Exam 2: Genetics Flashcards
Prenatal Testing
Standard of Care
Set in part by authoritative organizations.
Varies significantly by region.
- Offer “non-invasive” 1st and 2nd trimester screening to all patients
- Offer CF and SMA carrier screening to all patients
- Offer carrier screening for Tay Sachs, Canavan’s diease, and hemoglobinopathies based on population
- Offer invasive diagnostic testing to high risk, known carriers, prior affected, or those with abnormal serum screening
Advanced Maternal Age
35 years of age or older at the estimated date of delivery
ANA associated with higher risks of:
Infertility
Fetal aneuploidy
Gestational DM
Preeclampsia
Stillbirth
Advanced Paternal Age
Age 40 years or older at the time of conception
Increased risk likely due to genetic copying errors after repeated spermatogenesis cycles.
Includes:
Spontaneous abortion
New single gene defects
Some multifactorial diseases
Prental
Screening Tests
Only reveal the possibility of a problem.
Standard Carrier Screening
Testing individuals based on positive family history.
Population-Based
Carrier Screening
Testing individuals based on their ethnic background.
Universal Carrier Screening
All individuals should be screened.
Cystic fibrosis and spinal muscular atrophy should be offered to all.
Maternal Serum AFP
Screening
Alpha-fetoprotein Screening
- earliest non-invasive test
- evaluates for structural and chromosomal malformations
- done between weeks 16-21
- Reported as multiples of medians (MoM)
- Fetus makes AFP ⇒ can be found in maternal circulation
- Critical to know gestational age to interpret values
- Labs set positive threshold low
- Many “normal” fetal and maternal factors can alter AFP levels
- Elevated msAFP associated with fetal body wall defects
- Low msAFP associated with Down Syndrome
Factors Influencing
AFP Levels
- Gestational age
- Number of fetuses
- Maternal weight
- Maternal diabetes
- Race
Quad Test
Combines maternal age with serum screening factors.
Performed at 16-21 weeks
Results given in risk ratio
> 1:270 considered positive ⇒ same as 35 y/o F prior to other factors
ID euploid fetuses from those w/ Down syndrome and trisomy 18
ID pregnacies at risk for adverse outcomes.
- AFP
- Chorionic gonadotropin (hCG)
- Inhibin A
- Unconjugated estriol
First Trimester Screening
Includes maternal serum screening, US, or both.
US ⇒ nuchal translucency (NT)
1st trimester serum markers ⇒ free hCG and pregnancy-associated plasma protein (PAPP-A)
Results expressed as risk ratio ⇒ positive if > than 35 y/o F
Combined Screening
Combines both 1st and 2nd trimester screening.
Integrated test
Stepwise sequential testing
Integrated Test
Combines data from NT, PAPP-A, and quad screens BEFORE calculating risk.
Very sensitive ⇒ 95% detection rate
Policy of non-disclosure for 1st trimester data until quan screen completed in 2nd trimester.
Stepwise Sequential Testing
Same tests as integrated test ⇒ NT, PAPP-A, and quad screens
Discloses high risk first trimester results (>1:50 risk)
Allows option of CVS and earlier termination.
Low and moderate risk patients go onto second trimester screening.
Non-invasive Prenatal Testing
(NIPT)
- Goal to obtain info without CVS or amniocentesis
- Offered after 10 weeks
- is NOT diagnostic
- Analyzed fetal cell-free DNA found in materal circulation
- Used to detect overabundance of certain chromosomal material ⇒ trisomies
- Some labs offer rare trisomies and some microdeletion testing
- Covered only for patients with increased risk
Ultrasound
- Diagnosis and confirmation of early pregnancy
- Determination of gestational age and assess fetal size
- Diagnosis of certain structural anomalies
- Usually reliable between 18-20 weeks
- Cannot dx underlying cause
- Second trimester comprehensive ultrasound
- Detection of anomalies associated w/ Down syndrome and other chromosomal abnl
- Detection varies with person doing US, fetal position, and maternal body habitus
- Definitive dx only by amniocentesis
- ID soft markers for chromosomal abnormalities
- ID multiple pregnancies
- Localize placenta
- ID oligohydramnios or polyhydramnios
Prenatal
Diagnostic Tests
Determine with good certainty whether a fetus has a specific problem.
