Genetics and After Midterm Flashcards
Malaria Example
Malaria highly prevalent in Africa
»_space; Caused by a parasite transmitted by mosquitos. Invades mature RBCs (red blood cells)
First line of defense against malaria is genetic: alteration in hemoglobin or glucose-6-phosphate dehydrogenase (G6PD) deficiency
Hemoglobin = oxygen-transport protein
G6PD = critical enzyme that prevents free radical damage in RBCs by converting glucose to ribose-5 phosphate; NADPH produced, protects against build up of free radicals (reactive oxygen species)
G6PD deficiency can lead to premature destruction of RBCs (hemolysis)
However, G6PD-deficient individuals are somewhat resistant to malaria: parasite infects mature RBCs. In G6PD deficiency, RBCs are cleared rapidly before they mature, thus parasite dies as well
Sickle Cell is caused by a one amino acid substitution (glutamate replaced with valine) in hemoglobin
Sickle cell anemia
Changes in hemoglobin structure (due to mutation in the Hb gene) result in distortion of RBC shape from biconcave to half-moon (sickle) shape - Sickle cell anemia
Sickle cells get trapped in capillaries and cannot carry enough oxygen to body tissues. Parasite cannot live in sickle cells because 1) not enough nutrients, 2) body eliminates sickle cells, thus the parasite too.
Hardy-Weinberg Equilibrium Law
Genotype frequencies will remain in equilibrium (stable) from generation to generation in an infinitely large and randomly mating population in the absence of
mutations, migration and immigration of the population, and selection against a genotype
p = fr(A) frequency of dominant allele (A) q = fr(a) frequency of recessive allele (a)
Sum of allele frequencies p + q = 1 (100%)
Genotypic frequencies p2 + 2pq + q2 = 1
p2 = frequency of genotype AA 2pq = frequency of genotype Aa (carriers) q2 = frequency of genotype aa
Multiple Alleles: ABO Blood Groups
Multiple alleles: 3 or more alleles of the same gene
When multiple alleles exist, an individual diploid organism still inherits only 2 alleles
Example: ABO blood groups
ABO blood group system was discovered in 1901 by
Karl Landsteiner
Significant for blood transfusions: ABO incompatibility
between donor and recipient can result in life threatening situations
4 different blood groups/types: A, B, AB, and O
Each blood group characterized by the presence of an antigen on the surface of RBCs and the absence of its corresponding antibody in the serum
Different blood types due to different glycoproteins (sugars) on the surface of RBCs. Individuals inherit the gene which codes for the sugars to be added to the red cell
»_space;>A allele codes for an enzyme (transferase) that
adds N-acetylgalactosamine to the terminal sugar
> > > B allele codes for an enzyme (transferase) that
adds D-galactose to the terminal sugar
Landsteiner’s Law
If an individual possesses an ABO antigen, must not have the corresponding antibody
If an individual lacks an ABO antigen, must have the corresponding antibody
Type A – A antigen present; has anti-B
Type B – B antigen present; has anti-A
Type O – no antigen present; has both anti-A and anti-B
Type AB – A and B antigen present; has neither anti-A nor anti-B
Inheritance
IA and IB are dominant in a homozygous or heterozygous state
Both IA and IB are dominant to IO which is recessive
Both IA and IB are co-dominant to each other
AA or AO = Type A Classic Dominance
BB or BO = Type B Classic Dominance
OO = Type O Homozygous Recessive
AB = Type AB Co-Dominance
Hemolysis: If an individual is transfused with an incompatible blood group, destruction of the red blood cells will occur. This may result in the death of the recipient.
Rhesus (Rh) Blood Group System
most important blood group system after ABO
consists of 50 defined blood-group antigens. Most important are D, d, C, c, E and e
Rh system controlled by 2 closely linked genes: RHD and RHCE: Rh gene complex. (One Rh gene complex inherited from each parent)
RhD codes for presence of D antigen on the red blood cell surface (Rh+).
Rh+ individuals may be homozygous DD or heterozygous Dd
“d” is non-functional and produces no antigen (Rh-) - dd
all Rh- individuals must receive Rh- blood.
Production of Ab to D requires exposure to the antigen. A person with Rh- blood does not have Rh Abs naturally in the blood plasma
RHCE gene codes for presence of C, c, E, and e
At the RHCE gene locus, depending on the allele present, one of four antigenic combinations are produced: ce, Ce, cE or CE.
C or c --- co-dominant D or d --- classic dominance E or e --- co-dominant Most common Rh+ = CDe/CDe Most common Rh- = cde/cde
Any and all combinations are possible
Only D confers Rh+ blood
Hemolytic disease of the newborn (erythroblastosis fetalis)
If a Rh- mother during pregnancy is exposed to Rh (D) antigen, she will produce antibodies to this antigen that can cross the placenta and destroy Rh (D) positive fetal cells, resulting in death of the fetus
Preventive treatment = RhoGam Therapy (prevents mother from producing antibodies by binding to fetal Rh+ antigen)
Intrauterine fetal monitoring with repeated ultrasound examination and amniocentesis
Fetus Rh+:
Intrauterine blood transfusion of ‘Rh-’ blood into the baby’s circulation
Exchange transfusion to the baby after birth
In cases of severely sensitized women, medical termination of pregnancy is considered
Mendel’s Pea Plants
The pea plants’ traits were often similar to their parents (purebred)
However, sometimes they showed obvious differences in these traits
The passing of traits from parent to offspring is called heredity
Experiment: He cut away the pollen-bearing male parts of a flower and dusted that flower with pollen from another plant
Followed F1 generation and then let F2 generation self pollinate and noticed that 3/4 of seeds were yellow and 1/4 were green, even if none of the parents were green.
