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Adv Pathophysiology Unit 1: Cell, Inflammation, Immunity, Genetics Page 1 of 42

File: advpatho_unit1_3gene.pdf Source: C. DeCristofaro, MD

Learning Objectives for this file: 1. Review genetic code, transcription, translation, genetic material (DNA, RNA) 2. Review mitosis, meiosis, apoptosis, karyotype 3. Review metabolic pathways, proteins 3. Review allelic inheritance, mutations, somatic & sex-linked traits



METABOLIC PROCESSES & FEEDBACK CONTROL: 1. NEGATIVE FEEDBACK (necessary for MOST healthy systems): • this concept occurs over and over again in both health and pathologic states. • Negative feedback means that the production of a final metabolic product (structural protein,

enzyme, substrate) will feedback to the very beginning of the process that started this biosynthesis, and will TURN THE WHOLE SYNTHETIC PROCESS OFF.

• In the chromosome, there is negative feedback to the promotor area of the operon, which will turn off the operon, once the final product is made.

• So, production is stopped (which is fine, since the body already has some now !!). • You will see that pathologic states are almost always the result of normal negative feedback

being interrupted, so that production of the final product doesn't result in turning off the machinery. Example in physiology: production of thyroxine hormone “turns off” the system



2. POSITIVE FEEDBACK:

• where the final product further stimulates more of its own production, leading to poisonous buildup of substances or failure to take the next step in a metabolic pathway

• this can be PHYSIOLOGIC, but only a few processes in healthy systems use positive feedback:

o Example: platelet adhesion causing more platelet adhesion to establish rapid hemostasis inflammation producing more inflammatory chemicals to ensure rapid response of tissues to damage

• Often this is pathologic o failure to “turn off” a process leads to disease

3. PHYSIOLOGIC INTEGRATION OF FEEDBACK LOOPS TO CONTROL HOMEOSTASIS:

• typically, we need to stimulate some processes with positive feedback, but then “put on the brakes” to prevent the system accelerating to an extreme state

• Example: formation of a blood clot eventually creates a substance that will dissolve clot • Thus a balance is achieved – just enough clotting, not too much, not too little (the Goldilocks

sweet spot)



GENETICS: • based on work by Mendel (Punnett square), Darwin (natural selection), Watson-Crick (molecular

genetics & the double helix). • the gene is the basic unit of inheritance

o the human genome project – about 21,000 genes code for proteins, but many other types of genes (i.e., regulatory) increase the number to a much larger amount

o Link to: http://web.ornl.gov/sci/techresources/Human_Genome/index.shtml o And: http://www.genome.gov/Students/

• Deletions, additions and abnormal genes can contribute to human disease states • Genetic contribution for many previously defined “idiopathic” conditions

o Example: Multiple Sclerosis – genes conferring risk are at a locus (place) on chromosome 1 and the HLA class II region on chromosome 6.

• “Nature vs. Nurture” – is it our genetics (inheritable traits) or how we are raised (environment)? o New findings support that interaction of environment with genetic material starts as early as

the blastocyst stage o Genes that are active from fetal stage onwards are powerfully affected by environment o Answer: it is BOTH!

What exactly IS a gene? • Information contained in chemical sequences that code for ONE UNIQUE PROTEIN • remember – proteins may be:

o structural (e.g. collagen) o functional (e.g. hemoglobin) o hormone (e.g. insulin) o receptor (e.g. on cell membrane) o enzyme (e.g. acetylcholinesterase) o transporters (e.g. thyroglobulin) o special (e.g. pigment)

• thus, genes can also CONTROL actions in the cell, turn on/off cellular processes, begin/terminate metabolic pathways, etc. because they create enzymes and other types of proteins, not just structural proteins

Is all of our DNA made up of genes?

• No – much of the DNA is made of areas that control (turn on or turn off) genetic activity • The part of the gene that actually provides the information “code” to make the protein in the

cell is called the exon (that part of the genome formed by exons is called the exome) • For many years, people assumed that areas of DNA that don’t “code” for information were

“junk” or “dark” areas of DNA and had no purpose – but newer research indicates that these areas have impact on cellular function and also pathology; noncoding parts of the genome are called introns

• See NIH: https://www.nih.gov/news-events/nih-research-matters/genome-comparison-casts-light-dark-areas-dna

http://web.ornl.gov/sci/techresources/Human_Genome/index.shtml

http://www.genome.gov/Students/

https://www.nih.gov/news-events/nih-research-matters/genome-comparison-casts-light-dark-areas-dna

https://www.nih.gov/news-events/nih-research-matters/genome-comparison-casts-light-dark-areas-dna



GENOMIC MEDICINE & PHARMACOGENOMICS – “PERSONALIZED MEDICINE”: • See (New England Journal of Medicine): http://www.genome.gov/27541912 • More on clinical genome and exome sequencing: https://www.nih.gov/news-events/news-

releases/new-report-offers-primer-doctors-use-clinical-genome-exome-sequencing Some definitions: Genetics is the study of individual genes and their impact on inheritance and on single-gene and chromosomal disorders. Genomics is the study of the structure, function, and analysis of the human genome together. Epigenetics is the external modification of DNA that affect gene expression, and epigenomics is the study of the chemical compounds that instruct the genome where and when genes are expressed within a cell.

http://www.genome.gov/27541912

https://www.nih.gov/news-events/news-releases/new-report-offers-primer-doctors-use-clinical-genome-exome-sequencing

https://www.nih.gov/news-events/news-releases/new-report-offers-primer-doctors-use-clinical-genome-exome-sequencing



THE CENTRAL “DOGMA” (BELIEF SYSTEM) OF GENETICS: Prior basic understanding: • Remember this SEQUENCE when studying about TRANSCRIPTION and TRANSLATION • The flow of genomic information from DNA to RNA to protein remains the basis for understanding

genomic function Newly understood processes:

• A single gene can yield an extensive array of gene products, depending on the environment in

which it is expressed, thereby expanding the repertoire of the 20,000 or so genes in the human genome

• Translation is regulated by interactions between messenger RNA (mRNA) and proteins. • Processing of single-precursor RNA (preRNA) molecules can yield multiple RNA products,

including microRNA (miRNA) and small interfering RNA (siRNA) molecules. • MORE on this later in the file



ORGANIZATION OF GENETIC MATERIAL: DNA: DEOXYRIBONUCLEIC ACID (DNA). • There are 4 types of nitrogenous bases (cytosine, thymine, guanine, and adenine) that pair up as

A-T and G-C (complementary “base pairs”) • every sequence of three base pairs will “code” for one of the 20 amino acids used to make human

proteins (so, three base pairs = one codon) • the SEQUENCE of these codons therefore represents the SEQUENCE of amino acids in the final

protein product • thus, DNA is ALL ABOUT MAKING PROTEINS!!! It is the blueprint for your proteins –

nothing else!! CHROMOSOME: Where our DNA is in the nucleus of our cells. • All somatic (body) cells have 22 homologous pairs of somatic chromosomes and 1 pair of sex

chromosomes (i.e. 46 total chromosomes) and are diploid (have pairs of chromosomes) • All gonadal cells (that create sperm and ova) have half this number (only one of the chromosomes

of each pair) and are haploid (have ONE chromosome of each pair) • IMAGINE:

o 22 pairs of shoes, a left and a right in each pair, and these are called homologous autosomal (somatic) chromosomes (contain the same genes)

o for every pair of shoes, the left came from one parent (Mom – egg, or Dad – sperm) and the right came from the other parent (Mom – egg, or Dad – sperm)

o In addition to your 22 pairs of shoes, you have one special pair of shoes (the sex chromosomes) that are either XX (if you are a girl, also one from Mom and one from Dad) or XY (if you are a boy – the X from Mom, and the Y from Dad)

• SO: o autosomal (somatic) = 22 pairs (almost identical "sisters") = 22 x 2 = 44 o sex chromosomes = 1 pair = 2 chromosomes = one X + one Y o grand total = 46 chromosomes (somatic + sex).