CVS and amniocentesis
Directly assesses fetal status.
Increased risk ⇒ only recommended for high risk
Indications for Invasive Testing
Available to all women but not always covered.
- F/U for abnormal or positive screening test
- Advanced maternal age
- Parents with known chromosomal translocation or carrier for single gene disorder
- Previous affected pregnancy
- Positive family history
Chorionic Villus Sampling
(CVS)
- Performed between 10-12 weeks
- Tests chorionic villus ⇒ fetal tissue
- Risk of maternal contamination
- Few people can do the test
Amniocentesis
- Performed between 16-21 weeks
- Late results
- Earlier not recommended due to increased risk of pregnancy loss or deformation
- Cells must be placed in tissue culture media and incudated
- Used for karyotyping and genetic testing
-
Modified FISH for chromosomes 13, 18, 21, X, and Y
- Faster
- Must be confirmed by standard karyotype
- Chromosomal microarray
-
Modified FISH for chromosomes 13, 18, 21, X, and Y
- Can quantitate AFP levels and help detect neural tube defects
CVS versus Amniocentesis
- CVS more risky and more difficult to do
- early CVS associated with inc. risk of limb reduction anomalies
- due to disruption
- risk of mosaicism between extraembryoinc tissues and fetus
- does not quantitate AFP
- early CVS associated with inc. risk of limb reduction anomalies
Chromosomal Microarray
Uses tissue obtained from CVS or amniocentesis.
- Uses SNP analysis
- ID both the sequence and dosage
- Allows ID of
- copy number changes ⇒ deletions/duplications
- copy neutral changes ⇒ uniparental disomy, loss of heterozygosity
Preimplantation Genetic Diagnosis
- Invasive
- ID genetic defects in an embryo prior to implantation
- Done with in vitro fertilization
- Single cell taken from 8-cell stage
- Test with FISH or PCR
- Used when
- at least one parent carries a gene
- parent carries chromosomal abnormality
- women > 35 y/o
- repeated IVF failures
Prenatal Testing
Current Practices
- Everyone offered screening for Down syndrome before 20 weeks
- Invasive dx testing for aneuploidy available to all women seen before 20 weeks
- Counseling about screening vs dx tests
- NIPT first line screening test for high risk
- US for NT and serum markers in 1st trimester
- Let patients know screening test cannot detect all abnormalities
- Offer CF carrier screening to higher risk populations and those whom testing is most sensitive
- But available to everyone
- SMA in all pregnancies
- Offer Fragile X carrier screening
Prenatal Testing
Flow Chart
Germ-line
Mutations
A change in the DNA of the cells that form gametes.
Perpetuated from one generation to the next.
Responsible for inherited diseases.
Somatic Cell
Mutation
A change in the DNA of body cells.
Doesn’t affect germ cells.
Does not affect future generations.
Mutation Categories
Types of DNA Variations
- Point mutations ⇒ SNP
-
Errors in replication and repair
- Indel (insertion/deletion) variations
- Copy number variations (CNV)
- Satellite ⇒ 20-100K+ BP
- Minisatellite ⇒ 10-20K BP
- Microsatellite ⇒ 2-100 BP
Mechanisms of Mutations
-
Errors introduced during normal DNA replication
- ~ 1 mutation for every 2 cell divisions
- Mutation rate at given locus varies
- Mutational hot spots
- Base changes induced by endogenous or exogenous mutagens
Mutagen
An agent that increases the spontaneous mutation rate by causing changes in DNA.