Conclusion: individual factors must control the inheritance of traits that exist in pairs and remain separate instead of blending
they come in alternative versions resulting in variations in inherited characteristics, e.g. yellow peas and green peas
*genetic characteristics (such as height, color, shape, etc) are controlled by unit factors that exist in pairs and they are inherited as a unit
Mendel’s First Law of Segregation
During gamete formation, the paired unit factors (genes) for a specific trait separate (or segregate) randomly into their two alleles (MEIOSIS), so that each gamete receives one or the other with equal likelihood.
At fertilization, the two alleles – one from each parent – unite randomly.
Monohybrid cross
inheritance of a single trait
Ex) AA x Aa
Genotype = 1:1 (either AA or Aa) Phenotype = all dominant
Aa X Aa
Genotype = 1:2:1 ( 1 AA, 2 Aa, 1 aa)
Phenotype = 3:1 (3 dom, 1 recess)
Dihybrid cross
inheritance of 2 traits together
Ex) Yellow and Round seeded plants (YyRr) X Yellow and Round seeded plants (YyRr)
Genotypic ratio: 1YYRR 2YYRr 2YyRR 4YyRr 1YYrr 2Yyrr 1yyRR 2yyRr 1yyrr Phenotypic ratio: 9:3:3:1 9 yellow-round 3 yellow-wrinkled 3 green –round 1 green wrinkled
Mendel’s Second Law of Independent Assortment
the two alleles for seed color segregate independently of the two alleles for seed shape, producing four different pea phenotypes: yellow-round, yellow-wrinkled, green-round, and green-wrinkle
Therefore, each character is independently inherited
During gamete formation, segregating pairs (alleles) of unit factors (genes) assort independently of each other
genes for different traits are inherited independently of each other if they are on different chromosomes
Linkage
Linked genes cannot undergo independent assortment
Linkage: the failure of two genes to assort independently can occurs when genes are close to each other on the SAME chromosome and are inherited as a unit The closer the loci of the genes on the chromosome, the tighter the linkage
However, genes far apart from one another on the same chromosome can assort independently because
of recombination
Recombination (crossing over) = process in which exchange of genetic information occurs between 2 homologous chromosomes during meiosis
The frequency of crossing over (recombination rate) between any 2 loci on a single chromosome is proportional to the distance between them
The maximum rate of recombination is 50%
**A recombination frequency «_space;50% between 2 genes shows that they are linked***
A recombination frequency of 50% (1:1:1:1:1) shows that the transmission of 2 linked genes is indistinguishable from that of two un-linked, independently assorted genes
Example of linked genes
2 closely linked genes control the expression of ALL Rh antigens: RhD gene & RhCE gene
The genotype is determined by the inheritance of 2 pairs of 3 alleles
Each locus on each chromosome has its own set of alleles which are Dd , Cc , and Ee
There are 8 gene complexes at the Rh locus
CDe, Cde , cDE, CdE, cDe, cdE, CDE, cde
Double Crossovers
2 crossovers both occurring between the two loci being examined
First crossover changes the parental configuration of alleles to the recombinant configuration
Second crossover changes the recombinant configuration back to the parental
The net result is that the genes are in the parental configuration, SAME AS IF NO CROSSOVERS HAD OCCURED
Thus, any even number of crossovers is the same as 0 crossovers, and any odd number is the same as 1 crossover
Since we only see the offspring and not the actual crossovers, it is very easy to undercount the number of crossovers that occurred
The further apart 2 genes are, the more likely it is that undetected double crossovers will occur between them
Interference
Interference (I) is the phenomenon through which a crossover event in one region of a chromosome influences (inhibits) the occurrence of a crossover in an adjacent region of the chromosome
Interference is measured by calculating the coefficient of coincidence (C)
Crossovers in adjacent chromosome regions are usually not independent. This interaction is called interference.
A crossover in one region usually decreases the probability of a crossover in an adjacent region.
Inference = 1 - (observed # of double recombinants)/ (expected # of double recombinants)
SRY gene
Sex-determining region of the Y chromosome
Located near the end of the short arm of the Y chromosome
Plays a major role in causing the undifferentiated gonad to develop into a testis
Patterns of Inheritance
Autosomal Dominant:
affected individuals have an affected parent
either sex affected:
Familial hypercholesterolemia
Huntington disease
Neurofibromatosis
Marfan syndrome
Von Hippel-Lindau disease
Autosomal Recessive:
affected individuals usually have un-affected (carrier) parents either sex affected
Sickle cell anemia
Cystic fibrosis
Phenylketonuria
X-linked Dominant:
affected individuals have an affected parent of either sex affected; No male-to-male transmission!!!!
Fragile X syndrome
Hypophosphatemic rickets
X-linked Recessive:
affected individuals usually have un-affected (carrier) parents either sex affected (mother); No male-to-male transmission
Duchenne muscular dystrophy
G-6-PD deficiency
Hemophilia A and B
Mitochondrial Inheritance:
Inherited maternally because only mother contributes mitochondria during conception
Either sex affected
Usually neuropathies and myopathies because brain and muscle are highly dependent on oxidative phosphorylation
Sex-Linked Inheritance
Sex-linked trait: Traits only found on the X chromosome
Sex-linked disease: Disease or syndromes caused by recessive genes located on X-chromosome
Female -
Homozygous = disease
Heterozygous = variable expression (carriers)
Male – hemizygous
X linked alleles always show up in males whether dominant or recessive because males have only one X chromosome
EXAMPLES!!!!!!!!!!!!!!!!!!!!