Chromosome count: • Euploid cells have the correct number of chromosomes. • Aneuploidy means there is not an exact multiple of 23 chromosomes. Example: one extra

chromosome in a given pair some clinical syndromes include Down syndrome (trisomy-21) • Polyploidy: too many full sets of chromosomes (e.g. triploidy, tetraploidy -- these usually abort

spontaneously). Sex determination:

• A Y chromosome presence always results in male genitalia (the SRY region, or sex-determining region exists on the Y chromosome)

• if more than one Y chromosomes – sterility and retardation



Human DNA structure: • this forms the double helix, which looks like a twisted ladder, or double strand, that contains the

instructions for life. • DNA in the nucleus of the cell – in chromosomes packed together with histone proteins • In lower life forms (e.g. viruses), the DNA may be "single-stranded" & exist freely in the cell. DNA replication requires separation and replication of the strands:

DNA exists as two strands coiled around each other in a “double-helix” The strands can be separated by heat or chemicals



LOOK AT PICTURE ON NEXT PAGE WHILE READING THIS: HOW WE GET TRAITS: DNA is organized into codons, genes (with introns and exons), operons, alleles for traits to determine our heritable material CODON: • sequence of three base pairs that calls for one of the 20 amino acids used for human proteins • thus, the amino acids are linked in a specific order to make a specific protein • this is NOT done in the nucleus, but is done in the cytoplasm using another nucleic acid called

RNA (see below). • Codons may be start signs (promotors), stop signs (termination, nonsense codons), or

informational codons calling for an amino acid. • after protein synthesis, the protein is shipped throughout the cell via the endoplasmic reticulum. GENE – how does it result in a protein? • a sequence of codons (including promotor & terminator) that all together code for ONE UNIQUE

PROTEIN • remember – proteins may be:

o structural (e.g. collagen) o functional (e.g. hemoglobin) o hormone (e.g. insulin) o receptor (e.g. on cell membrane) o enzyme (e.g. acetylcholinesterase) o transporters (e.g. thyroglobulin) o special (e.g. pigment)

• thus, genes can also CONTROL actions in the cell, turn on/off cellular processes, begin/terminate metabolic pathways, etc. and NOT just make a structural protein such as collagen

• the genome contains introns (non-coding “nonsense” areas) and exons (coding areas that will make the RNA used to create proteins)(the part of the genome made up of exons is called the exome). Here are the sequential steps to making a protein from information on the genome:

o DNA pre-mRNA o pre-mRNA is excised (cut) so that the introns are removed and only the exons remain o the mRNA now is used to make proteins – travels to the cytoplasm where protein

synthesis takes place





OPERON: • a DNA sequence that contains one or more genes that code for enzymes that together form a

biosynthetic pathway leading to a final product • these enzymes are designed to peform sequentially • each OPERON starts with a PROMOTOR codon, and ends with a TERMINATION codon • Step 1: TRANSCRIPTION is DNA to RNA (nucleic acid to nucleic acid – the same “language”);

occurs in the NUCLEUS of the cell • Step 2: TRANSLATION is RNA to protein (nucleic acid to amino acids – different “languages”);

occurs in the CYTOPLASM of the cell

OPERON PROMOTOR codon TRANSCRIPTION process TRANSLATION process CODONS Gene mRNA Protein (enzyme#1 of metabolic path) CODONS Gene mRNA Protein (enzyme#2 of metabolic path) CODONS Gene mRNA Protein (enzyme#3 of metabolic path) TERMINATOR (stop transcribing) Final Protein Product (FROM METABOLIC PATHWAY) METABOLIC PATHWAY FROM THIS OPERON: A B C FINAL PRODUCT Enzyme 1 Enzyme 2 Enzyme 3 • A promoter would start the operon to give us proteins enzyme 1, enzyme 2, and enzyme 3 • Protein manufacture would be stopped by the terminator after these three enzymes are made • The three enzymes in the metabolic pathway would give us the final product • If the final product was responsible for a trait, then this sequence on the chromosome would be

called an allele (e.g. type of hemoglobin the person has).

A is a substrate for Enzyme 1, & its product, B, becomes the substrate for Enzyme 2.

Example of molecular biosynthetic pathway found on one operon



INHERITANCE OF TRAITS – ALLELES: • ALLELES are a collection of genes coding for a certain trait occupying a certain place on its

specific chromosome called a locus. • Most human traits are variable (e.g. eye color) & therefore called polymorphic – the trait exists in

a variety of forms (e.g. eye color). ALLELE = TRAIT: • genetic information to form observable traits is contained in an area of the chromosome called a

locus, containing all the necessary operons for that trait (eye color, blood type). • in our diploid cells, there are two alleles per person, since each chromosome in a homologous

chromosome pair has its own allele for this trait (one from Mom, one from Dad!). • Variations in the trait occur because there may be different types of alleles for this particular trait. • Traits may be very complex (e.g. intelligence) or more simple (e.g. eye color, type of Hemoglobin) Homozygous: the same allele for a trait is found on each chromosome of the pair. Heterozygous: different alleles for the trait are found on each chromosome of the pair (e.g. One allele could be for brown eyes, one for blue). Genotype: inherited genes on the chromosomes. Phenotype: outward expression of your genes (what you can observe, measure, quantify) – e.g. type of cholesterol abnormality in your blood, your eye color. Human traits: • Most human traits are multifactorial (polygenic) • several genes act together to produce the trait defined by an allele • e.g. height & cleft lip & cleft palate Variable penetrance: • percentage of times an allele will affect the phenotype (may not always “penetrate” to the

phenotype) • Example: retinoblastoma of children – every child with the gene may not develop this cancer. Variable expressivity: • even if expressed, may not be expressed to the same degree • thus, severity of the disease will vary. • Example: neural tube, neurofibromatosis, Hemophilia A. Mitochondrial Inheritance: genetic traits governed by DNA in the mitochondria (mitochondrial inheritance) is through the maternal line only, since only the egg has mitochondria. (see below) Pedigree chart: pictorial display of inheritance of traits throughout the generations (normal traits or illness)



Mendelian Inheritance: • uses a Punnett square to describe offspring outcomes. • there are other types of genetic inheritance (see below) that do NOT follow Mendelian laws.

o Autosomal (Somatic) traits: found on the non-sex chromosomes, equally inherited by both sexes, no skipping of generations and conditions exist in same proportion in both sexes

o Sex-linked traits: found on the sex chromosome (X chromosome) and result in different percentages of illness in male vs. female persons

• Autosomal dominance & co-dominance: o If the trait is found on one of the chromosomes, the trait will be expressed o Sometimes, the dominance relationship is “shared” and there is co-dominance o Example of dominance: brown eyes dominant over blue o Example of co-dominance: blood type antigens result in codominant expression of

homozygous A, homozygous B, homozygous O, and heterozygous AB (of interest is that A & B are codominant, but both A and B are dominant over O)

• Autosomal recessive: o only expressed if both chromosomes have the trait o since males & females inherit the trait equally, siblings are often affected o there is often consanguinity (increased amount of recessive disorders in families with a

history of intermarriage, also called "inbreeding") o both sexes can be carriers. o Example: cystic fibrosis

Mendelian Genetics & Punnett Squares: • Punnett Squares of autosomal allelic trait: offspring of parents with a particular genotype.

o If the allele is dominant, then this trait will appear in the phenotype for any genotype with this allele (homozygous or heterozygous).

o If the allele is recessive, then you must have a homozygous recessive genotype for this trait to be seen in the phenotype – if a disease trait, it is called recessive such as sickle cell anemia (abnormal Hemoglobin) with disease/trait (carrier) persons

Here, the parents are carriers (both mother and father in this example -- the top row and the side column) and their offspring may be normal (no recessive allele, homozygous dominant), a carrier (heterozygous) or have the disease (homozygous recessive).



• Punnett Squares of Sex-linked traits: o the allele is located on the X chromosome, & boys will be seen to show a higher proportion

of inheritance, since there are no corresponding alleles on the Y chromosome in loci that correspond to the known sex-linked diseases

o only females will be carriers. o Example: hemophilia.

