MMD Lecture 12 Medical Genetics Fall 2023.pptx

Molecular Biology and Medical Genetics Genetics • Is the study of biologically inherited traits determined by elements of heredity that are transmitted from parents to offspring in reproduction – AKA genes Gregor Mendel • Born in 1822 in Czechoslovakia. • Became a monk at a monastery in 1843. •...

Molecular Biology and Medical Genetics Genetics • Is the study of biologically inherited traits determined by elements of heredity that are transmitted from parents to offspring in reproduction – AKA genes Gregor Mendel • Born in 1822 in Czechoslovakia. • Became a monk at a monastery in 1843. • Taught biology and had interests in statistics. Basic physical and functional unit of heredity • Genes • Billions of nucleotides in the nucleus of a cell are organized linearly along the DNA (deoxyribonucleic acid) double helix in functional units . Most DNA is located in the cell nucleus on chromosomes – A small amount can also be found in the mitochondria (mitochondria DNA or mtDNA) Chromosomes are matched in homologous pairs • In humans, somatic cells have 46 chromosomes forming 23 pairs of homologous chromosomes. Chromosomes are matched in homologous pairs • Homologous chromosomes are matched in • length • centromere position • staining pattern • A locus (plural, loci) is the position of a gene. • Different versions of a gene (alleles) may be found at the same locus on the two chromosomes of a homologous pair. Chromosomes are matched in homologous pairs • The human sex chromosomes X and Y differ in size and genetic composition. • The other 22 pairs of chromosomes are autosomes with the same size and genetic composition. A karyotype is a photographic inventory of an individual’s chromosomes • A karyotype is an ordered display of magnified images of an individual’s chromosomes arranged in pairs. • Karyotypes • are often produced from dividing cells arrested at metaphase of mitosis • allowing for the observation of • • • • homologous chromosome pairs, chromosome number chromosome structure Therefore, karyotypes can render information on chromosomal abnormalities Tetrads Centromere Sister chromatids Pair of homologous chromosomes Sex chromosomes Changes/variability and Abnormalities • Modifications at level of chromosome • Phenotypic variations result from changes of individual genes (mutations) • Chromosome aberrations • • • • Total number of chromosomes vary Deletions Duplications Rearrangements Variations in Chromosome Number Variations from two haploid sets • Chromosome aberrations • Change in total number of chromosomes • Deletion or duplication of genes/segments of chromosome • Rearrangements of genetic material within or among chromosomes Aneuploidy • Aneuploidy • Variations in chromosome number • Individual may gain or lose one or more chromosomes (not entire set) • Monosomy: Loss of single chromosome in diploid genome • Trisomy: Gain of single chromosome Mechanism: Nondisjunction • Nondisjunction • Gives rise to chromosomal variation • Paired homologs fail to disjoin during segregation • Nondisjunction during meiosis I or II Normally, homologous chromosome segregate in meiosis I and sister chromatids in meiosis II Meiosis I Meiosis II Monosomy and Trisomy Result in a Variety of Phenotypic Effects Monosomy • Monosomy • Loss of one chromosome • Produces 2n 1 complement • Although one copy remains, can be lethal, organism is not viable • Monosomy unmasks recessive lethal • Haploinsufficiency: When one copy is not sufficient for organism to survive Trisomy • Trisomy • 2n 1 chromosomes • Addition of chromosome produces more viable organisms • Trisomies for autosomes are often lethal Trisomy 21 – Down Syndrome • Down syndrome: Trisomy of chromosome 21 • Extra chromosome (three number 21s) • Has 12 to 14 characteristics • Affected individuals express 6 to 8 on average An extra copy of chromosome 21 causes Down syndrome • A person with trisomy 21 has a condition called Down syndrome • Produces a characteristic set of symptoms, including characteristic facial features short stature heart defects susceptibility to respiratory infections, leukemia, and Alzheimer’s disease, and • varying degrees of developmental disabilities • • • • • The incidence increases with the age of the mother. 