosomes are treated with a dye that stains the DNA**. - **Euchromatin** = Lightly stained regions - **Heterochromatin** = Dark stained regions **G- BANDING (Giemsa Stain)** - G-banding technique uses the Giemsa stain. Most popular of all the banding techniques. **G-bands** - Dark bands - Heterochromatic - Rich in the base pairs: Adenine (A) and Thymine (T) **G negative bands** - Pale bands or Bright bands - Euchromatic - Guanine (G) and Cytosine (C) rich DNA **Q- BANDING (Quinacrine)** - Quinacrine bands uses fluorochrome stain and usually take on the same banding pattern as to the Giemsa stain. - The Q banding technique was the first one to be developed but because fluorescence is short-lived, it has been replaced by G banding technique. - **Dark bands** = Heterochromatin - **Pale bands** = Euchromatin **R- BANDING** - The reverse of Giemsa stain. - This technique may be used to detect deletion in the telomeres. - **Dark bands** = Euchromatic - **Light bands** = Heterochromatic **G banding** = Telomeres are light-stained. **R banding** = Telomeres have dark bands. **C- BANDING (C-BANDS)** - This technique stains the centromeres by treatment with an alkali solution and staining with Giemsa. - This sad on used to ide off sp citorososomes. - It also identifies the presence of dicentric chromosomes. **CHROMOSOME PREPARATION** **Cells in active division** - Natural cell division are made available by the use of cell culture with various mitogenic stimulating agents (phytohemagglutinin). **Metaphase plates are produced** - Mitotic arresting agent (colcemid). **Cells are subjected to hypnotic swelling (0.075M KCI)** - To further disperse the chromosomes within the cell and to lyse any red cell present. **Swollen cells are fixed** - Using the modified Carnoy's fixative. **Fixed cells are dropped on to a clean microscope slide** - Mechanically breaks the cell membranes and leaves the chromosomes separated slightly from each other. **Staining (aging at 65C, 30-60 min)** **Photomicroscopy** **Reporting** **MOLECULAR TECHNIQUES** **Nucleic Acid Amplification Technology** **Target amplification** - Usually use Single enzyme or multiple enzymes synthesize copies of target nucleic acid. **Polymerase Chain Reaction** - Most mature and widely used nucleic acid amplification method. **Probe amplification** - Amplification products contain a sequence only present in the initial probes. **Signal amplification** - The conc\'n of the probe or target does not increase, concin of label molecules attached to target nucleic acid increases; uses multiple enzymes and probes w/reduction of background noise to enhance target detection. **PCR** - Target DNA sequence - Two oligonucleotide primers - Flank the sequence to be amplified : Tomalenary to opposite strands of the target - A heat-stable DNA polymerase - An equimolar mixture of deoxyribonucleotide triphosphates (dATP, dCP, dGTP, and dTTP) - MgC12, KCI, and Tris-HCI buffer - Thin-walled PCR tube and thermal cycler **STEPS IN PCR** **Denaturation** (occurs 94-98°C for 20-30 secs) - Reaction mixture is heated to separate the two strands of target DNA. **Annealing** (occurs 50-65°C for 20-60 secs) - Cooled to permit the primers to anneal to the target DNA in a sequence-specific manner; polymerase enzyme binds to the primer- template hybrid and begins DNA synthesis. **Extension/Elongation** (72°C) - Dna polymerase synthesizes a new DNA strand complimentary to the DNA template in 5' to 3' direction. **RT-PCR** - Amplify ribonucleic acid (RNA) targets. - This involves Complementary DNA (cDNA) is first produced from RNA targets by reverse transcription, and then the CDNA is amplified by PCR. - This method is usually used in detection/quantification of hepatitis C virus (HCV) RNA and HIV-1. **RT (H)-PCR** - Target amplification and detection steps occur simultaneously in the same tube. - PCR product is detected as it is produced. - Flourescent dyes that preferentially bind to double-stranded DNA. - This method decreases the time required to perform nucleic acid assays because there are no post-PCR processing steps. **M-PCR** - Two or more/multiple primer sets are being used for amplification of different targets in the same reaction mixture. **HYBRIDIZATION ASSAYS/FISH** - PROBE - Utilizes probes which are well characterized fragments of nucleic acid - Large enough to hybridize specifically with its target - Can be applied to your chromosome spread **FISH** - Complements classical cytogenetic analysis - Used to form a diagnosis, to evaluate prognosis, or to evaluate remission of a disease, such as cancer - Numerical aberrations or translocations in chromosomes - Used to guide cancer treatment - Detection of infectious diseases caused by microbials and parasites **FLOW CYTOMETRY** - SPECIMEN (used for cytometry) - Whole blood - Bone marrow - Serous cavity fluids - CSF - Urine - Solid Tumors **FLOW CYTOMETRY IN HEMATOPOEITIC NEOPLASIA** - **Immunophenotyping** - **Major role** - classification of acute leukemias - **Neoplastic cells** express patterns of antigen that are distinctly different from those of their normal counterparts. **MEDICAL INDICATIONS FOR PERFORMING FLOW CYTOMETRY** **Diagnosis and classification of hematopoietic neoplasms** - Identify abnormal subpopulations of cells, assign lineage, and determine maturational stage **Assessment of biological parameters associated with prognosis** - CD38 or Zap-70 CLL - poor long-term survival indicator **Detections of Ags used as therapeutic targets** - CD33 in AML - anti-CD33 (gemtuzumab, ozogamicin) **Detection of residual neoplastic cells following therapy**