Punnett Square: X-linked recessive condition Demonstrates increased illness rate in males and usually carrier state in females Illness allele: X-h (recessive) Normal allele: X-H (dominant) Carrier state: X-h, X-H Disease state: X-h, X-h (rare) X-h, Y



MITOCHONDRIAL INHERITANCE (from Mitchondrial DNA, mtDNA: • does not use Mendelian genetics • the pedigree chart will be maternal because ALL your mitochondria come from MOM!! Atypical inheritance patterns: • result from mitochondrial DNA mutations, inherited through the maternal line • in general, ALL mitochondria in a person’s body come from the mother’s egg, none from the

father’s sperm (only rarely does the sperm can contribute mitochondria and thus contribute to mitochondrial genetic diseases)

o mitochondrial DNA (mtDNA) is in the form of circular chromosomes o also contains tRNA (called mtRNA) & rRNA (called mrRNA) o mitochondrial DNA (mtDNA) encodes for mitochondrial proteins & enzymes, as well as the

13 proteins of the respiratory chain, necessary for aerobic metabolism (oxidative metabolism).

• Usually ONLY comes from the mother (“maternal genetic” pedigree chart) Mutations: • Most of the mutations are deletions • Results in in diseases of: brain, eye & muscle. • All offspring (both sexes equally) of an affected female will be affected, and no offspring of an

affected male (since it is passed via the maternal line). • You will note that most of these syndromes are so severe as to be nearly incompatible with life,

therefore diagnosed in the neonatal or infancy period. Threshold effect: • since more than one mitochondria is inherited and passed along with the developing fetal cells,

there is a mosaicism of normal & abnormal mitochondria • differ among the different tissues of the body – some tissues get good mitochondria, others get

the affected mitochondria • It may be that muscle & brain cells are particularly affected, due to their high energy requirements

& dependence on the mitochondria for energy production. Aging and mutations?

• Some theories that accumulated genomic mutations in mitochondria play a role in aging • Other theories that the normal structure and function of the genome is designed to cause

eventual cell death (more below)



EXAMPLES OF MITOCHONDRIAL GENETIC DISEASES: • Most of the mutations are deletions & often result in disease of brain, eye & muscle. • All offspring (both sexes equally) of an affected female will be affected, and no offspring of an

affected male (since it is passed via the maternal line). • Most of these syndromes are so severe as to be nearly incompatible with life

o therefore diagnosed in the neonatal or infancy period and are considered “pediatric” diagnoses

Syndrome examples: • MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes):

o devastating disease affecting young children, progressive hemiparesis & hemianopsia, cortical blindness, and dementia due to multiple strokes & encephalomalacia.

o Short stature & seizure disorder, positive muscle biopsy (ragged-red fibers). • MERRF (myoclonus epilepsy & ragged-red fibers): devastating, affects young children

(progressive ataxia/nystagmus/dysarthria & myoclonic seizure disorder, sometimes dementia). o Positive muscle biopsy for ragged-red fibers & other abnormalities.

• CPEO (chronic progressive external ophthalmoplegia) & a variant called KSS (Kearns-Sayre syndrome:

o progressive ophthalmoplegia & pigmentary retinopathy in persons < age 20 y/o. o This is accompanied by hearing loss, short stature, endocrinopathies (including DM &

hypoparathyroidism), & dementia. o Again, muscle biopsy often positive for ragged-red fibers & other abnormalities.

• ATPase 6 mutation syndrome: similar to above syndromes. • Leigh disease (subacute necrotizing encephalomyelopathies):

o variable, many of the above symptoms o plus brainstem (e.g. respiratory) findings.

• Leber hereditary neuroretinopathy: bilateral blindness occurring in teenage years. • Pearson syndrome: lethal in infancy (anemia, progressive pancreatic & liver failure).



TRANSCRIPTION (DNA RNA): • the amino acids being strung together happens in the CYTOPLASM & NOT in the nucleus • So, the information on DNA has to somehow get out to the cytoplasm to get this job done • Another nucleic acid is made (RNA) that will travel to the cytoplasm to carry the instructions

regarding protein synthesis • This is the same language (nitrogenous base to nitrogenous base) so it is called “transcription” RNA (RiboNucleic Acid): • Base pairs are adenine, cytosine, and guanine, plus a fourth base that is different from DNA,

Uracil • The base pairing occurs on the DNA strand and is A - U, G - C. • RNA is made from only one of the paired helical (double-stranded, ds) DNA strands, and

therefore RNA is single-stranded (ss) • The enzyme that does this is DNA-dependent RNA-polymerase, also called transcriptase. • mRNA (messenger RNA) goes out from the nucleus to the cytoplasm

o is a copy of the DNA blueprint for protein manufacturing o its codons calls for the correct sequence of amino acids, just like on the DNA

In the NUCLEUS — TRANSCRIPTION (DNA to mRNA) codon codon codon codon DNA --|--|--|-----|--|--|-----|--|--|-----|--|--|-- A T C G C T A A T G C G copying DNA RNA done by the transcriptase enzyme U A G C G A U U A C G C in the nucleus mRNA --|--|--|-----|--|--|-----|--|--|-----|--|--|-- codon codon codon codon

NOW THE mRNA goes to the cytoplasm to make proteins (this next protein manufacturing process is called translation)



RNA TRANSLATION = PROTEIN BIOSYNTHESIS (DNA RNA Proteins): • We are going from nucleic acids (DNA & RNA) to proteins (made up of amino acids) • The process is therefore called "TRANSLATION" since we are using a new chemical

"language" (amino acids, not nucleic acids). • The mRNA has traveled to the cytoplasm & is now the template (blueprint), since it contains the

information copied from the DNA. • Each codon calls for one of 20 different amino acids (some amino acids have more than one

codon – since you can make 64 codons out of grouping 4 letters into groups of three) • The tRNA carries the amino acids to the mRNA, and matches up its ANTICODON to the mRNA

codon. • The rRNA is part of the ribosome, which travels down the mRNA to “read” the instructions on the

mRNA – the ribosome also has the enzymes that can link up the amino acids into a protein molecule

• Remember – proteins are also called “polypeptides” – they are formed by many amino acids linked together by peptide bonds.

• The mRNA is like a "zipper" with the ribosome serving as the zipper-pull (enzyme factory); the start position is always methionine (AUG) and the STOP position is coded by UAA, UAG, and UGA (don’t correspond to an amino acid).

• As the amino acids are brought in close proximity along the mRNA strand, enzymes on the ribosome catalyze the formation of peptide bonds, forming the peptide (protein).

In the CYTOPLASM — TRANSLATION (mRNA to Protein) Occurs on the Rough Endoplasmic Reticulum (RER) codon codon codon codon codon mRNA start--|--|--|-------------|--|--|--------------|--|--|-------------|--|--|------|--|--|----stop tRNA 1 2 3 Ribosomal enzymes are connecting the amino acids to make proteins RIBOSOME is traveling in this direction along the mRNA tRNA anticodons match the mRNA codons, each codon calls for one particular amino acid, carried by the tRNA for that codon rRNA together with special proteins, makes up the ribosome

aa aa aa aa

anti-codon

anti-codon

anti-codon anti-

codon



ANIMATIONS AND VIDEOS: Great animations on these concepts – central dogma, transcription, translation: http://www.wiley.com/college/test/0471787159/biology_basics/animations/fromGeneToProtein.swf (click on the tabs at the top of the page for each animation) Interactive animation – transcribe & translate your own protein: http://learn.genetics.utah.edu/content/basics/transcribe/TranscribeTranslate.swf Good website on genetics: Starting page: http://learn.genetics.utah.edu/ Video of actual DNA molecule being transcribed: https://www.dnalc.org/resources/3d/12-transcription-basic.html Animation of transcription AND translation: http://learn.genetics.utah.edu/content/evo/tandt/

http://www.wiley.com/college/test/0471787159/biology_basics/animations/fromGeneToProtein.swf

http://www.wiley.com/college/test/0471787159/biology_basics/animations/fromGeneToProtein.swf

http://learn.genetics.utah.edu/content/basics/transcribe/TranscribeTranslate.swf

http://learn.genetics.utah.edu/

https://www.dnalc.org/resources/3d/12-transcription-basic.html

http://learn.genetics.utah.edu/content/evo/tandt/



REMINDERS about PROTEINS: • Joining amino acids together by peptide bonds results in a protein. • Proteins may be:

o structural (e.g. collagen) o enzymes that “catalyze” (speed up) chemical reactions in the cell o receptors & carrier proteins o hormones o functional (e.g. pigments)