Colorblindness: inability to distinguish the differences between certain colors
most common type is red-green colorblindness, where red and green are seen as the same color
Color vision genes are located on X chromosome
Hemophilia (The Royal disease): inability of blood to clot
absence of clotting factors VIII and IX, located on X chromosome
prolonged bleeding time in affected individuals can lead to death
Sex-linked Syndromes
of chromosomes is not an exact multiple of the haploid set -aneuploidy’
XO – Turner’s Syndrome (45 chromosomes, missing one X)
YO – Lethal Mutation
XXY – Klinefelter’s Syndrome (47 chromosomes, extra X)
XYY – Jacobs Syndrome (47 chromosomes, extra Y)
XXX – Triple X Syndrome (47 chromosomes, extra X)
Multiple XY – more than 2 X with a Y – like Klinefelter’s syndrome
Sex Influenced Traits
When the sex of an individual influences the expression of a phenotype that is not limited to one sex or the other.
Traits controlled by autosomal genes that are usually dominant in one sex but recessive in the other sex e.g. Pattern baldness
(Acts like a dominant trait in males and a recessive trait in females; different testosterone levels)
Sex-Limited Traits
When the expression of a specific phenotype is absolutely limited to one sex
Traits expressed only in females because males die before birth
Example: Male-lethal X-linked dominant traits such as Rett syndrome
Traits expressed only in males
Example: Duchenne muscular dystrophy (X-linked recessive); life expectancy 25 years
-Therefore X recessive not passed on from father onto daughters, very rare in females
Causes of Mutations
Substances Inducing Mutations
- Mutagens (Substances that alter DNA structure in various ways and can cause DNA damage; cause bases to mispair and bond with the wrong base (most common mutation); like UV light and chemicals like Benzene)
- Teratogens (Agents that cause harm or birth defects to an embryo or fetus)
- Carcinogens (Chemicals or ionizing radiation that cause or promote cancer)
Aneuploidy
of chromosomes is not an exact multiple of the haploid set (related to increasing maternal age)
Monosomy
Monosomy – loss of a single chromosome from a diploid genome (2n - 1 chromosomes)
Trisomy
gain of one chromosome from a diploid genome (2n + 1 total chromosomes)
Euploidy
of chromosomes is exact multiple of the haploid set
humans are euploidy
Polyploidy
more than 2 complete sets of chromosomes
Triploid
one extra complete set of chromosomes (3n)
Tetraploid
2 extra complete sets of chromosomes (4n)
Cause of numerical chromosome abnormalities
Nondisjunction =
cell division error in which homologous chromosomes fail to separate during segregation in meiosis I or the sister chromatids of a chromosome fail to separate during meiosis II or mitosis
Consequence of nondisjunction: imbalance in the number of chromosomes, one daughter cell has two chromosomes or two chromatids, and the other has none
Mosaicism
presence of different chromosome constitution (karyotype) within cells of a single individual
condition in which a tissue may contain cells that have some chromosome abnormality and other cells with normal karyotype
females are naturally mosaics for genes on the X chromosome because one X chromosome (paternal or maternal) in every cell is randomly inactivated (Barr body). Therefore, some cells express maternally-derived and others paternally-derived alleles
Types of numerical chromosomal abnormalities
Sex chromosome abnormalities
Autosomal (non sex-linked) chromosome abnormalities
Turner’s Syndrome
XO – Turner’s Syndrome (45 chromosomes, missing one X)
CLASSIFIED AS FEMALE
Most XO fetuses die before birth
Surviving individuals are females – 1 in 5,000 female births
noticeable at puberty, when secondary sexual characteristics fail to develop
immature ovaries- sterile poorly developed breasts short stature webbed necks heart problems retardation unaffected lifespan
Klinefelter’s syndrome
(XXY, 47 chromosomes)
CLASSIFIED AS MALE
Male – incidence 1 in 1,000 male births Small testes, low testosterone - sterile Breast development in about 50% of XXY individuals Sparse facial hair Subnormal intelligence in some cases 48, XXXY 49, XXXXY
Jacobs Syndrome (XYY)
Jacobs Syndrome (XYY)
Male – 1 in 1,000 male births
Excessively tall
sometimes retardation – not severe
usually fertile but sometimes fertility affected
antisocial behavior patterns e.g. sociopathic, aggressiveness
Trisomy X (XXX)
Females – 1 in 1,500 female births usually normal underdeveloped secondary sex characteristics sterility mental retardation
Autosomal chromosome abnormalities
Monosomies – loss of one chromosome (2n - 1) – lethal
Trisomies – addition of an extra chromosome (2n + 1):
Down Syndrome – Trisomy 21
Patau Syndrome – Trisomy 13
Edwards Syndrome – Trisomy 18
Down Syndrome
Caused by an extra chromosome 21 (trisomy 21);
nondisjunction in meiosis I;
father contributing extra chromosome in 15% of cases
The incidence of trisomy 21 is about 1 in 800 live births but SHARPLY INCREASES WITH MATERNAL AGE
Patau Syndrome
Trisomy 13
Incidence 1 in 15,000 live births; maternal disjunction
50% of these babies die within the first month, 90% die within the first year, and very few survive beyond the first year
multiple dysmorphic features: Small head Small or missing eyes Cleft lip/palate Extra fingers Heart defects Abnormal genitalia Severe developmental and mental retardation
