Exam 1: Genetics

Genetics Overview
• Describe the organization of the human genome and how DNA is packed into human
chromatin
o Combines with proteins/histones to “wind around” and become inaccessible
o Distinct territory between chromatin
• Describe the organization and distribution of the mitochondrial genome
o mtDNA contains 37 genes (~16.5K nucleotides)
o mtDNA only inherited from mother
o Most mitochondrial genome still located in nucleosome DNA (aka: chances are
even mitochondrial disorders are dad’s fault, too)
• Describe the function and structure of genes
o Watson and Crick double helix (1953); aka Rosalind Franklin is a bossette and old
white guys stole her work for credit
o Phosphate (-) and sugar backbone attached to base form nucleotides
o Bases hydrogen bond to each other (A-T is 2, G-C is 3)
o Extends from 3’ always
• Identify the components of DNA
o Purines

o Pyrimidines

o Genetics as a science is 160 yrs old (DNA in 1940’s); 1990’s human genome project
o Griffith: genetic material can be transferred (bacteria in mice)
o Avery, McLeod, McCarty: genetic material carried in DNA (compared with protein/RNA),
but they were nobodies, so no one cared
o Hersey-Chase: confirmed Avery et. al w/fancy respectable name and radioactive
virus/phage DNA (sexy pictures will convince anyone)

Epigenetics
• Describe the mechanisms and regulation of gene expression including transcription,
translation, cis and trans acting factors, micro RNAs, and chromatin structure
o Genomic expression is regulated at every step and is highly controlled
o Chromatin structure (mechanical accessibility) – repositioning or removal of
nucleosomes on DNA; promotes accessibility and costs ATP
 o Gene transcription (chemical sliding-scale switches):
§ Activators – bind to enhancers and speed up transcription
§ Repressors – bind to silencers and slow down transcription
§ Coactivators – process signals from activators and repressors in
transcription
§ Tata Box – holds the secrets of life, the universe, and everything else; 42;
does the actual transcription
o Processing and editing of RNA:
§ Chemical modification (says what or what won’t be transcribed – see
above)
§ Cutting (gets rid of introns and keeps exons during transcription)
§ Splicing (stitches together exons); can alternate exon order to create
different proteins (each exon acts as a unique building module/block with
reordering creating different final products; exons = base ingredients in a
cake recipe and splicing = unique selection/order for different types of
finished cakes)
§ End modification (adds a cute poly A tail like a ponytail on a lil’ girl before
picture day; aka: it’s ready)
• Define the concept of epigenetics and explain the role of epigenetic mechanisms in
regulation of gene expression and development
o Epigenetics = heritable changes in gene expression or cellular phenotype caused
by mechanisms other than changes in the underlying DNA sequence (aka: not
the base trait, but the code that influences how much that trait is expressed like
when you decide 2 cloves of garlic in the recipe is obviously not enough)
o Some examples:
§ Histones (DNA wraps around becoming inaccessible)
• Acetylation (on); catalyzed by HATs; makes accessible by
neutralizing Lys (+) charge
• Phosphorylation (on)
• Methylation (off); common on C and contributes to structure
§ Chromosome structure (tight winding for mechanical inaccessibility; see
above for remodeling)
§ DNA methylation (represses gene transcription)
§ Imprinting (gene expression regulated by gender of parent)
§ Gene silencing (genetic code that “silences” expression)
§ RNA interference (induces cleavage, promotes degradation, or blocks
translation by binding to other RNA)
§ Prions? (transfer genetic expression from environment to organism and
promote unhealthy protein folding; definitely/hopefully not planned or
as intentional as the others, but accomplishes the same end goal of
throwing a wrench in the works)
• Explain how patterns of gene expression vary during development and differentiation
 o Signaling chemicals can induce silencers or activators on various genes so as to
promote or diminish growth during development of anatomical features (for
example, eye growth is promoted in utero, but falls off after initial growth)
o In stem cells, epigenetic mechanisms determine differentiation
• Explain how epigenetic mechanisms regulate normal gene expression and the
implication for normal phenotype variation
o Degree of regulation will result is variation of phenotype expression for
individuals that have similar or identical genetic code; examples:
§ Variation in pigmentations of eyes, hair, skin, etc.
§ Identical genetic material for bone growth plate development will result
in various bone growth plates that lead to individuals of various heights
§ Sex organs (the penis and the clitoris) originate from same genetic origins
with variability in epigenetics through phenotype development and
hormonal signaling resulting in vastly different anatomical appearance
and mechanism
• Explain how altered epigenetic mechanisms contribute to diseased phenotypes
o Over expression can lead to things like cancer or prion diseases
o Under expression can result in enzymatic deficiencies
o Genome = all the DNA in an organism
o Transcriptome = all the RNA in an organism
o Proteome = all the proteins expressed by a genome (actions speak louder than words; if
it’s not expressed, it doesn’t count)