Ex. hydrolytic and oxidative damage, chemicals, UV or ionizing radiation
Mutation Classification
Based on effect on DNA sequence OR the encoded protein.
-
Classified by effect on DNA
- Insertions
- Deletions
- Substitutions
- Inversions ⇒ 180° rotation of DNA segment
- Translocations
- Chromosomal rearrangements
-
Classified by effect on the gene or protein’s function
- Transcription
- Translation
- Protein function
Consequences of Base-Pair
Alterations
-
∆ in promotor ⇒ ∆ in mRNA expression
- decrease or “prevent” expression
- complete or partial
- heterochronic expression ⇒ wrong time
- ectopic expression ⇒ wrong place
- decrease or “prevent” expression
-
∆ in mRNA ⇒ impacts RNA processing and half-life
- RNA splicing mutations
- interfere with transcription/translation
- alter RNA stability
-
∆ in protein coding regions ⇒ ∆ translation and protein function
- missense
- nonsense
- frameshift
Missense Mutation
A point mutation in DNA alters the codon ⇒ replacement of one AA by another
Conservative ⇒ no significant change in function
Nonsense Mutation
Mutation causes codon to go from coding for AA to a stop codon.
Leads to truncated protein.
Ex. nonsense mutation in NF1 gene in neurofibromatosis
Frameshift mutations
Caused by deletions and insertions not in a multiple of 3.
Alters the reading frame of all downstream codons.
Usually leads to a stop codon and truncation of the protein.
Examples:
ABO alleles ⇒ 1 bp deletion ⇒ changes A to O
Tay-Sachs ⇒ 4 bp insertion
Cystic Fibrosis
Most Common Mutation
∆F508
3 BP deletion
Lost residue prevents normal folding ⇒ retrograde destruction.
Clinical picture is the same as the “null” allele phenotype.
Changes in Promoter
or
Alteration of mRNA Expression
- Promoter mutation ⇒ inc or dec RNA polymerase affinity
- ↓ mRNA production ⇒ ↓ [protein]
- Mutations in transcription factors or enhancer sequences
- ∆ expression pattern of the gene
Changes in mRNA
Processing and Translation
RNA processing mutations
-
RNA integrity/function
- cap sites
- polyadenylation sites
-
RNA splicing
- ∆ in consensus sequences
- splice donor or splice acceptor site
- interfere with or abolish splicing
- activation of cryptic sites
- create new alternative donor or acceptor site
- Ex. Tay-Sachs
- mutation in donor site of Hexosaminidase A gene
- translation into the intro often resulting in termination at stop codon
- ∆ in consensus sequences
β-Thalassemia
Inheritance Mechanism
Bo allele from nonsense and frameshift mutations have no function.
Creation of a cryptic splice site
G→A of first intron creates abnormal splice acceptor site
90% of mRNA made using incorrect splice site
10% made with correct splice site
“Leaky” mutation results in B+ allele
Hemophilia B
Inheritance Mechanism
Base substitutions outside of coding sequences can interfere with transcription/translation.