Edwards Syndrome
Trisomy 18
90% die within the first year, and very few survive beyond the first year
The surviving individuals have severe mental and developmental retardation
Small face
Small sternum
**Clenched fingers and toes*****
Variations in chromosome structure
Due to break(s) along the axis of a chromosome
Deletion
Duplication
Inversion
Translocation (Transposition)
Unilateral
Reciprocal
Robertsonian TranslocationDeletions
A chromosomal deletion occurs when a chromosome breaks and a fragment is lost. This chromosome will be missing certain genes (can be a terminal deletion or an internal deletion; ABCDEF»_space; ABCEF or ABC/
Deletion Example: Cri du Chat Syndrome (46, 5p)
length of short arm of chromosome 5 deleted varies: longer deletions = greater impact
Duplications
A duplication occurs when a chromosome fragment becomes attached as an extra segment to a sister chromatid and thus it is present more than once in the genome
It is usually caused by unequal crossing over between synapsed chromosomes during meiosis or through a replication error prior to meiosis
ex) ABCDEF»_space;> ABCBCBCDEF
Examples: Bar-eyed Drosophila- Instead of normal
oval-eye shape, narrow slit-like eyes
Charcot-Marie-Tooth disease in humans –
neurological disease, chromosome 17 duplication
Inversions
An inversion occurs when a piece of chromosome has been lifted out, turned around and reinserted. Thus a chromosomal fragment reattaches to the original chromosome but in the reverse orientation
No loss of genetic information
If the rearranged chromosome segment includes the centromere then the inversion is termed pericentric
If it excludes the centromere then it is a paracentric inversion
ABCDEF»_space; ADCBEF
Individuals with one copy of a normal chromosome and one copy of an inverted chromosome are called inversion heterozygotes
For 2 such chromosomes to pair properly during meiosis I, an INVERSION LOOP is formed
The homologs will segregate resulting in 2 normal and 2 inverted chromatids
Inversions, unlike the other structural chromosomal abnormalities, do not affect gene dosage.
Translocations
the movement of a chromosomal segment to a new location in the genome
A translocation can be UNILATERAL in which a section of a chromosome breaks off and is inserted into the end of another chromosome (either homologous or non-homologous)
Reciprocal Translocations
exchange of segments between 2 non-homologous chromosomes (when a portion of one chromosome is transferred to another chromosome)
Usually generate so-called “balanced translocations”
In balanced translocations, there is NO NET GAIN OR LOSS of chromosomal material, just rearrangement of genetic material
Usually without phenotypic consequences
Philadelphia Chromosome
Reciprocal Translocation with phenotypic consequences;
Philadelphia chromosome = translocation between chr. 9 and chr. 22 leading to chronic myeloid leukemia
altered chromosome 22 is called Philadelphia
Burkitt’s Lymphoma
translocation between chr. 8 and chr. 2, 14 or 22
Translocations and Gamete Production
Individuals carrying translocations have a greater risk of producing gametes with unbalanced combinations of chromosomes
This depends on the segregation pattern during meiosis I
During meiosis I, homologous chromosomes synapse with each other
For the translocated chromosome to synapse properly, a translocation cross is formed which contains 2 normal chromosomes and 2 with translocated parts
3 Types of Meiotic Segregation
- Alternate segregation
- Chromosomes on opposite sides of the
translocation cross segregate into the same cell - Leads to normal and balanced gametes
- Both contain a complete set of genes and are
thus viable
- Chromosomes on opposite sides of the
- Adjacent-1 segregation
- Adjacent non-homologous chromosomes
segregate into the same cell- Leads to unbalanced gametes
- Both have duplications and deletions leading to
birth defects
- Adjacent-2 segregation
- Adjacent homologous chromosomes segregate
into the same cell
- Leads to unbalanced gametes
- Both have duplications and deletions leading to
birth defects
Robertsonian Translocation
Most common type of chromosomal rearrangement in humans
This translocation occurs as such:
Breaks occur at the extreme ends of the short arms of two non-homologous acrocentric chromosomes
The small acentric fragments are lost
The larger fragments fuse at their centromeric regions to form a single chromosome
Example:
Familial Down Syndrome
Familial Down Syndrome
Caused by translocation between chromosomes 14 and 21 - the majority of chromosome 21 is attached to chromosome 14
Chance of the same parents having a second affected child is extremely low
However, there are cases where the syndrome occurs in a much higher frequency over several generations and this is known as Familial Down Syndrome
Down Syndrome vs. Familial Down Syndrome
Down Syndrome: Nondisjunction during meiosis 47: (+21) No association with prior pregnancy loss Older mother Very low recurrence rate
Robertsonian translocation
46: -14, +t(14;21)
May be associated with prior pregnancy loss
May be a younger mother
Recurrence rate 10-15% if mom is translocation carrier; 1-2% if dad is translocation carrier
Spontaneous Mutation
changes in the nucleotide sequence of genes that appear to have no known cause (accidental)
Result from abnormalities in cellular/biological processes (ex. error in DNA rep.)