• Once they are formed, they fold up into 3D space based on internal attractive and repulsive forces • the final shape (morphology) has indentations & protrusions, as well as different electrical

charges, that determine the proper function of the protein • so remember, the function of the protein is really determined by its shape • AND the shape is determined by:

o the proper homeostatic environment (pH, temperature) o the proper sequence of amino acids that created the protein o if there is a problem with the DNA or RNA, an incorrect sequence will result in a non-

functional or dysfunctional protein • Xray-crystallography can be used to actually look at the 3D shape of proteins (experiments often

done on the Space Station because they are more easily done in null-gravity situations). THE PROTEASOME AND METABOLOMICS: Proteomics: • Proteomics is the science of how proteins are modified and interact to promote normal cellular

physiology o AFTER proteins are made there is STILL MORE done to the protein by the cell o e.g. insulin is not made in active form, but must be further “cleaved” after initial production

to form the active hormone • NIH research program: https://www.nih.gov/news-events/news-releases/nih-announces-new-

program-metabolomics What is the proteasome? • the proteasome normally is a complex of enzymes that are the “trash-removal” system of the cell

– it breaks down and removes old proteins Pharmacology correlate: • Proteasome inhibitors: first of a brand-new class of drugs • in cancer cells, if the proteasome can’t work, the cancer cell seems to undergo apoptosis

(programmed cell death) – however, normal cells can recover from inhibition of the proteasome • bortezomib (Velcade) available for multiple myeloma (bone marrow cancer) • other effects of this drug are to prevent the expression of adhesion molecules in the cancer cells,

as well as to turn off nuclear gene expression that accelerates cancer growth • this is an exciting new addition to cancer chemotherapy and will probably be used in many types

of cancer, both alone and as adjuvant therapy for synergistic effect when added to other drugs

https://www.nih.gov/news-events/news-releases/nih-announces-new-program-metabolomics

https://www.nih.gov/news-events/news-releases/nih-announces-new-program-metabolomics



Transcription occurs in the nucleus. The DNA is copied as mRNA, so the directions for making proteins can be carried to the cytoplasm. Codons (3 base pairs) will call for an amino acid. Translation occurs in the cytoplasm, by ribosomes on the Rough Endoplasmic Reticulum). tRNA brings the unique amino acids to the mRNA based on the information in the codons. The ribosome contains enzymes that will link the amino acids together to form the protein. This process is called translation since you are going from one language (the nucleic acids of the codons of the mRNA) to another language (the amino acids of the protein). Usually, when transcription occurs, entire lengths of the chromosome are read to produce multiple proteins that are the sequential enzymes of a metabolic pathway that will lead to a final product. The length of codons that determines the sequence for a unique protein is called a gene. The length of the chromosome for one metabolic pathway is the operon.



VARIATIONS TO THE “CENTRAL DOGMA” OF GENETICS: OTHER types of RNA: • rRNA (ribosomal RNA):

o combines with structural and enzyme proteins in the cytoplasm to form a structure called the RIBOSOME

o ribosomes are found on the ROUGH ENDOPLASMIC RETICULUM (RER) o this is the ACTUAL SITE of protein synthesis in the cell (factory & shipping center) o more on this below

• tRNA (transfer RNA): o transports (carries) amino acids to the mRNA to manufacture proteins at the ribosome o 20 types of tRNA since each corresponding to 20 amino acids used in making human

proteins o On one end of the tRNA is the anticodon (matches up with the opposite complementary

nucleic acids that are on the mRNA codon) o At the other end of the tRNA is a site that attracts and temporarily holds only one type of

specific amino acid. Interfering RNA? • Now that you think you know everything about DNA, RNA and so forth, what about “interfering

RNA (RNAi)? • These RNA are formed from individual genes

o They go BACK to the DNA and REGULATE how the DNA functions • Different types of RNA:

o small nuclear RNA (snRNA) – involved in RNA splicing o small nucleolar RNA (snoRNA) – modifies ribosomal RNA o small interfering RNA (siRNA) – bind to RNA and degrades it o micro-RNA (miRNA) – goes BACK into the nucleus and regulates gene expression

• more on “small RNA”: o small nuclear RNA (snRNA) – involved in RNA splicing o small nucleolar RNA (snoRNA) – modifies ribosomal RNA o micro-RNA (miRNA) – goes BACK into the nucleus and regulates gene expression

• these miRNA may alter histone methylation on the DNA, thus either enhancing or reducing DNA expression

• this is a way for cells to REGULATE genetic expression without actually changing the DNA genome

• this is called epigenetics (see below) “Jumping” genes:

• Literally, sections of DNA that insert themselves into the DNA strand • May be associated with many different disease states • Sometimes also called “mobile” DNA • Long known in plants & bacteria, but only recently recognized in human cells • See (Kazazian & Moran, 2017): http://www.nejm.org/doi/full/10.1056/NEJMra1510092

http://www.nejm.org/doi/full/10.1056/NEJMra1510092



Epigenetics – Heritable Gene Control! • the epigenome remembers what happened in the individuals lifetime and affects what portions of

the DNA are expressed, fun interaction: http://learn.genetics.utah.edu/content/epigenetics/control/ • this is also hereditable and can be passed along to the offspring • the histone proteins that wrap around the chromosome create a nucleosome • the nucleosome coils on itself to form chromatin (collapsed DNA-protein) • on the histone proteins there are different types of tags

o Some tags tend to loosen the histone proteins and allow transcription (e.g., acetyl tags from a chemical reaction called acetylation

o Other tags tend to prevent transcription (e.g., methyl tags from a chemical reaction called methylation)

• Thus, “acetylation” or “methylation” of the DNA histones can turn on transcription OR silence the genome

Clinical correlates of epigenetics: • Knowledge of how these small RNAs work has therapeutic potential

o we could alter how ribosomes and/or chromosomes work • Nice review article: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4055025/ • Clinical example: cocaine exposure represses an enzyme called G9A, altering histone

methylation, and creating a “preference” for cocaine o has implications for the understanding of how addiction develops o has implications for pharmaoclogic targets in treatment of addiction o knowledge of how these small RNAs work has large therapeutic potential (alter ribosomes) o http://www.drugabuse.gov/news-events/nida-notes/2008/03/epigenetics-promise-new-

science • Obesity and epigenetics: evidence of methylation in obesity that may be heritable

o The Lancet (2014); http://www.ncbi.nlm.nih.gov/pubmed/24630777

Collapsed DNA-protein (chromatin) from https://www.nih.gov/news-events/news-releases/new-imaging-technique-overturns-longstanding-textbook-model-dna-folding

http://learn.genetics.utah.edu/content/epigenetics/control/

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4055025/

http://www.drugabuse.gov/news-events/nida-notes/2008/03/epigenetics-promise-new-science

http://www.drugabuse.gov/news-events/nida-notes/2008/03/epigenetics-promise-new-science

http://www.ncbi.nlm.nih.gov/pubmed/24630777

https://www.nih.gov/news-events/news-releases/new-imaging-technique-overturns-longstanding-textbook-model-dna-folding








DNA REPLICATION & MUTATIONS: DNA Replication : • DNA replicates by "uncoiling" and "unpairing" (helicase enzyme does this)

• DNA (deoxyribonucleic acid) with nucleic acid bases of Cytosine, Adenine, Guanine, Thymine as the “backbone” of the molecule

• Cytosine pairs with Guanine, Adenine pairs with Thymine as “complementary” base pairs to form two new chains identical to the original parent DNA

• DNA polymerase matches up the base pairs and creates a new strand – BUT it can only move in one direction (5prime to 3prime due to energy requirements of the process) – on the “leading” strand, the DNA is synthesized in one continuous strand

• But on the other strand (the “lagging” strand), another enzyme (primase) creates RNA fragments, then DNA polymerase builds segments of DNA discontinuously (Okazaki fragments) that are later joined together into one full strand

See Video: Simple baby steps: https://www.youtube.com/watch?v=5qSrmeiWsuc Another video: https://www.youtube.com/watch?v=2LnXpaVUG0Y

https://www.youtube.com/watch?v=5qSrmeiWsuc

https://www.youtube.com/watch?v=2LnXpaVUG0Y



Mutations: • are often the result of this DNA replication process becoming damaged • can also be deliberately induced by enzymes in the cell • Mutations – how things go wrong:

o the wrong base pair is inserted (base pair substitution) o frameshift mutations (additions or deletions)

• Are all mutations a cause of disease? NO !! o mutations may be silent (abnormal DNA but doesn’t cause illness) o mutations may actually cause disease o mutations can also be part of the normal functioning of a cell!!