- Mutation in 5’ UTR of factor IX gene
- Results in 1/3 the normal amount of clotting activity
Trinucleotide Repeat Expansion
Mutations
- Dynamic mutations
- expansion of a segment of DNA containing a repeat nucleotide sequence
- shows amplification
- can be in coding sequence or 5’ and 3’ UTR
Trinucleotide Expansion
Examples
Non-homologous Recombination
- deletion or duplication of hightly similar or identical DNA sequences
- usually a multigene family with members in a tandem head-to-tail organization
- members of the family misalign during sister chromosome pairing during meiosis
- causes unequal crossing over ⇒ gene loss or duplication
α-thalassemia
Inheritance Mechanism
Complete/partial gene deletion:
-
Silent carrier (alpha-thalassemia minima) ⇒ aa/a-
- small amount of abnormal Hb detected in peripheral blood
- possible mild hypochromia and microcytosis
- no anemia and/or clinical manifestations
-
Alpha-thalassemia trait (alpha-thalassemia minor) ⇒ aa/– or a-/a-
- mild anemia
- RBC hypochromic and microcytic
- clinical sx usually absent
- detected by Hgb electrophoresis and microscopic exam of peripheral RBCs
- important to know phenotype because can have offspring with a-/– or –/–
-
Hemoglobin H disease (alpha-thalassemia intermedia) ⇒ a-/–
- moderate to severe anemia
- elevated reticulocyte count
- HbH inclusion bodies
- Most live normal life
- 25% need transfusion at some point
-
Hemoglobin Bart syndrome (alpha-thalassemia major) ⇒ –/–
- profound anemia
- hydrops fetalis ⇒ abnormal accumulation of fluid in two or more fetal compartments
- often leads to intrauterine death or death shortly after birth
- increased complications of pregnancy for mother
Charcot-Marie-Tooth Disease
Inheritance Mechanism
Gene duplication:
- 70% with type I CMT have 1.5 x 106 duplication ⇒ 3 copies of PMP22 gene instead of 2
- increased gene dosage ⇒ demyelination ⇒ progressive atrophy of distal limb muscles
Chronic Myelogenous Leukemia (CML)
Inheritance Mechanism
Reciprocal translocation (9;22 - Philadelphia Chromosome)
Chimeric protein formed from Bcr gene and c-Abl gene
Aberrant expression of Abl and altered intracellular localization
Mutations
Summary
Loss-of-Function
Mutations
The result of non-wild type gene products having less or no function.
- Recessive traits
- Heterozygotes ⇒ no discernible abnormal phenotype
- Wild-type allele can mask effects of abnormal allele
- Dominant traits
- Heterozygotes ⇒ has a discernible abnormal phenotype
- Called haploinsufficiency
- Normal phenotype requires protein product of both alleles
- Reduction of 50% of gene function ⇒ abnormal phenotype
Dominant-Negative
Diseases
A mutation whose gene products adversely affects the normal, wild-type gene product within the same cell.
- Usually occurs when abnormal product can still interact with the same elements as the wild-type.
- Usually with multimeric proteins.
- Usually have dominant phenotypes.
Gain-of-Function
Mutations
Mutation that alters the biochemical pathways by increasing one or more of the normal functions of the gene product.
- Usually have dominant phenotypes
- Can be due to enhancing one function or increasing production/half-life of the gene product
- Dec. degradation
- Inc. catalytic activity
- Inc. copy number of gene
Novel Property
Mutation
Mutation that confers a new property on the gene product, without necessarily altering the normal function.
Can occur with AD or AR diseases.
Abnormal Gene Expression
Mutations
Mutations that alter regulatory regions causing gene to be inappropriately expressed.
Expression at the wrong place ⇒ ectopic mutation
Expression at the wrong time ⇒ heterochronic mutation
Loss of Heterozygosity
Germ-line inheritance of a non-functional + somatic mutation of the only functional copy in 1 cell.
“Two-Hit” mechanism
Explains why individuals can inherit diseases in an autosomal dominant manner despite needing the loss of both alleles.
Ex. neurofibromatosis and BRCA-1 or -2
Familial Hypercholesterolemia
Pathogenetics
Lack of LDL receptors
AD
Displays haploinsufficiency
- hh ⇒ normal [LDL receptors] ⇒ normal phenotype
- Hh ⇒ 1/2 [LDL receptors] ⇒ atherosclerosis in 30’s and 40’s
- HH ⇒ no LDL receptors ⇒ atherosclerois very early, heart attack in teens to 20’s, Xanthomas
Osteogenesis Imperfecta
Pathogenesis
Mutation in gene for one of the two α1-chains of collagen.