47, XX, +21 DSCR • DSCR: Down syndrome critical region • Critical region of chromosome 21 • Genes are dosage sensitive • Responsible for many phenotypic-associated syndromes Origin of Extra 21st Chromosome • Origin of extra 21st chromosome • • • • Nondisjunction of chromosome 21 during meiosis Homologs do not disjoin in anaphase I or II Leads to n 1 gametes Ovum is source of 95% of trisomy cases • Translocation of the DSCR to another chromosome • Shows increased incidence with increasing maternal age © 2017 Pearson Education, Ltd. Diagnostic Testing • Diagnostic testing • Amniocentesis or chorionic villus sampling (CVS) • Fetal cells obtained from amniotic fluid or chorion of placenta • NIPGD: Noninvasive prenatal genetic diagnosis • Fetal cells and DNA obtained from maternal circulation Human Aneuploidy • Patau syndrome (trisomy 13) • Edwards syndrome (trisomy 18) • Both trisomies survive to term • Manifest severe malformations and early lethality • Shows abnormal karyotype Aneuploid Conditions • Trisomies • Often found in spontaneously aborted fetuses • Autosomal monosomies are seldom found • Suggests monosomic gametes may be functionally impaired Nondisjunction of the sex chromosomes • Can lead to a variety of nonlethal genetic disorder • Klinefelter syndrome- most common (1 out of 700 to 1000). An ovum with an extra X chromosome is fertilized by a sperm with a Y chromosome- XXY genotype • Turner syndrome- 1 out of every 10,000 female births ( more likely to spontaneously abort)- XO Klinefelter vs Turner Klinefelter Syndrome XXY Turner Syndrome XO Klinefelter Syndrome XXY Turner Syndrome XO Gen es • Polygenic- Human characteristics/ genetic disorder resulting from the combined action of alleles of more than one gene • Mendelian • Environment • Monogenic-controlled by a single gene, especially by either of an allelic pair • Mendelian genetics • Particulate hereditary determinants ( genes) – Segregation of unchanged hereditary determinant in the reproductive cells – Random union of gametes Autosomal Dominant Inheritance • Vertical pattern is observed with multiple generations affected • Heterozygotes for the mutant allele show an abnormal phenotype • Males and females are affected with equal frequency and severity • Only one parent must be affected for an offspring to be at risk for developing the • When an affected person mates with an unaffected one, each offspring has a 50% chance of inheriting the affected phenotype. Pedigr ee Human traits #1 • Some human traits are controlled by a single gene • Some of these exhibit dominant and recessive inheritance • Pedigree analysis is used to track inheritance patterns in families • Dominant pedigree – juvenile glaucoma • Disease causes degeneration of optic nerve leading to blindness • Dominant trait appears in every generation Autosomal Recessive Inheritance • Horizontal pattern is observed – a single generation being affected • Males and females are affected with equal frequency and severity • Inheritance is from both parents ( each of whom is a heterozygote carrier) and each of whom is usually clinically unaffected by his or Human traits #2 • Recessive pedigree – albinism (TYR gene) predominant form. • Condition in which the pigment melanin is not produced • Pedigree for this form of albinism due to a nonfunctional allele of the enzyme tyrosinase • Males and females affected equally • Most affected individuals have unaffected parents • Each offspring of two carriers has a 25% chance of being affected, a 50% chance of being a carrier, and a 25% chance of inheriting neither mutation alleles. Use the punnette square. • In mating's between individuals, each with the same recessive phenotype, all offspring will be affected • Affected individual who mate with unaffected individuals who are not carriers have only unaffected offspring • The rarer the recessive phenotype, the more likely is it that the parents are consanguineous ( related) Carrier Parents Hereditary hemochromatosis One Affected Parent HFE gene: Human homeostatic iron regulator protein X-Linked Inheritance • There is no male-to male transmission of the phenotype • Unaffected males do not transmit the phenotype • All daughters of an affected male are heterozygous carriers. • Some mothers of affected males will not themselves be heterozygotes (they will be homozygous normal )- but will have a germinal mutation. • Heterozygous women transmit the mutant gene to 50% of their sons, who are affected, and to 50% of their daughters, who are heterozygotes Always double check • If an affected male mates with a heterozygous female, 50% of the male offspring will be affected, giving the false impression of male- to male transmission. • Among the female offspring of such mating, 50% will be affected as the average hemizygous male (XY). • In small pedigrees this pattern may simulate autosomal dominate inheritance • Why? Because the mother is not a known carrier Hemophilia A • Hemophilia A is an X chromosome-linked recessive coagulation disorder Hemophilia A • Hemophilia A is an X chromosome-linked recessive bleeding disorder, and is due to mutations or deletions in the Factor VIII gene (F8). • Ratio in population: 1:5,000 males. • The severity (mild to severe) of disease is defined by the percentage of FVIII levels to normal levels. Severe:<1% Moderate:2-5% Mild: 5-30% Intracranial hemorrhage Joint hemorrhage SEC From outside Current Treatment • Regular intravenous infusion of VIII concentrates --Isolated from blood serum --Recombinant technology --Combination Problems: a) Short half life of FVIII b) Safety concerns of infectious diseases c) Very costly ($150,000-$300,000) per year d) Immune response: Inhibitor development Gene therapy is an alternative approach which my overcome these shortcomings. ---Restoring 1-5% of normal FVIII prevents spontaneous bleeding Cancer Unrestrained, uncontrolled growth of cells • Failure of cell cycle control • Two kinds of genes can disturb the cell cycle when they are mutated 1. Tumor-suppressor genes 2. Proto-oncogenes Tumor-suppressor genes • p53 gene and many others • Both copies of a tumor-suppressor gene must lose function for the cancerous phenotype to develop • First tumor-suppressor identified was the retinoblastoma susceptibility gene (Rb) • Predisposes individuals for a rare form of cancer that affects the retina of the eye Tumor-suppressor: p53 gene • p53 plays a key role in checkpoint • p53 protein monitors integrity of DNA • If DNA damaged, cell division halted and repair enzymes stimulated • If DNA damage is irreparable, p53 directs cell to kill itself • Purpose: Prevent the development of many mutated cells • p53 is absent or damaged in many cancerous cells Rb gene • Inheriting a single mutant copy of Rb means the individual has only one “good” copy left • During the hundreds of thousands of divisions that occur to produce the retina, any error that damages the remaining good copy leads to a cancerous cell • Single cancerous cell in the retina then leads to the formation of a retinoblastoma tumor • Rb protein integrates signals from growth factors • Role to bind important regulatory proteins and prevent stimulation of cyclin and cyclindependent kinase BReast CAncer susceptibility protein • Tumor suppressor gene: BRCA1 and BRCA2 • Expressed in breast tissue • Repair of damaged DNA and apoptosis • Mutation: Improper repair of DNA • Increased risk of breast cancer Proto-oncogenes • Involved in signal transduction and mitogenic signals, triggering mitosis • Normal cellular genes that become oncogenes when mutated • Oncogenes can cause cancer • Some encode receptors for growth factors • If receptor is mutated in “on,” cell no longer depends on growth factors • Some encode signal transduction proteins • Only one copy of a proto-oncogene needs to undergo mutation for uncontrolled division to take place It is the accumulation of mutations • Inherited mutations seen in 5% of all breast cancers and 10% of ovarian cancers • Developing these cancers is higher than in the general population without the mutation(s). • • • • 10% women will develop breast cancer during life time 50% if BRCA1 or BRCA2 is mutated 1% women will develop ovarian cancer during life time 40 % if BRCA1 or 15% if BRCA2 is mutated Autosomal Dominance ditary Breast and Ovarian Cancer (HBOC) Syndrome