Example: B-lymphocytes make antibodies for normal immune function; the antibodies (immunoglobulin proteins) that they make are directed against specific substances in the environment. Every time this happens, the B-lymphocyte is said to be “committed” – it will ONLY make that specific type of immunoglobulin for the rest of it’s cellular lifespan. The way this happens is that a specific cellular enzyme (activation-induced cytidine deaminase, or AID) creates a selective mutation on exactly the correct stretch of DNA in the cell.

• Mutations can be spontaneous, and some genes are more prone to mutation ("hot spots") & occur at rate of 1:10,000 or higher.

• MUTAGENS: o environmental factors that initiate or promote mutations o inlcude ionizing radiation, drugs, chemicals

• TERATOGENS: o produce fetal abnormalities o these problems may or may not be traced to genetic mutation

• Pharmacological Correlate: o the PARP enzyme (poly-ADP ribose polymerase) normally regulates DNA transcription. o Inhibition of PARP causes double-strand DNA breaks, which then activates another

process called homologous recombination (HR) repair. o HOWEVER, if someone has the BRCA mutation, the normal HR repair doesn’t occur and

an error-prone repair process occurs instead. o This leads to cell death – thus, PARP inhibitors are drugs that are cytotoxic to cancer cells,

especially those associated with BRCA mutation such as breast and ovarian cancer.



Knudson’s Two-Hit Hypothesis:

• Some mutations are inherited – germline mutations • Others are acquired – somatic mutations • fffThe first “hit” is the inherited (germline mutation) that may be silent • The second “hit” is the acquired mutation (somatic mutation) from environmental mutagens,

and now the effect of the mutation is seen • Famous statement by Dr. Judith Stern (UC Davis):

o “genetics loads the gun, environment pulls the trigger” o For a nice overview of the interaction of environment and intrinsic genetic susceptibility,

leading to human disease, see (Olden, 2008): http://www.mdpi.com/1660-4601/5/1/4/htm

o National Cancer Institute website on cancer & genetics (includes inherited syndromes, genetic counseling, and more): http://www.cancer.gov/about-cancer/causes-prevention/genetics/overview-pdq

National Cancer Institute page on cancer and genetics: http://www.cancer.gov/about-cancer/causes-prevention/genetics

http://www.mdpi.com/1660-4601/5/1/4/htm

http://www.mdpi.com/1660-4601/5/1/4/htm

http://www.cancer.gov/about-cancer/causes-prevention/genetics/overview-pdq

http://www.cancer.gov/about-cancer/causes-prevention/genetics/overview-pdq

http://www.cancer.gov/about-cancer/causes-prevention/genetics

http://www.cancer.gov/about-cancer/causes-prevention/genetics



Proto-oncogenes and oncogenes – mutations and cancer:

• in the normal (non-mutated) state these genes are called proto-oncogenes and control cell division

o Either accelerate cell division o OR o Take the brakes “off” by turning off genes that slow division

• In the mutated state, we call them oncogenes o Implicated in cancer – they are cancer-promoting genes

• How does the proto-oncogene become and oncogene? o Usually acquired via environmental triggers o Can also be inherited

• TP53 oncogene: o A mutated p53 gene is an oncogene o This is also called “tumor protein 53” gene, or TP53 o P53 is actually a gene that normally is a tumor suppressor gene! o It makes a protein (TP53) that suppresses cell division o This has been called the guardian of the genome o If DNA is damaged, the TP53 protein activates genes to repair the genetic damage o If the damage cannot be repaired, it tells the cell to undergo apoptosis (programmed

cell death) o When mutated it allows the formation of cancer and acts as an oncogene o Many very aggressive cancers are associated with p53 mutations:

http://ghr.nlm.nih.gov/gene/TP53 See more on apoptosis below…

http://ghr.nlm.nih.gov/gene/TP53



What about apoptosis? Apoptosis vs. Necrosis? Response to stress? • Apoptosis is natural cell death, sometimes called programmed cell death (term literally means

“dropping off”) o Important in NORMAL development – in early organ formation, more cells than are needed

are created, and they the organ is formed by loss of excess cells as part of differentiation o Important in protecting the body against damaged cells – cells are told to “commit cell

suicide” if they have damage that cannot be repaired o Pro-apoptotic factors include free radicals and some viruses o This "cell suicide" may also be due to lack of tonic hormonal factors that stimulate

continued healthy activity (e.g. as in denervation atrophy, dementia) • Adaptation to stress:

o Mild stress results in compensatory adaptation that allows further ability to survive additional stress – this adaptation is called hormesis

o Part of this response may be apoptosis if the cell cannot adapt to the mild stress • Mechanism of apoptosis – two pathways:

o Enzymatic pathways (caspase enzyme system) & autophagy (cell self-digestion) • Necrosis is cell death via other mechanisms in response to extreme stress

o Some theories assume this is chaotic and uncontrolled; other theories have evidence of some control of the process and use the term “necroptosis”



Regenerative medicine & stem cell therapy: • Regenerative medicine involves using stem cells to replace cells in degenerative disorders • Used in conditions such as Alzheimer’s dementia, Parkinson’s disease, etc. • Examples of some sources of stem cells:

o mesenchymal stem cells (MSCs) from blood o embryonic stem cells (fetal tissue) o adult stem cells that are “induced” to become pluripotential

“inducing” adult stem cells often includes “stressing” the cells by chemical applications before transplantation, to take advantage of hormesis

Use of adult stem cells has removed much of the stigma of stem cell therapy o Direct programming of fibroblast cells “turn them” into the type of cell you want

Has been used to convert cells into pacemaker cells in the heart and treat sick sinus syndrome, see (Marban & Cingolani, 2015, JAMA): http://jama.jamanetwork.com/article.aspx?articleid=2382986

• Background information: o Nice discussion of adult stem cells at NIH:

http://stemcells.nih.gov/info/basics/pages/basics4.aspx (if the link asks for login information just click “cancel” and it will go to the website)

o Nice overview of hormesis, regenerative medicine, apoptosis & necrosis (2013): http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3682200/

o Nice overview of regenerative medicine in JAMA (Atala & Murphy, 2015): http://jama.jamanetwork.com/article.aspx?articleid=2247152

http://jama.jamanetwork.com/article.aspx?articleid=2382986

http://stemcells.nih.gov/info/basics/pages/basics4.aspx





CELLULAR REPRODUCTION (THE CELL CYCLE) – OVERVIEW: Mitosis: • replication of all the chromosomes to make up another diploid cell, exactly like the first cell. • The original cell is the "mother" which gives rise to two "daughters." • This happens in all somatic cells. Meiosis: • replication and division of the "mother" cell in the gonads (germ plasm) to create HAPLOID cells

that only contain 23 chromosomes — ½ of each pair. • When the two haploid cells (ovum, sperm) combine, the number jumps back to diploid, and

undergoes mitosis (above) to create another complete organism (fetus). • This is sexual reproduction (more below). • This happens in the cells of the gonads (germ plasm). Organs: • made up of specialized cells in specialized structures • Non-dividing cells:

o Some specialized cells (nerve, lens cells, muscle cells) can't divide any longer. o Clinical correlate:

damage to these specialized cells results in fibrosis healing (scar tissue), rather than renewal of original architecture & cellular function.