Genetic Assays
• Describe the structure and function of chromosomes
o Chromosomes contain cellular DNA and their structure is notably organized and
compact, needing conformational structural changes both in analysis and cell
replication
• Compare and contrast their replication and segregation in mitosis and miosis (only
mitosis was covered in lecture)
o Somatic (Body; aka: non-sex) Cell Cycle
§ Interphase
• G0 – growth and repair
• G1 – checkpoint near end; can revert cell to G0
• S (DNA replication)
• G2 – checkpoint near end to check for replication quality (can
force apoptosis)
§ Mitotic Stage
• Mitosis (division of nucleus)
o Prophase; chromosomes pair up
o Metaphase; chromosomes are grabbed by spindles and
centered, checkpoint for spindle assembly and alignment
 o Anaphase; chromosomes are split by spindles
o Telophase; nucleus splits
• Cytokinesis (division of cytoplasm)
• Define the concept of epigenetics and explain the role of epigenetic mechanisms in the
regulation of gene expression and development
o G1 checkpoint heavily regulated by growth factors and quality of DNA
o Epigenetic factors can force cell into G0 phase for growth and repair
• Describe the common molecular diagnostic techniques used in molecular diagnostic
testing and how they are used in genetic testing
o Polymerase Chain Reaction (PCR)
§ copies (aka: amplifies) DNA
§ only requires trace amounts
§ doubles amount each cycle
§ primer attaches 5’ and moves to 3’ of template (the primer is reverse and
DNA only grows form 3’ to 5’
§ incorporates endonucleases (palindromic sequences that marks specific
sequence by cutting and binding inside normal DNA sequence)
§ Restriction Fragment Length Polymorphism (RFLP) = different alleles will
result in different cleavage sites from enzymes during PCR
o Electrophoresis
§ Used to determine size (small length travels faster) and/or charge (-
travels faster)
o Southern Blot
§ Chromosomal DNA (larger sections)
§ Blot on membrane and attach radioactive probe then x-ray
§ Can be used to show homozygous vs. heterozygous and genetic heritage
o Northern Blot
§ RNA (small sections)
§ Similar process to Southern, but only with RNA instead of DNA (aka:
expressed phenotypes vs. genotypes)
§ Can be used to analyze tissue differences in the same body
o DNA Microarray
§ mRNA converted to cDNA using viral reverse transcriptase
§ Analysis of active transcription (healthy vs. unhealthy cell transcription)
§ Simultaneous analysis of many genes
§ DNA chip is the micro version
o Western Blot
§ Proteins isolated
§ Proteins mixed with denaturing agent and detergent (SDS) to normalize
negative charge across entire length
§ Antibody for section of protein is labelled and mixed with protein
 § Protein is ran on electrophoresis gel
o Enzyme-Linked Immunosorbent Assay (ELISA)
§ Similar to Western Blot
§ Less specificity, but faster
o Eastern Blot
§ No one cares; post-translational modifications (PTM)
o Chain Termination DNA Sequencing (Sanger Method)
§ No -OH group in 3’ nucleotide (ddNTP) forcing termination
§ Several chains of variable lengths with terminating ddNTP fluorescent
§ Computer sequencing by length and fluorescence for full genome
§ Used in conjunction with PCR
o Immunoassays
§ Measures concentration of macromolecule in solution using antibody
with marker
§ Applied in blood typing