Autosomal Dominant
-
Type I
-
null mutation in one of the α1 genes
- nonsense, frameshift
- abnormal protein unable to combine with α2
- 50% normal procollagen made
- haplosinsufficiency
-
null mutation in one of the α1 genes
-
Type II
- missense mutation in one of the α1 genes
- slightly abnormal protein still able to interact with α2
- make only 25% of normal protein
- dominant-negative effect
- all de novo mutations because disease is lethal in infants
Charcot-Marie-Tooth Disease Type IA
Pathogenesis
Duplication of PMP22 gene on chromosome 17.
Autosomal dominant
Gain-of-function mutation of peripheral myelin protein
Progressive demyelination of peripheral nerves ⇒ peripheral muscle weakness and atrophy
Achondroplasia
Pathogenesis
Point mutation in FGFR3 gene ⇒ ligand independent activation
Autosomal dominant
Gain-of-function mutation of FGFR3
Inhibits chondrocyte proliferation within growth plate.
Huntington Disease
Pathogenesis
Trinucleotide expansion in HTT gene
Autosomal dominant
Novel property mutation
Shows genetic anticipation
>36 copies ⇒ Huntingtin protein aggregation ⇒ nuclear inclusions in neurons ⇒ neuronal atrophy and death ⇒ uncontrolled movements, emotional disturbance, loss of cognition, death
Chronic Myelogenous Leukemia
Pathogenesis
Translocation of BCR from chromosome 9 to 22
Chimeric BCR/ABL Philadelphia chromosome
Autosomal dominant
Heterochronic & ectopic gene expression
Gain-of-function mutation
Novel property mutation
Sickle Cell Disease
Pathogenesis
Missense mutation in β-hemoglobin gene
Autosomal recessive
Novel property mutation
Aggregation of globin in deoxygenated state ⇒ sickle shaped RBCs
Gene Discovery
The process of identifying disease-associated/causing genes.
Genetic Linkage
Loci are physically connect to one another along the chromosome.
Independent assortment does not apply.
Haplotype
The actual combination of the individual alleles on the chromosome.
Alleles are often inherited together.
Haplotype arrangements designated: A1 B2 / A2 B2
Genetic Linkage
Analysis
Uses the rate of recombination between two linked loci to estimate the distance between them.
Used to ID disease-causing genes and to track single gene disorders within a family.
- Two genes are close together on a chromsome ⇒ usually inherited together, unless a recombination event separates them
- Odds of a recombination event between two linked genes are inversely proportional to the distance between them
- Measured using centiMorgans (cM)
- 1 cM ⇒ distance where two loci recombine 1% of the time
- 1 cM equivalent to ~ 1 million base pairs
- Crossing over more frequent with oogenesis
Lod Score
Logarithm of the odds score.
Tool used to analyze the probability of co-segretation and linkage.
Recorded as log10.
Strong evidence for linkage ⇒ Lod score +3 ⇒ 1000:1 odds for linkage
Strong evidence against linkage ⇒ Lod score -2 ⇒ 100:1 odds against linkage
Genetic Markers
DNA marker located near the disease-causing gene.
Allows tracking of the disease-causing allele through families.
- RFLPs (restriction fragment length polymorphisms)
- SNPs (single nucleotide polymorphisms)
- VNTR (variable number tandem repeats)
Track Disease-Causing Allele
via
Linkage Analysis
- Know the disease and its inheritance pattern
- Use informative marker(s) near the disease-causing gene that allows tracking
-
Determine the linkage phase for the disease
- Which marker is associated with the disease-causing allele
Identification of Disease-Causing Genes
Methods
- Functional cloning
- Candidate gene cloning
- Positional gene cloning
- Positional-candidate gene cloning
Functional Cloning
Limited to diseases where a biochemical defect can be determine and the protein isolated.
Limitation ⇒ need to know deficient protein or enzyme to identify the gene.
Candidate Gene Cloning
Begins with an educated guess of which genes are involved.
Look for mutations that are associated with the disease.
Limitation ⇒ need to know the normal mechanism and all of the genes in the pathway.
Compare gene sequences or gene expression patterns between affected and unaffected individuals.