o New news: recently discovered that cardiac muscle cells can actually regenerate under the

right conditions – it was previously thought that it was impossible for these specialized cells to divide and regenerate tissue

• Rapidly dividing cells: o intestine, skin, lung o May be especially susceptible to ionizing radiation (DNA breakage leading to cancerous

mutations). Great animations of mitosis/meiosis (click on button “start the animation”): Mitosis: http://www.cellsalive.com/mitosis.htm Meiosis: http://www.cellsalive.com/meiosis.htm

http://www.cellsalive.com/mitosis.htm

http://www.cellsalive.com/meiosis.htm

news:%EF%82%A7

news:%EF%82%A7



DIVISION (CELL CYCLE) – MITOSIS & CYTOKINESIS: cycle takes 12-24 hours • the cell that is going through division is called the “mother cell”

o replicates its DNA & undergo nuclear division (mitosis) o grows in size and split the cell (cytoplasmic division is cytokinesis) o splits to form two diploid daughter cells

• necessary so the body can maintain cell mass (growth, replace dead cells). • NOT part of sexual reproduction, no sharing of DNA from one individual to another • Phases of mitosis & cytokinesis:

o Interphase: growth, replication of chromosomes (DNA), then each replicated chromosomes collapses into two chromatids (each a complete chromosome) attached by centromere; nuclear membrane disappears, spindle fibers originate from centrioles at opposite ends of the cell. Phases: G1, G2, S phases. Slowly dividing cells have a long interphase.

o Metaphase: spindle fibers pull centromeres to the center (to form the equatorial plate -- also called metaphase plate) & "line up" the replicated chromosomes, anchored by their centromere.

o Anaphase: centromeres split and chromatids are pulled apart into a complete set of chromosomes (46) on each half of the cell; this is division of the chromosomes.

o Telophase: nuclear membrane forms, spindle fibers disappear, chromosomes uncoil, cytoplasm divides, and two diploid daughter cells are formed.

Great animation of cell cycle (click on button “start the animation”): http://www.cellsalive.com/cell_cycle.htm

Hormonal control: • growth factors. • Examples:

o PDGF -- platelet derived growth factor for connective tissue o IL-2 -- interleukin 2 for T-lymphocytes o erythropoietin for RBC

Genetic control: • control genes. • example: oncogenes that may promote cancerous tumors

http://www.cellsalive.com/cell_cycle.htm



The mitosis “molecular clock” – the telomere and telomerase enzyme: • The telomere:

o after each cell division, the chromosome becomes shortened t the telomere (the tip of the chromosome).

o Eventually, the shortening is so great that the replicating enzymes can't "read" the chromosome to replicate it, and the cell can no longer divide – this is sometimes called the “Hayflick limit”

o This prevents cells from indefinite reproduction, otherwise called the "molecular clock” o Recent evidence that the length of the telomere is associated with cancer

(Willeit P, et al. Telomere length and risk of incident cancer and cancer mortality. JAMA July 7, 2010;304(1):69-75. Abstract: http://jama.jamanetwork.com/article.aspx?articleid=186171

• Telomerase is an enzyme that prevents the normal shortening of the chromosome at the telomere, and therefore allows indefinite cell division, i.e. a cancerous type of growth.

o Blocking the telomerase enzyme might inhibit cellular division (returning the cell to a normal "clock"), might prevent cellular division of cancerous cells once a normal limit has been reached.

o Other uses might be harnessing the telomerase to enhance cellular divisions, such as in repair of traumatized tissues (can we grow a new arm if we lose our arm in trauma?)

• Another example of how the telomere affects pathophysiology – telomere shortening in T-lymphocytes may impair their ability to prevent infection: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3786437/

• For examples of research targeting telomerase and telomeres in cancer therapy, see: http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0085155 and http://www.nejm.org/doi/full/10.1056/NEJMoa1515319#t=article

• Can we turn back the hands of time? Lengthen our telomeres with a better diet? See the Mediterranean diet and telomere length (Crous-Bou, et al., 2014) in the BMJ: http://www.bmj.com/content/349/bmj.g6674



http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0085155

http://www.nejm.org/doi/full/10.1056/NEJMoa1515319#t=article

http://www.bmj.com/content/349/bmj.g6674



CONTROL OF CELL REPLICATION: Complete cycle: 12 - 24 hours.

GROWTH FACTORS & CELL REPLICATION: • Cells respond to growth factors by interaction with cell receptors. • By definition, any chemical that can react with a cell receptor is called a

ligand. • The receptor protein has a particular shape due to the way it is folded up in 3-

D space • The function of the receptor, just like the function of any protein, is therefore

based on this unique shape and the ligand can interact with the receptor because it “fits in” to the shape and also interacts based on similar attractive/repulsive forces between the chemical components of the ligand and the receptor.

• Once the ligand/receptor interaction takes place, “signal transduction” occurs – the signal is the actual interaction of the ligand & receptor and transduction means converting that signal into something else (e.g. intracellular enzyme activity).

• In the case of growth factors, and sterol (steroid) hormones, the effect is to turn on chromosome transcription of mRNA, which in turn is sent to the cytoplasm to make new proteins (translation).

Hormonal control: • growth factors. • Examples:

o PDGF -- platelet derived growth factor for connective tissue

o IL-2 -- interleukin 2 for T-lymphocytes

o erythropoietin for RBC Genetic control: • control genes. • example: oncogenes that may

promote cancerous tumors



SEXUAL REPRODUCTION REQUIRES MEIOSIS: creating haploid cells Why meiosis? • two haploid cells (from meiosis) join to make diploid cell, that can divide and become a new

organism – this is how genetic material is SHARED among members of a species • hopefully, this enhances survival of the offspring, leading to improved species survival • If the trait is advantageous, eventually the offspring with this trait will become more successful at

survival & reproduction, increasing the numbers of individuals with this trait in the population • This may be confined to a geographical area, since the trait may be advantageous because of

climate or other conditions (e.g. endemic infectious diseases, etc.). Meiosis: • haploid germ cells are egg (ova produced in ovary) & sperm (produced in testes) • the mother cell (diploid cell with 23 PAIRS of chromosomes) will produces 4 haploid daughter cells

(with 23 chromosomes -- 22 autosomes, & only one sex chromosome, either X or Y). • Process occurs in well defined steps:

o #1: mother cell replicates all the chromosomes, each becoming two chromatids joined at a central centromere. At the beginning of meiosis, when the chromatids line up in the mother cell,

the pairs of homologous chromosomes originally from Mom & Dad have a chance to exchange genetic material (the genes/alleles) since they are very close together.