Autosomal Inheritance
• Mendelian Principles
o Principle of Segregation: sexually-
reproducing organisms possess genes
that occur in pairs (alleles) and alleles of
a gene segregate equally among all the
gametes produced by this organism
o Principle of Independent Assortment:
genes at different loci are transmitted
independently of each other (must not
be adjacent on the same chromosome)
• Pedigrees (strongly suggested to see examples
provided in slideshow for applications)
o Graphical representations of family relationships and the occurrence of a
particular disease among family members
o Proband – indicated by an arrow; first individual displaying disease
o Circle – female
o Square – male
o Diamond – unspecified sex
o Roman numerals – generations
o Horizontal line (single) – mating pair
o Horizontal line (double) – consanguineous mating (incest)
o Vertical line – parent to offspring
o Dot – carrier
o Slash – deceased
o Darkened shape – affected
 o Vertical angled triangle w/o line – fraternal twins
o Vertical angled triangle w/ line – identical twins
o SB – still birth (usually accompanied with gestation period)
• Factors that affect expression and inheritance pattern of disease-causing genes
o De novo mutation (new mutation); seen in offspring
o Germline mosaicism (carrier in only gametes – not somatic cells)
o Reduced penetrance (carry diseased allele, but exhibit no phenotype despite it)
o Age-dependent penetrance (delay of disease phenotype until later age)
o Variable Expression (range of phenotype expression; influenced by environment,
heterogeneity, mutations, etc.)
o Locus heterogeneity (when multiple genes contribute to a disease and which are
affected decide severity)
o Pleiotropy (genes that have multiple effects on the body)
o Consanguinity (inbreeding increases diseased allele frequencies and diminishes
variation in gene pool)
o Coefficient of relationship= (1/2)^(n-1); n=number of individuals in pedigree path

Genetic Variation
• Describe the types and extent of variation seen in the human genome, including both
sequence and structural variation in coding and non-coding sequences
o Codon Commandments
§ The sequence of bases in a codon must follow the direction of translation
§ The code is non-overlapping
§ The code is read in a fixed reading frame
§ The code is unambiguous
§ The code is degenerate
o Mutation rate is ~ 1 in a billion per cell division with male gamete rate 4x higher
o ~100 mutations/generation
o Mutation in somatic (body) cell aren’t inherited
o Mutation in germline (sex) cell are inherited
• Explain the difference between a polymorphism/non-pathogenic and a pathogenic
alteration (mutation)
o Polymorphism – a common mutation that more than 1% of the population has
o Mutation – a less common mutation that less than 1% of the population has
o Can be different for geographic territories and specific populations
• Describe the nature of mutation and the mechanisms that produce different types of
mutations
o Mutation – a heritable change in the genetic material; typically spontaneous;
increased rate of frequency with chemical or radiation treatment (induced
mutations)
o Base pair substitution – 3 types
§ Silent – makes no amino acid change; typically, the last letter in a codon
 § Missense – results in a different amino acid; typically, the first or second
letter in a codon
§ Nonsense – no protein developed; stop codon inserted by replaced letter
o Deletion or Insertion
§ frameshift when only one or two letters
§ expanded repeats when unregulated repeating codon
§ duplications when an entire gene is repeated
o Promoter mutation – decrease affinity for a polymerase at promoter site leading
to reduced production of protein
o Splice site mutation – interferes with designation of introns and exons slowing
down transcription and resulting in less protein production
o Mutation classifications based on effects of gene function
§ Gain of function mutation: alters the action of a gene enabling it to do
more than originally coded for (not always good)
§ Dominant negative mutation: a protein product that doesn’t work and
gets in the way of other stuff working
§ Loss of function mutation: alters the action of a gene disabling it to do
what it was originally coded for (often making it completely knocked out)
§ Haploinsufficiency: genes from one parent or the other don’t work and
the other half isn’t enough to keep up with production demands
• Explain genetic variation with respect to geographic ancestry and evolution
o Genetic variation is guided by natural selection among populations in different
geographic areas in order to promote survivability within a given environment
(ex: sickle cell gives resistance to malaria and is common in African ancestry
populations)
o Gene flow occurs from interbreeding of populations (ex: African Americans are
less likely to have sickle cell than Africans due to European ancestry and no
health benefit in the US geographically)
o Genetic drift occurs from a random change of frequency in alleles and is
observed in smaller populations (ex: sickle cell anemia has no benefits to African
American populations and results in higher mortality rates, resulting in less of
the recessive genotype being present in the gene pool and a shift in allele
frequency for the disease in the US); usually triggered by a population bottleneck
event (in the example above, higher mortality rates for sickle cell)
o Founder effect – similar to population bottleneck; limitations in genetic diversity
result in higher frequencies of mutations within an isolated population
• Describe the implications of genetic variation due to geographic ancestry for disease
risk, prevention, and treatment
o Genetic variation in populations means various strengths and weaknesses
towards different illnesses among different ancestral groups
o As populations continue to mingle, those distinguishing factors become more
muddled
Sex Linked and Non-Traditional Inheritance
• Sex Determination in Humans and Dosage Compensation
o ~1100 genes localized on X (153 mil base pairs) and a couple dozen on Y (50 mil)
o PAR1 and PAR2 – pseudo autosomal regions shared between X and Y
chromosomes (at the ends)
o Females are mosaics of X chromosome (Lyon hypothesis); males are hemizygous
§ Shown in phenotypic evidence (calico cats)
§ Biochemical evidence (alternate versions of enzymes in woman’s body)
§ Cytogenic evidence (Barr bodies)
§ Inactivation in 7-10 days after fertilization and is incomplete (which is
why it doesn’t become pathological like Turner syndrome, etc.)
• Sex Linked Inheritance (including screencast)
o Typically X linked
o Sex limited traits – occurs only in one sex because the other lacks anatomy
o Sex influenced traits – occurs more strongly in one sex because of hormonal
development
o X linked recessive
§ Preference in phenotype for males
§ Never passes father to son
§ Women are typically carriers
§ “Manifesting Heterozygotes” – carriers will exhibit partial phenotype
o X linked dominant
§ Observed in both genders
§ Males can suffer lethal situation due to hemizygous
o Y linked inheritance – passes from father to son exclusively
o Mitochondrial inheritance
§ Inherited exclusively through maternal line
§ Various degrees of expression due to multiple copies of mtDNA (often
seen as “deficiencies”)
o Imprinting
§ Similar to X inactivation
§ One parent’s genes are transcriptionally deactivated
§ 15q deletion
• In father = Prader-Willi syndrome (prader > pater > father)
• In mother = Angelman syndrome (mother is an angel)
o Uniparental disomy
§ Double set of chromosomes from one of the parents in zygote (sperm
with 2 or ovum with 2)
§ Can result in duplicates of diseased chromosomes
§ Can result in trisomy when a single set from the other parent is added
o Anticipation
 § Each generation has a stronger phenotype for disease
§ Caused by repeating expansion that grows in each generation
§ Ex: Huntington and Fragile X