Underlying cause originates within the cell
The rate is exceedingly low and varies considerably between different organisms (even within the same species, the spontaneous mutation rate varies from gene to gene)
Hot spots = specific bases or regions that are more likely to be the site of a mutation within a gene
some genes have locations within the chromosome that make them more susceptible to mutation and some can be found within a single gene
Induced mutations
Caused by mutagens: agents that have potential to damage DNA
These can be chemical or physical agents (uv light, ionizing radiation, toxins)
Mutations can occur in germ-line or somatic cells
Germ-line mutations are those that occur in a sperm or egg cell - transmitted
Somatic mutations are those that occur in any cell in the body, except germ cells - not transmitted
Autosomal mutations are those within genes located on the autosomes
X-linked mutations are those within genes located on the X chromosome
Forward Mutation v. Reversion
In a population, the wild-type (normal) is the relatively prevalent genotype
A forward mutation changes the wild-type genotype into some new variation
A reverse mutation changes a mutant allele back to the wild-type
It is also termed a reversion
Loss or Gain of Function Mutations
Loss-of-function mutations: reduce/eliminate function of the gene product
When result in complete loss of function – null mutation
Gain-of-function mutations: result in a gene product with enhanced or new functions
ex) hereditary pancreatitis: mutation results in activation of a digestive enzyme (trypsin) that is normally inactivated in the pancreas
Sickle cell disease: mutation results in resistance to malaria (new function)
Single Gene Mutations
Change in the nucleotide sequence of a gene
May only involve a single nucleotide
Point mutation = a change of one base pair to another in a DNA molecule
Point Mutations may be:
> Base Pair Substitutions
- Silent
- Missense – new protein (amino acid substitutions)
- Nonsense – stop codon
> Base Pair Inversions
> Base Pair Insertions & Deletions
- Frameshift Mutations
- Triplet Repeats
Base substitution
Change in single base pair
A transition is a change of a pyrimidine (C, T) to another pyrimidine or a purine (A, G) to another purine
A transversion is a change of a pyrimidine to a purine or vice versa
Transitions are more common than transversions
These mutations affect protein synthesis. If one nucleotide of a DNA triplet is changed, the corresponding mRNA codon will also be altered, resulting in the creation of a new triplet that codes for a different amino acid and thus codes for a different protein
Silent Mutation
Silent mutations are those base substitutions that alter a codon but do not alter the amino acid sequence of the protein
Example: a change from normal codon CTC to CTT still codes for the same amino acid (Glu) in the protein, and thus, there is no alteration in function
Missense Mutations
Base substitutions in which an amino acid change does occur due to altered codon
Example: Normal CTC»_space; CAC leads to different amino acid (Glu»_space; Arg) in polypeptide, so different function
Sickle Cell Anemia is the result of one nucleotide substitution - Glutamic Acid is replaced by Valine - in the hemoglobin gene
Nonsense Mutations
those base substitutions that change a normal codon to a stop codon (UAG, UAA, or UGA) resulting in the termination of translation of the protein
Base Insertion or Deletion
Mutations may also involve the insertion or deletion of one or more nucleotides at any point in the DNA sequence within the gene
Example: AACGTCTGCAAAT»_space;> AACGTCTG AACGTCTGCAAAT»_space; AACGTCTGCAGAGACAAAT
Frameshift mutations
When loss or addition of nucleotides occurs, there is a shift in the reading sequence (frame) of DNA during protein synthesis
Change in the 3-letter codons - different amino acid sequence - completely different protein formed – frameshift mutation
If one of the many altered triplets is UAA, UAG, or UGA (the translation termination codons; stop codons) at that point polypeptide synthesis is terminated
Therefore, frameshift mutations can have severe consequences
Inversion
may also occur in the DNA sequence and result in altered amino acid sequence
Mutations Due to Trinucleotide Repeats
The term refers to the phenomenon that a tandem sequence of 3 nucleotides within a gene is repeated many times expanding the size of the gene
In normal individuals, these sequences are transmitted from parent to offspring without mutation
However, in persons with TNRE disorders, the length of a trinucleotide repeat is increased above a certain critical size
The number of repeats continues to increase in future generations
Occurs within the coding sequence of a gene:*
Trinucleotide repeats lead to increased presence of a particular amino acid (e.g. CAG expansion which codes for glutamine)
The encoded protein will contain long tracks of this amino acid
This causes the proteins to aggregate with each other; protein aggregates are usually toxic and implicated in many diseases
Occurs in a noncoding region of a gene
Trinucleotide repeats can become chemically modified and cause changes in RNA structure
This can result in gene inactivation/silencing
Examples of Trinucleotide Repeats mutations
Fragile X syndrome (CGG repeat - arginine)
> X-linked dominant trait
> When the number of repeats reaches over 230,
the CGG regions of the gene become chemically
modified (methylated) –
causing inactivation of the gene and this part of
the chromosome where this gene is located
becomes susceptible to breakage.