https://s3.amazonaws.com/myriad-library/images/myriad_brca-mutation-cancer-risk-graph.jpg Cancer and Viruses • Oncogenic viruses • Activated oncogenes transform normal cells into cancerous cells • Transformed cells have increased growth, loss of contact inhibition, tumor-specific transplant antigens, and T antigens (nucleus) • The genetic material of oncogenic viruses becomes integrated into the host cell’s DNA • Cancer may develop years after initial infection Oncogenic Viruses • Oncogenic DNA viruses • Adenoviridae • Herpesviridae • Poxviridae • Papovaviridae • Hepadnaviridae • Oncogenic RNA viruses • Retroviridae • Viral RNA is transcribed to DNA, which can integrate into host DNA • Human T cell lymphotropic viruses • HTLV-1 • HTLV-2 Diagnostic Tools • Family history – All first-degree relative ( parents, siblings and offspring • Second-degree (grandparents, aunts, uncles, nieces and nephews and grandchildren) • Third-degree (first cousins) • This information can be further analyzed utilizing a pedigree diagram to identify mode of inheritance for a disease process Cytogenetic studies • Study of chromosomes utilizing light microscopy • Obtained from peripheral blood, amniotic fluid, trophoblastic cells ( chorionic villus) bone marrow and cultured fibroblasts ( usually obtained from a skin biopsy) Klinefelter Syndrome and Jacob’s Syndrome Many Analytical Techniques Have Been Useful during the Investigation of DNA and RNA Scientific Applications • Understanding DNA • Sequencing patients’ genomes • DNA fingerprinting for identification (Restriction Enzymes) to generate and RFLP Selected Restriction Enzymes Used in DNA Technology Techniques • Analytical techniques useful during DNA and RNA investigation • • • • • Absorption of UV light Denaturation and renaturation of nucleic acids Molecular hybridization FISH: Fluorescent in situ hybridization Electrophoresis of nucleic acids Absorption of UV Light • Nucleic acids • Absorb UV light strongly at 254–260 nm due to interaction between UV light and ring systems of the bases • Use of UV critical to isolation of nucleic acids following separation Molecular Hybridization • Molecular hybridization • Denaturation and renaturation of nucleic acids are the basis for molecular hybridization • Example: Single strands of nucleic acids combine duplex structures, yet are not from same source • If DNA is isolated from two distinct sources, double- stranded hybrids will form © 2017 Pearson Education, Ltd. Fluorescence in situ hybridization (FISH) • Fluorescent in situ hybridization (FISH) • • • • Uses fluorescent probes to monitor hybridization Mitotic cells fixed to slides and subjected to hybridization ssDNA is added and hybridization is monitored Probes are nucleic acids that will hybridize ONLY with specific chromosomal areas Fluorescence in situ hybridization (FISH) • Fluorescence in situ hybridization (FISH) has made it easier to visualize and map chromosomal (gene) abnormalities and the presence of infectious agents FISH • Fluorescent in situ hybridization • Using fluorescent dye-labeled RNA or DNA • Quick test • Clinically: used in diagnostics and in genetic counseling • Circulating tumor cells • Infectious particles FISH, or fluorescent in situ Examined phase contrast microscopy Electrophoresis • Nucleic acid electrophoresis • Separates DNA and RNA fragments by size • Smaller fragments migrate through gel at faster rate than large fragments • Agarose gel • Porous matrix restricts migration of larger molecules more than it restricts smaller ones Agarose Gel Electrophoresis • Gel electrophoresis is a widely used technique for the analysis of nucleic acids and proteins. Agarose gel electrophoresis is routinely used for the preparation and analysis of DNA. • Gel electrophoresis is a procedure that separates molecules on the basis of their rate of movement through a gel under the influence of an electrical field. • DNA is negatively charged. • When placed in an electrical field, DNA will migrate toward the positive (anode). • pole An agarose gel is used to slow the movement of DNA and separate by size. • Polymerized agarose is porous, allowing for the movement of DNA © 2017 Pearson Education, Ltd. How fast