Linkage and Gene Mapping

• Be able to interpret a pedigree
o Allows for tracking of marker on chromosome that is closely related to gene, but
isn’t the gene itself
• Differentiate between gene mapping and physical mapping
o Gene mapping – using crossover information to determine distance between
genes (genes are shown by cM’s rather than true distance)
o Physical mapping – various methods to determine the actual physical location of
genes on chromosomes (genes are shown by true distance rather than cM’s)
• Define linkage
o Genes in close proximity will show a degree of dependence during assortment
and cross-over
o Crossing over occurs during prophase I of meiosis
o Polymorphisms used as markers
§ RFLPs (Restriction Fragment Length Polymorphisms)
§ VNTRs (Variable Number Tandem Repeats) – genetic fingerprinting
§ STRPs (Short Tandem Repeat Polymorphisms)
o Qualities that make a good marker
§ Codominant
§ Close to target gene
§ Highly polymorphic
§ Marker IS NOT the gene
o Used to localize disease carrying genes
• Determine recombination frequency
o Measured in centimorgans (cM) = distance in which there is a 1% chance of
crossover in a single generation; ~ 1 mil base pairs
o Frequency of recombination (cM) = (total number of recombinants)/(total
number of recombinants and non-recombinants)
• Define LOD scores in relation to genetics
o LOD = logarithm of the odds
o Used to determine whether two loci are linked are randomly assorted
o LOD = Z = log(10) (likelihood of observing pedigree data if recombination
frequency is .1/likelihood of observing pedigree data if recombination frequency
is .5)
§ If Z is 3 or more, then linked
§ If Z is -2 or less, then non-linked
§ If Z is between, then inconclusive
• Differentiate between linkage and association
 o Linkage is conceptually based on probability of penetrance in a pedigree (ex: all
people related to this person have X because of diseased gene)
o Association is conceptually based on probability of penetrance based on a
genome (ex: all people with blue eyes have X because blue eye gene associates
with x diseased genes)
o Linkage and association are not mutually inclusive or exclusive
o Topic not formally addressed in slideshow – independent study suggested
• Know physical mapping techniques
o Physical mapping = base pair analysis (see Sanger Method in genetic assays for
example)
o Linkage is based on traits rather than distance (only 4.5% of DNA maps or
influences genes)