Thus, each ovum (sperm) is NOT LIKE ALL the other produced by Mom (Dad). o #2: first division each set of homologous chromosomes to daughter cells. o #3: sister chromatids separate cell division yielding 4 cells, each with a haploid

chromosome number (23 chromosomes). Great animations of mitosis/meiosis: Mitosis: http://www.cellsalive.com/mitosis.htm Meiosis: http://www.cellsalive.com/meiosis.htm Meiosis errors: • Non-disjunction Errors means non-separation of the sister chromosomes during meiosis. • Other errors

o breakage, deletions, inversions, duplications & translocations of parts of the chromosome resulting in specific clinical syndromes.

o Some areas are particularly fragile in lab cultures and are related to clinical syndromes (fragile-X, second most important cause of genetic mental retardation, mostly in boys).

http://www.cellsalive.com/mitosis.htm

http://www.cellsalive.com/meiosis.htm



CHROMOSOME ABNORMALITIES & DISEASE SYNDROMES: 1. Chromosome count: • Euploid cells have the correct number of chromosomes. • Aneuploidy means there is not an exact multiple of 23 chromosomes.

o Examples: o Trisomy-21: Down syndrome, the #1 cause overall of mental retardation (see:

http://ghr.nlm.nih.gov/condition/down-syndrome ) o Monosomy-X: loss of one X-chromosmome, causing Turner's syndrome (see:

http://ghr.nlm.nih.gov/condition/turner-syndrome ) o 47,XXY: Klinefelter syndrome (see: http://ghr.nlm.nih.gov/condition/klinefelter-syndrome )

• Polyploidy: too many full sets of chromosomes (e.g. triploidy, tetraploidy -- these usually abort spontaneously, although may be found in the liver; commonly found in plants)

2. Sex determination:

• A Y chromosome presence always results in male genitalia (the SRY region, or sex-determining region exists on the Y chromosome)

• if more than one X chromosomes – sterility and retardation

3. Meiosis errors: • Non-disjunction Errors means non-separation of the sister chromosomes during meiosis. • Other errors

o breakage, deletions, inversions, duplications & translocations of parts of the chromosome resulting in specific clinical syndromes.

o Some areas are particularly fragile in lab cultures and are related to clinical syndromes – these areas of DNA tend to “break” easily and are often called “hot spots” because they are associated with genetic abnormalities Example: fragile-X, second most important cause of genetic mental

retardation, mostly in boys – may be associated with methylation of histone proteins that allow “hot spots” for genetic mutations to occur, see http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3851448/

Example: autism, see http://www.ncbi.nlm.nih.gov/pubmed/24859339 and http://www.dnalc.org/view/881-Hotspot-for-Autism-Genes.html

4. Imprinting errors:

• We know that inheritance is driven by inheriting two copies of a gene – one gene on one homologous chromosome from mother, one gene on homologous chromosome from father

• Usually, both genes are active, but in some genes, one is inactivated – this deactivation is determined by the “parent of origin” (there is a “stamp” on the gene that says “from father” or “from mother”)

• This process is called genomic imprinting and the “stamp” is dependent on methylation of the histone proteins (recall from epigenetics)

• Some human diseases, such as Prader-Willi syndrome and Angelman’s syndrome, result from improper genomic imprinting – the person has two copies of a gene from only one parent; this is called uniparental disomy (UPD)

• For a discussion, see http://ghr.nlm.nih.gov/handbook/inheritance/updimprinting

http://ghr.nlm.nih.gov/condition/down-syndrome

http://ghr.nlm.nih.gov/condition/turner-syndrome

http://ghr.nlm.nih.gov/condition/klinefelter-syndrome


http://www.ncbi.nlm.nih.gov/pubmed/24859339

http://www.dnalc.org/view/881-Hotspot-for-Autism-Genes.html

http://ghr.nlm.nih.gov/handbook/inheritance/updimprinting



KARYOTYPE See more next page • An ordered & numbered display of chromosomes • Identification accomplished by staining and "band" identification & matchup. • What is often confusing is the pictures we see of chromosomes – they look like pairs of "double

chromosomes" • Chromosomes are easier to see in the lab when the cells are stained during metaphase, when

each chromosome in the pair has just replicated itself into two chromatids joined by a central centromere, just prior to the equal chromosome division and cell division of mitosis.

Karyotype: an ordered, numbered display of the chromosomes • The chromosomes are harvested from cells undergoing replication (usually from cultures of

WBC), caught at the stage of the replication where the duplicated chromosome is attached to its duplicate by the chromatid (easier to stain at this stage); cells harvested via amniocentesis or chorionic villus sampling.

• Amniocentesis: (invasive test) o withdrawing amniotic fluid at 16 weeks gestation to harvest fibroblast cells that have

been sloughed off by the fetus) provides cells to grow in culture o They are karyotyped (ordered, numbered display of chromosomes) to determine if

chromosome count and gross morphology of chromosomes is normal • Chorionic villus sampling (CVS): (invasive test)

o earlier at 8 weeks o but,rates of fetal loss are greater than for amniocentesis.

• Cell-free fetal DNA testing: (newer non-invasive test) o Fetal DNA does cross over from the fetus into the mother’s bloodstream via the

placenta so a maternal prenatal blood test can look for “cell-free” DNA o Now can test for three common chromosomal disorders (Down/trisomy 21,

Patau/trisomy 13, Edwards/trisomy 18). • Why are we doing this diagnostic test? Therapeutic choices:

o termination of pregnancy (TOP) based on abnormal findings (thus, the CVS would be preferred since the TOP is easier to perform at an earlier stage of gestation)

o awareness of potential for neonate needing intensive care at birth o mostly, if TOP would not be done under any circumstances, there is no reason to

perform invasive procedures (just places the fetus at risk)



Clinical procedures using karyotyping: an ordered, numbered display of chromosomes.

• Amniocentesis o Withdrawing amniotic fluid at 16 weeks gestational age to harvest fibroblast cells that

have been sloughed off by the fetus) provides cells to grow in culture o They are karyotyped (ordered, numbered display of chromosomes) to determine if

chromosome count and gross morphology of chromosomes is normal • Chorionic villus sampling (CVS)

o earlier at 8 weeks gestational age o cells taken directly from a structure in the placenta o rates of fetal loss MAY be greater than for amniocentesis (although in centers that do a

lot of these procedures, rates of fetal loss are approaching equal) • Cell-free fetal DNA testing: (newer non-invasive test)

o Fetal DNA does cross over from the fetus into the mother’s bloodstream via the placenta so a maternal prenatal blood test can look for “cell-free” DNA

o Now can test for three common chromosomal disorders (Down/trisomy 21, Patau/trisomy 13, Edwards/trisomy 18)

o Not for low-risk testing, see: https://labtestsonline.org/understanding/analytes/cell-free-fetal-dna/tab/test/

o A positive test would require one of the invasive tests to confirm due to high false positive rate

• Why are we doing this? o termination of pregnancy (TOP) based on abnormal findings (thus, the CVS would be

preferred since the TOP is easier to perform at an earlier stage of gestation) o awareness of potential for neonate needing intensive care at birth o mostly, if TOP would not be done under any circumstances, there is no reason to

perform these invasive procedures (just placing the fetus at risk) Good websites on chromosomal disorders & karyotyping: http://www.biology.iupui.edu/biocourses/N100/2k2humancsomaldisorders.html and http://anthro.palomar.edu/abnormal/abnormal_5.htm Other methods to analyze the genome in prenatal testing: • Chromosomal microarray testing identifies additional information in addition to what is seen in

the karyotype; may help better identify abnormalities that may be more subtle, see (Wapner, et al., 2012, N Engl J Med): http://www.nejm.org/doi/full/10.1056/NEJMoa1203382?viewType=Print&viewClass=Print

• FISH test: Fluorescence in situ hybridization (FISH) rapidly detects chromosome number and specific DNA sequences, including small deletions or structural abnormalities that are not seen in standard karyotyping. A probe that has the target DNA sequence will attach to the chromosome if the sequence is present. A fluorescent tag on the probe signals when the DNA sequence has been identified. For example, if FISH test shows three signals for a chromosome 21 probe, then this indicates trisomy 21 (Down syndrome). Similarly, failure of the probe to attach indicates a chromosome deletion.