will the DNA migrate? strength of the electrical field, buffer, density of agarose gel… Size of the DNA! *Small DNA move faster than large DNA …gel electrophoresis separates DNA according to size Southern blotting. Gel Restriction enzyme Larger Gene of interest DNA containing the gene of interest is extracted from human cells and cut into fragments by restriction enzymes. Paper towels Smaller The fragments are separated according to size by gel electrophoresis. Each band consists of many copies of a particular DNA fragment. The bands are invisible but can be made visible by staining. Nitrocellulose filter Sponge Salt solution Gel Human DNA fragments Nitrocellulose filter The DNA bands are transferred to a nitrocellulose filter by blotting. The solution passes through the gel and filter to the paper towels by capillary action. Gel DNA transferred to filter This produces a nitrocellulose filter with DNA fragments positioned exactly as on the gel. Labeled probes Sealable plastic bag The filter is exposed to a labeled probe for a specific gene. The probe will base-pair (hybridize) with a short sequence present on the gene. The fragment containing the gene of interest is identified by a band on the filter. DNA fingerprints used to track an infectious disease. E. coli isolates from patients whose infections were not juice related E. coli isolates from patients who drank contaminated juice Apple juice isolates DNA chip. (a) A DNA chip can be manufactured to contain hundreds of thousands of synthetic single-stranded DNA sequences. Assume that each DNA sequence was unique to a different gene. (b) Unknown DNA from a sample is separated into single strands, enzymatically cut, and labeled with a fluorescent dye. DNA chip. (c) The unknown DNA is inserted into the chip and allowed to hybridize with the DNA on the chip. (d) The tagged DNA will bind only to the complementary DNA on the chip. The bound DNA will be detected by its fluorescent dye and analyzed by a computer. In this Salmonella antimicrobial resistance gene microarray, S. typhimurium-specific antibiotic resistance gene probes are green, S. typhi-specific resistance gene probes are red, and antibiotic-resistance genes found in both serovars appear yellow/orange. Genetic Tools • PCR • Primer for a specific organism will cause application if that organism is present • Real-time PCR: newly made DNA is tagged with a fluorescent dye; the levels of fluorescence can be measured after every PCR cycle • Reverse-transcription (RT-PCR): reverse transcriptase makes DNA from viral RNA or mRNA Polymerase Chain Reaction (PCR) • Invented in the 1984 as a way to make numerous copies of DNA fragments in the laboratory • PCR is a means to amplify a particular piece of DNA • Amplify: making numerous copies of a segment of DNA • Generates identical copies of a target sequence of DNA in a short period of time • Many applications DNA Replication vs. PCR • PCR is a laboratory version of DNA Replication in cells • The laboratory version is commonly called “in vitro” since it occurs in a test tube while “in vivo” signifies occurring in a living cell. PCR: the in vitro version of DNA Replication The following components are needed to perform PCR in the laboratory: 1) DNA (your DNA of interest that contains the target sequence you wish to copy) 2) A heat-stable DNA Polymerase (like Taq Polymerase) 3) All four nucleotide triphosphates 4) Buffers 5) Two short, single-stranded DNA molecules that serve as primers 6) Thin walled tubes 7) Thermal cycler (a device that can change temperatures dramatically in a very short period of time) PCR The DNA, DNA polymerase, buffer, nucleoside triphosphates, and primers are placed in a thin-walled tube and then these tubes are placed in the PCR thermal cycler PCR Thermocycler The Three steps of PCR • The basis of PCR is temperature changes and the effect that these temperature changes have on the DNA. • In a PCR reaction, the following series of steps is repeated 20-40 times Step 1: Denature DNA At 95 C, the DNA is denatured (i.e. the two strands are separated) Step 2: Primers Anneal At 40 C- 65 C, the primers anneal (or bind to) their complementary sequences on the single strands of DNA