Clinical Cytogenetics
• Chromosome Abnormalities
o Common in live births (1/150)
o Leading known cause of mental retardation and pregnancy loss
o Abnormality in chromosomal number
§ Euploidy – “good set;” multiple of 23 in humans
§ Polyploidy – complete extra sets of chromosomes beyond 2 (tri or tetra
and typically results in miscarriage)
§ Aneuploidy – extra or lacking individual chromosomes
• Monosomy: only one chromosome (ex: Turner syndrome)
o Most common: 21, 18, 13
o Almost always incompatible with life
o Caused by non-disjunction during meiosis
• Trisomy: three chromosomes (ex: Down syndrome)
o Abnormalities in chromosomal structure
§ Occur in prophase 1 (when crossing over occurs)
§ Unbalanced – gain or loss of genetic material affects individual
§ Balanced – mis-combination with net zero on genetic material affects
individual’s offspring
§ Most result in mental and anatomical disfunction during development
§ Robertsonian translocations – two acrocentric chromosomes lose short
arms and fuse long arms together; short arms are trashed and
chromosome number drops by 1
§ Deletions – chunks get cut out and lost; most common of clinically
significant chromosomal structure abnormalities (aka: it happens the
most when life and death are on the line); can create ring chromosomes;
if centromere is lost, issues with cell reproduction and lost (leads to
monosomy)
§ Inversion loops – keeps all genetic material in parent, but creates issues
in offspring
• Chromosome Structures and Staining
o Short arm (top) – p; long arm (bottom) – q

o
o Banding used to localize arms – G Band most common (and easiest to use)
• Karyograms
o Karyogram = picture of chromosomes arrested in metaphase
o Karyotyping = interpretation of chromosomes by genes present (aka: numbering)
o Bands/regions are numbered from centromere (p10 or q10) to telomeres; first
number is region and second is band (ex: 14p32; 14th chromosome, short arm
(p), 3rd region, 2nd band; sub-bands can be shown after decimal)
• Fluorescent in situ Hybridization (FISH)
o Can be used on any fetal cell
o Applied in microarrays for marking chromosomes/arms/genes/alleles
o Applied to classic Karyograms for more refined imagining
o Detects microdeletions
• Characteristics of Key Chromosomal Disorders
o Most syndromes require surgical intervention for unseen anatomical defects
o Down syndrome (Trisomy 21)
§ Phenotype: flattened nose and face, upward slanting eyes, single palmer
crease with pinky shortened and curving in, wide separation between big
toe and other toes, mental retardation, intestinal blockages, congenital
heart disease
§ Factoids: 10% mental retardation cases in the US, 50% survive to 50 yo,
men are sterile, women are not (and 50% offspring can have trisomy 21),
de novo mutation, mosaicism can result in milder phenotype
§ Maternal age effect
o Edwards syndrome (Trisomy 18)
§ Phenotype: clenched hand with overlapping fingers (most distinct),
prominent back of skull, malformed ears, small mouth/jaw/neck,
congenital heart defects
§ Factoids: more common than Down syndrome at conception, high
mortality rate (5% live to 1 yo), more severe development difficulties
than Down syndrome, 90% cases extra 18 comes from mother
§ Maternal age effect
o Patau syndrome (Trisomy 13)
§ Phenotype: oral facial cleft, extra finger (polydactyl), small eyes
(microphthalmia), CNS/heart/renal abnormalities, abnormal testes, no
eyebrows
 § Factoids: high mortality rate (5% live to 1yo), severe mental retardation,
80% full trisomy and 20% translocation/partial trisomy
§ Maternal age effect
o Turner syndrome (Monosomy X)
§ Phenotype: manly girls (no female sex organ development including
ovaries and breasts), normal intelligence, shorter, distinct neck
musculature
§ Factoids: 99% do not survive to term, 60-80% lack second x from
paternal, extreme phenotype variation due to mosaicism
o Klinefelter syndrome (Disomy X – aka: XXY)
§ Phenotype: womanly boys (males with female traits), mild phenotype,
taller with longer extremities, small testes (sterility is common), breast
development (gynecomastia), regular intelligence (with learning
disabilities)
§ Factoids: typically diagnosed in adulthood after puberty, testosterone
treatment is helpful
o XYY syndrome (Disomy Y)
§ Phenotype: taller, reduced IQ, normal testosterone levels, normal sexual
development and fertility, mild phenotype
§ Factoids: originally associated with violent behavior that was ultimately
debunked