Positional Gene Cloning
“Reverse Cloning”
Look for disease-causing genes/alleles by identifying areas in the genome that appear to be linked to a specific trait or disease.
Uses linkage mapping.
DNA from families with affected members ⇒ track thousands of markers ⇒ ID chromosomal regions that cosegregate with disease/trait via LOD score ⇒ ID region chromosome ⇒ look for recombination events ⇒ DNA sequence analyzed and sequenced
Strength ⇒ track phenotypic trait without knowing the cause underlying the trait.
No biochemical or functional info about the gene needed.
Weakness ⇒ works well on high-penetrance, single gene traits and diseases.
Need pedigrees of related individuals and a phenotype that is present in some but not all family members.
Positonal-Candidate Gene Cloning
- Use the same technique as positional gene cloning
- Track markers in families with affected people
- Enter chromosomal region into DNA database
- ID potential genes of interest
- ID genes in that area and extrapolate which genes are likely responsible
- Once potential gene identified and cDNA identified, sequence both affected and unaffected members for differences
Genome-Wide Association Studies
(GWAS)
Compare DNA from people with the disease and similar people without the disease.
Used for low penetrant genes responsible for multifactorial disorders.
- Do not require large pedigrees
- Search subject’s genomes for markers that occur more frequently in people with disease than without
- SNP or other variations
- Uses microarrays or SNP chips
- Variations are said to be “associated” with the diease
- may not directly cause the disease
- might be marking the nearby causal variant
- need more research to confirm role of variant
Disease-Causing Gene
Determination
Once one or more potential genes identified, need to determine if gene is involved in disease development:
-
ID if gene expressed in an expected tissue pattern
-
assess mRNA exression pattern
- northern blot
- PCR of cDNA
- high-throughput RNA sequencing
-
assess protein expression
- western blot
- immunofluorescence
- biochemical assay
-
assess mRNA exression pattern
- ID mutations in patients but not unaffected family members
- Dissecting role of gene in normal function
-
Determine how different mutations affect gene function and cause disease
- evaluation of patients with the disease
- creating genetically modified animals
Restriction Enzymes
Specific endonucleases that recognize certain recognition sequences and cut the sugar-phosphate backbone of DNA.
Sequences are typically palindromic.
Southern Blotting
Technique
Detect specific sequences in genomic DNA.
- DNA digested with a restriction enzyme
- Seperated via gel electrophoresis
- DNA transferred onto blotting membrane
- Probe labeled and allowed to hybridize with DNA
Southern Blotting
Uses
- Detect significant changes in DNA
- translocations
- insertions/deletions
- Analysis of RFLP
Northern Blotting
Technique
Analyzes RNA.
- RNA extracted
- Seperate by gel electrophoresis
- Oligo (dT)-coated beads often used to seperate out mRNA via poly(A) tails
- Gel contents transferred to a blotting sheet
- Labeled probes used to visualize bound targets
Western Blotting
Technique
Analysis of proteins.
- Protein samples obtained
- Seperated via gel electrophoresis
- Transferred to blotting sheets
- Proteins visualized with labeled antibodies.
Sanger Dideoxy Termination Sequencing
- Four deoxy nucleotides and small amount of single dideoxy nucleotide used
- Chain elongation continues until dideoxy nucleotide incorporated
- Obtain DNA chains of differing lengths
- DNA chains seperated by size using electrophoresis
- Sequence is determined by reading gel from smallest to largest
Automated Sequencing
(Dye termination sequencing)
- Single reaction with all four dideoxy nucleotides
- Each ddNTP has a different fluorescent group attached
- Reaction allowed to run to completion
- Mixture seperated by capillary electrophoresis
- Labeled chains detected by fluorometer
- Read colors as they pass through the detector
PCR
Method
Uses oligonucleotide primers that flank target DNA region.