Step 3: DNA polymerase Extends the DNA chain At 72 C, DNA Polymerase extends the DNA chain by adding nucleotides to the 3’ ends of the primers. Heat-stable DNA Polymerase • Given that PCR involves very high temperatures, it is imperative that a heat-stable DNA polymerase be used in the reaction. • Most DNA polymerases would denature (and thus not function properly) at the high temperatures of PCR. • Taq DNA polymerase was purified from the hot springs bacterium Thermus aquaticus in 1976 • Taq has maximal enzymatic activity at 75  C to 80  C, and substantially reduced activities at lower temperatures. Denaturation This occurs at 95 ºC mimicking the function of helicase in the cell. Annealing or Primers Binding Reverse Primer Forward Primer Primers bind to the complimentary sequence on the target DNA. Primers are chosen such that one is complimentary Step 3 Extension or Primer Extension extension extension DNA polymerase catalyzes the extension of the strand in the 5-3 direction, starting at the primers, attaching the appropriate nucleotide (A-T, C-G) • Exponential Expansion of DNA End Cycle 1 End Cycle 2 The Size of the DNA Fragment Produced in PCR is Dependent on the Primers • The PCR reaction will amplify the DNA section between the two primers and their location of binding. • If the DNA sequence is known, primers can be developed to amplify any piece of an organism’s DNA. Forward primer Reverse primer Size of fragment that is amplified The DNA of interest is amplified by a power of 2 for each PCR cycle For example, if you subject your DNA of interest to 5 cycles of PCR, you will end up with 25 (or 64) copies of DNA. Similarly, if you subject your DNA of interest to 40 cycles of PCR, you will end up with 240 (or ) copies of DNA! PCR powerful tool in molecular biology • One can start with a single sperm cell or strand of hair and amplify the DNA sufficiently to allow for DNA analysis and a distinctive band on an agarose gel. • One can amplify fragments of interest in an organism’s DNA by choosing the right primers. • One can use the selectivity of the primers to identify the likelihood of an individual carrying a particular allele of a gene. More about Primers • PCR primers are short, single stranded DNA molecules (15-40 bp) • They are manufactured commercially and can be ordered to match any DNA sequence • Primers are sequence specific, they will bind to a particular sequence in a genome • As you design primers with a longer length (15 → 40 bp), the primers become more selective. • DNA polymerase requires primers to initiate replication Selectivity of Primers • Primers bind to their complementary sequence on the target DNA • A primer composed of only 3 letter, ACC, for example, would be very likely to encounter its complement in a genome. • As the size of the primer is increased, the likelihood of, for example, a primer sequence of 35 base letters repeatedly encountering a perfect complementary section on the target DNA become remote. PCR and Disease • Primers can be generated that will specifically bind to and amplify certain alleles of genes or mutations of genes • This is the basis of genetic counseling and PCR is used as part of the diagnostic tests for genetic diseases. • Some diseases that can be diagnosed with the help of PCR: • Huntington's disease • cystic fibrosis • Human immunodeficiency virus PCR and Agarose Gel Analysis of HIV-1 DNA amplified from seminal fluid nes 5 and 11 are positive control equence Chromatogram of a woman with early onset ovarian cancer A. WT B. Mutation Human Immunodeficiency Virus (HIV) • HIV is a retrovirus that is linked to immune suppression. • HIV tests rely on PCR with primers that will only amplify a section of the viral nucleic acid found in an infected individual’s bodily fluids. • Therefore, if there is a PCR product, the person is likely to be HIV positive. If there is no PCR product the person is likely to be HIV negative. • RT-PCR and qPCR • Clinically: analysis of gene expression and quantification of viral RNA • How many copies of genes • Protein detection-based tests are available as well, but all US blood is tested by PCR. This qPCR technique is sensitive and accurate Threshold cycle Ct