Huntington disease (HD)
> Autosomal dominant neurodegenerative disorder
(CAG repeat-glutamine; HTT gene)
> 36-39 repeats: later onset of the disease/slower
progression of symptoms; 40+ repeats seriously
affected
> affects normal protein expressed in the brain;
increased number of repeats = altered protein =
increased decay rate of certain types of neurons
Myotonic muscular dystrophy
> Autosomal dominant (CTG repeat – leucine; MDPK
gene)
> normal protein expressed in skeletal muscle;
increased number of repeats = altered protein =
muscle wasting
Types of Genetic Disorders
Chromosome disorders
> Sex chromosome abnormalities
> Autosomal chromosome abnormalities
Single – gene disorders
> Autosomal Dominant
> Autosomal Recessive
> X-linked dominant
> X-linked recessive
Multi-factorial disorders
> Multiple genes
> Environmental factors
Mitochondrial DNA disorders
> Damage to mitochondrial DNA
Autosomal Dominant Diseases
caused by an autosomal dominant allele
often encode structural proteins
both homozygotes and heterozygotes are affected
either sex is affected, exhibit the trait in approximately equal proportion, and equally likely to transmit the trait to their offspring
no skipping of generations: disease phenotype is usually seen in one generation after another – vertical transmission
if an individual has the disease, one parent must also have it
if neither parent has the trait, none of the children has
it
offspring usually heterozygous for the dominant allele (inherited from one parent)
50% of children inherit it
Examples: Huntington disease
Familial hypercholesterolemia
Von Hippel – Lindau disease
Neurofibromatosis
Marfan Syndrome
Familial hypercholesterolemia
AUTOSOMAL DOMINANT
(= subgroup of hyperlipoproteinemia)
**most frequent Mendelian disorder - 1:500
mutation in gene encoding low-density lipoprotein (LDL; bad cholesterol) receptor
high levels of LDL in blood because too little taken up by cells
narrowing of arteries (atherosclerosis), heart attacks at young age
heterozygotes 2-3× elevated plasma cholesterol level – heart attack by 40-50
homozygotes 5-10× elevated plasma cholesterol levels – heart attack as early as 5 years of age and before 20 and death likely by that age
Von Hippel – Lindau disease
AUTOSOMAL DOMINANT
Rare mutation in VHL (tumor-suppressor gene) on chr. 3
growth of tumors in the body (e.g. CNS, brain, spinal cord, kidney) and formation of cysts in internal organs
higher risk for renal cell carcinoma
Neurofibromatosis
AUTOSOMAL DOMINANT
disorder of the nervous system
mutation in NF1 or NF2 gene on chr. 17
affects how nerve cells form and grow causes tumors to grow on nerves/tan or dark spots on skin
“elephant man”
Marfan Syndrome
AUTOSOMAL DOMINANT
connective tissue disorder
caused by mutation in fibrillin-1 gene on chr. 15
defect results in too much growth of the long
bones of the body – arachnodactyly (spider-like fingers)
risk for aortic aneurism due to stretching of the aorta
Autosomal Recessive Disorders
rare - disease occurs only when both recessive alleles are present
females and males affected equally
parents are usually normal / unaffected (heterozygotes)
25% of offspring of 2 heterozygous parents (carriers) will be affected
often encode catalytic proteins
nearly all inborn errors of metabolism are recessive
onset is frequently early in life
Examples: Sickle cell anemia
Cystic Fibrosis
Phenylketonuria (PKU)
Tay-Sachs
Cystic Fibrosis
Most common lethal genetic disorder
AUTOSOMAL RECESSIVE
Mutation in CFTR gene on chromosome 7
1 in 25 Caucasian Americans is a carrier
Thick mucus builds up in lungs and digestive tract
Difficulty breathing & lung infections, pneumonia, bronchitis
Death by lung complications – average lifespan 37 years
Phenylketonuria (PKU)
AUTOSOMAL RECESSIVE
Can’t break down amino acid phenylalanine (missing critical enzyme)
Phenylalanine builds up and interferes with nervous system leading to mental retardation (IQ below 50) and even death
Early screening»_space; phenylalanine restricted diet for children with disorder
Tay-Sachs
AUTOSOMAL RECESSIVE
Lethal disease of the nervous system
Mutation in HEXA gene on chr.15 encoding hexosaminidase (enzyme that breaks down a fatty substance found in nerve tissue called ganglioside)
Fatty substance builds up in neurons
Newborns appear normal first few months; gradual paralysis and loss of nervous function, mental retardation, blindness by age 4-5 – no cure
Heterozygote carriers do not have disorder, but are protected from tuberculosis – mechanism unknown
more prevalent in Ashkenazi Jews (1 in 30)
glucose-6-phosphate dehydrogenase (G6PD)
X-LINKED RECESSIVE
G6PD = critical enzyme for preventing free radical damage in RBCs
Converts glucose to ribose - NADPH production
NADPH prevents building up of free radicals within cells
G6PD deficiency can lead to rupture and break down of RBCs (hemolytic anemia)
fava beans and napthalene contain compounds that produce free radicals
G6PD deficiency = resistance to malaria
Reason = malaria parasite infects mature RBCs and cannot survive in immature RBCs. In G6PD deficiency, RBCs are cleared rapidly before they mature, thus parasite dies as well
400 million people worldwide affected
Duchenne Muscular Dystrophy
X-LINKED RECESSIVE
Caused by a mutation in the dystrophin gene, an important structural component within muscle tissue
both sexes can carry the mutation, but females rarely exhibit signs of the disease
Affected individuals have learning difficulties and mental retardation
Most importantly: they have muscle weakness leading to muscular deterioration which starts as early as 3-5 years of age
ability to walk is lost by the age of 12 and have to use a wheelchair
They rarely survive past 20; death from breathing difficulty and heart disease
DUCHENNE = X-LINKED RECESSIVE DISORDER WHEREAS MYTONIC MUSCULAR DYSTROPHAGY IS AN AUTOSOMAL DOMINANT DISORDER CAUSED BY TRINUCLEOTIDE REPEATS
Multi-factorial Disorders
Caused by combination of multiple genes and environmental factors (e.g. diet and lifestyle) which can directly influence the expression of the genes involved
Examples:
Heart disease
Cancer
Mitochondrial DNA Disorders