A site with good patient brochures on these procedures: https://www.bcm.edu/research/medical-genetics-labs/index.cfm?PMID=24709

https://labtestsonline.org/understanding/analytes/cell-free-fetal-dna/tab/test/

https://labtestsonline.org/understanding/analytes/cell-free-fetal-dna/tab/test/

http://www.biology.iupui.edu/biocourses/N100/2k2humancsomaldisorders.html

http://anthro.palomar.edu/abnormal/abnormal_5.htm

http://www.nejm.org/doi/full/10.1056/NEJMoa1203382?viewType=Print&viewClass=Print

https://www.bcm.edu/research/medical-genetics-labs/index.cfm?PMID=24709



Examples of DNA mutations and genetic variations:

FROM: Feero WG, Guttmacher AE, Collins FS. Genomic Medicine – An Updated Primer. N Engl J Med May 27, 2010;362(21):2001-11. AT: http://www.nejm.org/doi/pdf/10.1056/NEJMra0907175 (Used by permission)

http://www.nejm.org/doi/pdf/10.1056/NEJMra0907175

http://www.nejm.org/doi/pdf/10.1056/NEJMra0907175



GENOME-WIDE ASSOCIATION STUDIES, GENE THERAPY, AND GENE EDITING: Recombinant DNA: • Combining different DNA sequences in the lab (investigational or clinical). • Enzymes called ENDONUCLEASES cleave (cut) DNA into pieces • these can now be inserted into another cell. Clinical correlates: • Drug manufacture by genetically engineered bacteria:

o human DNA is incorporated into bacterial DNA in the form of cytoplasmic plasmids (circular DNA fragments) or insert directly into the bacterial DNA.

o The bacteria are cloned (allowed to multiply from a single colony), proteins made from the inserted DNA can be recovered & purified for medical use.

o This is how insulin was originally mass produced in the "human" insulin (not pork or beef extracted) form.

o Note that whenever a drug is produced by recombinant technology, you will see a small letter “r” in front of the drug (e.g. rHGH for recombinant human growth hormone).

• Drug manufacture by CLONED mammals: o DNA to make a particular protein is inserted into a fetal cell o this is brought to term via in vitro fertilization (surrogate animal mother). o The mature infant born of this process may now produce the desired protein product. o Currently, being tried in sheep ("Dolly") in order to obtain clotting factor IX from the milk of

cloned sheep that have been genetically engineered in this manner. Where did we get the DNA for this process? • mRNA can be extracted, and "REVERSE TRANSCRIPTASE" enzyme will make "complementary

DNA = cDNA" that can be studied using the plasmid method in bacteria • identify what portion of the DNA is desirable to purify & later insert into cells for cloning. How do we get enough DNA? • the POLYMERASE CHAIN REACTION (PCR) uses cycles of temperature to cause uncoiling of

DNA in the presence of the nitrogen base pairs, thus replicating the DNA. • This will copy one small tissue or blood sample millions of times, for diagnosis or forensic

medicine (e.g. semen analysis, blood analysis).



Older gene therapies: • Usual method:

o cells are harvested from the patient o a retrovirus or adenovirus is used to INSERT genes into cells o cells are put back into the patient (e.g. bone marrow)

• Examples: o in cystic fibrosis, giving them gene therapy via aerosolized nebulizer to help them make

enzymes that degrade the proteins that build up in their lungs o in severe combined immunodeficiency disease (SCID) using patient bone marrow cells that

are modified using a retrovirus, providing patients with the missing gene (a success in 2009)(Auiti, et al., 2009, N Engl J Med):

http://www.nejm.org/doi/full/10.1056/NEJMoa0805817 • An nice overview (2013) review article (Kaufman, et al., EMBO Molecular Medicine):

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3840483/ • Some of these older methods resulted in unexplained deaths and slowed the research for many

years What about newer genetic engineering? See next page…

http://www.nejm.org/doi/full/10.1056/NEJMoa0805817




Newer CRISPR-Cas9 gene editing (gene engineering): • CRISPR-Cas9 gene therapy is still experimental

o inserting "correct" DNA into the patient's DNA, either at the somatic cell level (fully grown fetus or neonate), or at the embryo level of development (in utero)

o OR removing sequences of DNA that might contribute to disease • CRISPR is a protein that searches for specific sequences on the DNA & then untwists DNA and

cuts out that sequence (CRISPR stands for “clustered regularly spaced short palindromic repeats”) – now another segment of DNA can be inserted

• Good overview (New England BioLabs): https://www.neb.com/tools-and-resources/feature-articles/crispr-cas9-and-targeted-genome-editing-a-new-era-in-molecular-biology

• Video from MIT (where one of the original scientists works): https://www.youtube.com/watch?v=2pp17E4E-O8

• Video from World Science Festival: https://www.youtube.com/watch?v=UCC2oILE7i0 • how CRISPR-Cas9 was discovered by the scientists: https://mpnforum.com/cascade/ • Current investigations:

o Editing plant genomes to reduce browning of vegetables o Editing mosquito DNA to produce more male mosquitoes (those don’t bite) and make them

more malaria resistant o Editing HIV virus OUT of an infected cell o Editing human T-cells to make them more aggressive cancer fighters (then infuse back into

the patient with cancer)

https://www.neb.com/tools-and-resources/feature-articles/crispr-cas9-and-targeted-genome-editing-a-new-era-in-molecular-biology

https://www.neb.com/tools-and-resources/feature-articles/crispr-cas9-and-targeted-genome-editing-a-new-era-in-molecular-biology

https://www.youtube.com/watch?v=2pp17E4E-O8

https://www.youtube.com/watch?v=UCC2oILE7i0

https://mpnforum.com/cascade/



GWAS (Genome-wide Association Studies), Whole Genome Studies, and Personalized (Precision) Medicine: • Overview:

o human genome contains 3x109 base pairs o genetic variance at one locus (location) predicts genetic variance at an adjacent locus o survey the genome for risk of disease by genotyping 500,000 markers in the genome o can identify common, low-risk variants that are present in more than 5% of the population

which confer a small risk of disease (typically RR 1.2 to 5.0) (such as type 2 diabetes) • Good fact sheet on GWAS from the NIH: http://www.genome.gov/20019523 • Ways of thinking about genetic inheritance in human disease:

o single-nucleotide polymorphisms (SNPs) one nucleotide difference in sequence • variations in the DNA sequences of humans affect disease development, response to

drugs & pathogens • compare differences in matched cohorts (with & without a disease) • What makes up our individual differences is mainly due to SNPs • Opens the door to personalized medicine such as pharmacogenomics – tailoring

drug therapy to the individual • SNP fact sheet: https://www.broadinstitute.org/education/glossary/snp • Most drug response differences between individuals are due to SNPs:

http://www.ebmconsult.com/articles/single-nucleotide-polymorphism-snp-drug-therapy • Public access databases exist to look up SNPs:

http://www.ncbi.nlm.nih.gov/sites/entrez?db=snp o monogenic: caused by a mutation in a single gene (also called a Mendelian disease)

• Examples in clinical work: o GWAS of susceptibility to leprosy (Zhang, et al., 2009):

http://www.nejm.org/doi/full/10.1056/NEJMoa0903753 o GWAS and cancers (the “genetic lineage” of the cancer cell):

• Study of gene sequencing in uterine leiomyomas (Mehine, et al, 2013): http://www.nejm.org/doi/full/10.1056/NEJMoa1302736

• Review (Hardy & Singleton, 2009): http://www.nejm.org/doi/full/10.1056/NEJMra0808700

o Genomics and drug response (Want, et al., 2011): http://www.nejm.org/doi/full/10.1056/NEJMra1010600#t=article

o Personlized (Precision) Medicine (Collins & Varmus, 2015): http://www.nejm.org/doi/full/10.1056/NEJMp1500523#t=article

o FDA publication on this topic: http://www.fda.gov/downloads/ScienceResearch/SpecialTopics/PersonalizedMedicine/UCM372421.pdf

o Finding pathogenic variants in the human genome (Evans, et. al, 2017): http://jamanetwork.com/journals/jama/article-abstract/2625307

http://www.genome.gov/20019523

https://www.broadinstitute.org/education/glossary/snp

http://www.ebmconsult.com/articles/single-nucleotide-polymorphism-snp-drug-therapy

http://www.ncbi.nlm.nih.gov/sites/entrez?db=snp



http://www.nejm.org/doi/full/10.1056/NEJMra0808700

http://www.nejm.org/doi/full/10.1056/NEJMra1010600#t=article

http://www.nejm.org/doi/full/10.1056/NEJMp1500523#t=article

http://www.fda.gov/downloads/ScienceResearch/SpecialTopics/PersonalizedMedicine/UCM372421.pdf

http://www.fda.gov/downloads/ScienceResearch/SpecialTopics/PersonalizedMedicine/UCM372421.pdf

http://jamanetwork.com/journals/jama/article-abstract/2625307

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