Human mitochondrial DNA (mtDNA): 37 genes (13 encode proteins, 2 rRNAs and 22 tRNAs)
mtDNA is vulnerable to mutations because:
> High concentration of highly mutagenic free radicals
generated by cell respiration accumulate in such a
confined space
> Reduced ability to repair mtDNA damage than nuclear
DNA
> Inherited maternally: only mother contributes
mitochondria during conception; mitochondria in
sperm destroyed by the egg cell after fertilization
(mature oocyte >100,000 mtDNA copies, sperm <1000)
Either sex is affected
Factors Affecting Genetic Diseases
Decreased Penetrance Variable Expressivity Delayed Onset of Genetic Expression Genetic Anticipation Genomic Imprinting Germline Mosaicism
Penetrance
% of individuals that exhibit some degree of expression of a mutant genotype
Reduced penetrance =
an individual who has the genotype for a disease but
not exhibit the disease phenotype at all, even though
he or she can transmit the disease gene to the next
generation
Example: familial cancer syndromes; many people with a mutation in breast cancer genes BRCA1 and 2 will develop cancer during their lifetime, but some people will not
Cause of reduced penetrance: combination of genetic, environmental, and lifestyle factors
Challenging to predict who will develop the disease and the risk of transmitting it to future generations
Expressivity
Range of expression of mutant genotype
Penetrance may be complete, but severity of disease can vary greatly
Variable expressivity: range of signs and symptoms that can occur in different people with the same genetic condition
Example: Marfan syndrome;
although mutation in the same gene some people have only mild symptoms (tall and thin with long, slender fingers), while others experience life-threatening complications involving the heart and blood vessels
Cause of variable expressivity: combination of gene interactions, environmental, and lifestyle factors
Onset of Genetic Expression
many inherited disorders are not manifested until after birth and even later in life thus not possible until later in life to determine whether an individual carries a mutation – delayed onset
Can cause difficulty in deducing mode of inheritance
Examples: Huntington’s, Tay Sachs, Duchenne Muscular Dystrophy
Genetic Anticipation
The phenomenon whereby a genetic disorder exhibits a progressively earlier age of onset and an increased severity in each successive generation
Anticipation is common in trinucleotide repeat disorders:
Fragile X syndrome,
Huntington disease,
myotonic dystrophy
They all have:
variation in the severity of symptoms (mildly to severely affected and death)
Correlation between increased severity and earlier onset with successive generations
Genomic Imprinting
the expression of a gene depends on whether it has been inherited from a male or a female parent and determines whether specific genes depending on their parental origin will be expressed or remain genetically silent
Examples:
Prader-Willi syndrome (PWS; results when paternal
chr. is deleted)
Angelman’s syndrome (AS; results when maternal
chr. is deleted)
Both caused by deletion of an identical region of chr. 15, different phenotypes:
PWS: mental retardation, eating disorder, obesity, diabetes
AS: mental retardation, involuntary muscle contraction, seizures
Therefore: same region in chr. 15 is imprinted differently in male and female gametes
Gene Therapy
the therapeutic delivery of nucleic acid polymers into
a patient’s cells as a drug to treat disease
Is an approach to treat diseases based on modifying the expression of a person’s genes toward a therapeutic goal.
▪ Therapeutic genes are usually deliberately carried into the patient through a vector that transports the genes into target cells.
▪ Is potentially more effective, long lasting than protein therapy*****
gene delivery is much more controlled, sustaining,
and therapeutically benign
Goal of Gene Therapy
• Replace a mutated inactive gene that causes disease with a healthy copy
• Inactivate, “knock out,” or suppress expression of a mutated gene that is functioning improperly.
Dominant negative, gain of function or overexpression mutations.
1. Target the mutated gene to make it inactive
CRISPR/Cas9
2. Introduce silencing RNA that target the mRNA of the gene to block protein production (using interfering RNA which is naturally produced by the body and have it silence bad gene production)
• Introduce a new gene into the body to help fight a disease (not a replacement of the bad gene, but a new gene that codes for like an “antigene”)
• Correct a defective gene sequence - gene editing
CRISPR/Cas9
Disease Treatment
Fix a genetic defect
1. SCID - Severe Combined Immunodeficiency 2. Cystic fibrosis 3. Type 1 diabetes
• Treat cancer
1. Introduce a gene into immune cells to make them
fight the tumor.
2. Introduce a gene into tumors to make them die.
• Treat an infectious disease such as HIV/AIDS
1. Introduce a gene into immune cells to make them
fight HIV better.
2. Introduce a gene that makes cells resistant to
infection
3. Knock out CCR5 - the coreceptor for HIV to make
CD4 T cell uninfectible.
To Fix the Cells (Somatic v. Stem)
You may have to fix the gene in all the cells, if it’s a disease such as Huntington’s, an
autosomal dominant disease, where the defective protein remains in cells - so just replace the defective gene with healthy ones in the cells of the body
However, the gene product may be released from cells and affects other cells, such as Type 1
diabetes. In this case you only have to “fix” a sufficient number of cells to generate enough of the gene product to restore health. The cells may or may not survive the life span of the patient- you may need to keep injecting yourself since the cells won’t stay alive
**Stem Cells**
You can replace the stem cells with genetically modified one so that they repopulate the
body with healthy cells - blood cell diseases.
Stem cell modification potentially lasts longer
than somatic cell editing.
Correcting immune system disorders is a likely use for this. Bone marrow stem cells could be modified and used in a bone marrow autologous transplant.
Transgenic Animals
Contains a gene for a fluorescent protein in their germline
Example: Zebrafish
Added a gene for fluorescent protein, added a controlling promoter so that each cell of the body has this gene to express fluorescence, add UV light, and BAM! glowing fish!
In vivo Gene Therapy
Take a vector (some mode of transmission of the synthesized gene) and directly inject it into the individual where they find their target cells
Ex vivo Gene Therapy
Obtain host cells from individual (draw blood), purify them, and then combine with vector. Then, take the engineered cells containing the therapeutic gene and inject them back into the individual
Non-viral “transfection”
Cationic Polymers - which bind directly to the negatively charged DNA and allow for it to diffuse across lipid mem.
Lipids - which form a micelle around the DNA and fuses with lipid mem. to allow for DNA to enter cell
Naked DNA- into muscle cells where myocytes take up DNA at useful rate
*None allow specific targeting to cells and are
inefficient compared with viruses
Basically you just take DNA, mix it with the polymers or liposomes and wait a few min.
Then you mix this with the cells and the DNA is delivered into the cells’ nuclei where
transcription begins.
Naked DNA can be injected into muscle tissue which takes it into the cells.
Viral “transduction”
Viruses are the most common way to deliver genes into the body*
Adenovirus (Ad) = High cytotoxicity/immunogenicity
(problem is your body might have
seen this virus already which means
you will develop an immune response
to it which 1) inactivates vector you’re
trying to use therapeutically and 2)
you might add so much and the body
produces such a strong immune
response that it KILLS you..)
Retrovirus = Integration into the genome and long-term
gene expression; can cause cancer
Lentivirus - form of a retrovirus = HIV-derived vectors
“Lentivectors;” Don’t
seem to cause cancer
(can get into cells that
are not proliferating)
Adeno-associated virus(AAV) = Low toxicity/, low
immunogenicity
Gene insert must be
short
Problems with viral vectors – Viral vectors carry the risks of toxicity, inflammatory
responses, and gene control and targeting issues
Jesse Gelsinger Case
Ornithine transcarbamylase deficiency, an X-linked genetic disease of the liver, the symptoms of which include an inability to metabolize ammonia – a byproduct of protein breakdown.
Entered a clinical trial at the University of Pennsylvania in 1999 usingadenoviral vectors to deliver a corrected gene.
The vector triggered a cytokine storm and he died from multiple organ failure - the first death from human gene therapy.
He probably had an earlier natural exposure to the virus used to develope the vector so that he made a memory response that was very strong when a lot of vector was injected into him.
The team led by James Wilson was faulted for not following proper procedures:
- Gelsinger’s ammonia levels were too high for inclusion in the study
- Failure of the team to report two prior similar but nonfatal incidents in other patients
- Failure to disclose similar incidents and death in monkey trials
This incident set back human gene therapy for about a decade
Viral Vectors used outside the body
- Remove the viral genes required for continual virus replication
- Remove any genes that are toxic
- Inert the gene you want to express into the viral genome (needs to be short DNA)
- Generate a stock of virus in the lab
- Infect cells
- Select for successfully infected cells that make the factor you want
- Put the cells back into the patient
Vectors that do not integrate their DNA into the cell’s chromosomes will only last for weeks or months. Retroviral vectors integrate into the cell DNA and will last for the life of the cells. However, cell’s protect themselves from retrovirus integration, and often
shut down the inserted DNA through epigenetic silencing.
ADA deficiency
ADA deficiency was an ideal target for the first set of gene therapy trials for a number of reasons:
• The effects of the disease are reversible and do not cause irreversible, long term damage in the individual.
• The disease results from the loss of function of a single gene. It is much more difficult to treat multiple genetic defects.
• The adenosine deaminase gene is very small and easy to manipulate in the laboratory.
• The target cells for the therapy are stem cells for lymphocytes (white blood cells), that are accessible, easy to grow and easy to put back into the body of
a patient.
• Defect is autosomal recessive, so you don’t have to repair either or both genes, just put a new good gene into a cell to fix the problem.
• You don’t have to replace all the defective cells, just a few is enough.
• The alternative treatments are expensive and/or hazardous
Studies on Retrovirus
Retroviruses can cause cancer by inserting near
oncogenes and turning them on.
However, a retrovirus therapy for ADA-SCID has not
caused cancer in test and has recently been approved
for use in Europe
Treating Cancer or AIDS
(i) Collection of PBMC’s and transfer to GMP manufacturing facility
(ii) viral gene transfer of TCR
or CAR into PBMCS
(genetically modified cell)
(iii) propagate gentitically
modified tumour-reactive T cells
(iv) transfer cells from manufacturing centre to
patient
(v) precondition patient (chemotherapy) and transfuse T-cell therapy
CRISPR/Cas9 aka CRISPR
Bacteria have a defense mechanism against viruses that targets their DNA in a sequence-specific manner and cleaves it using the enzyme Cas9.
This system has been modified to allow the targeting of any gene in animal genomes, allowing any gene to be disabled or edited.
• Bad sequences can be corrected by editing or removal.
• New DNA sequences can be inserted.
CRISPR/Cas9 aka CRISPR
Bacteria have a defense mechanism against viruses that targets their DNA in a sequence-specific manner and cleaves it using the enzyme Cas9.
This system has been modified to allow the targeting of any gene in animal genomes, allowing any gene to be disabled or edited.
• Bad sequences can be corrected by editing or removal.
• New DNA sequences can be inserted.
A problem with CRISPR is that is has “off target” effects: Sometimes the wrong DNA target is modified