Three step cycle:
-
Denaturation
- Reaction heated to melt dsDNA in ssDNA
-
Annealing
- Reaction cooled to allow primers to bind to DNA
-
Elongation
- DNA synthesis using primers to start replication
- Uses thermostable DNA polymerases
Reverse Transcription PCR
(RT-PCR)
Detection and quantification of mRNA in a sample.
- Use reverse transcriptase and primer to anneal and extend mRNA
- Transcribes a complimentary strand of DNA (cDNA)
- PCR used to amplify targer sequences in cDNA
Allele-Specific Oligonucleotide Hybridization
(ASO)
PCR-based technique.
Used to detect SNPs, short deletions or insertions.
Need to know the exact mutation and where to look.
- PCR amplifies DNA containing site of interest
- DNA spotted many times onto solid matrix
- Sometimes beads used
- Hybridized with ASO probes
- Short probes of 15-20 BP
- Sensitive to even single-base pair mismatches
- Matrix analyzed
Monitoring Trinucleotide Repeats
Primers flank the unstable region containing trinucleotide repeat.
DNA amplified using PCR.
DNA seperated by size via gel electrophoresis.
Correlate size to phenotype/risk.
Microsatellite Analysis
-
Microsatellites and simple sequence repeats
- number of copies highly variable
- highly polymorphic between homologous chromosome and between individuals
- Primers used that flank regions
- PCR ⇒ gel electrophoresis
- Used for:
- genetic mapping
- linkage analysis
- trace inheritance patterns
- ID uniparental disomy
- ID specific individuals through DNA fingerprinting
Multiplex PCR
Simultaneous amplification of multiple exons in a single PCR.
Used to detect the loss of one or more exons in the gene of interest.
Does not pick up missense, nonsense, or frameshift mutations.
Always start with the affected individual.
- Primer pairs used ⇒ covers different regions of the gene
- Produce PCR products of differing sizes
- Lack of a particular PCR product usually means significant structural changes in DNA
Gene Expression Microarray
Expression of multiple genes simultaneously monitored.
Steps:
-
Create/obtain DNA microarray
- Contains multiple ssDNA sequences from coding strand of multiple genes
-
Isolate and purify mRNA from multiple samples
- One usually serves as a control for comparison
- Reverse transcribe, PCR, and label each mRNA with a different color
- Cohybridize equal amounts of cDNAs on array
- Scan array and quantitate signal to relative amount of cDNA binding
- Interpretation
Chromosomal Analysis
Resolutions
Bacterial Expression Systems
Preferred for non-glycosylated, non-post-translationally modified recombinant proteins.
Typically uses a plasmid vector.
Ex. human insulin
Generics called biosimilars.
Transgenic Animals
-
Transgene injected into pronucleus of embryo
- Shortly after fertilization
- Before fusion of male and female pronuclei
- Transgene randomly integrated into genome
- Genetic changes occur in a random manner
- Creates a transgenic animal (usu mouse)
- Mouse mated with other mice to create a transgenic line of “genetically identical” animals
Embryonic Stem Cell Derived Animals
-
Pluripotent embryonic stem cells (ES) modified in tissue culture
- Via homologous recombination into site of interest
- Modified ES cells injected into an early embryo ⇒ chimeric embryo
- Embryo implanted into uterus of female mouse and allowed to develop into mouse pups
- Some tissues derived from modified ES cells ⇒ chimeric animal
- If changed gene is in the germ-line, it will propogate through generations
- Selective breeding can result in animals homozygous for transgene
Gene Therapy
Overview
The transfer of genetic material into the patient’s cells to treat disease.
Process known as transfection.
Can be broken down into type of tissues impacted and how procedure is done.
Several approaches are being tested:
- Replacing a mutated gene with a normal one
- Inactivating the abnormal gene
- Introducing a new gene
Somatic Gene Therapy
Modifies the genes in the somatic cells of a single individual.
Not passed on to future generations.
Currently practiced.
Germline Gene Therapy
The permanent modification of genes found in germ cells.
- Techniques like transgenics, knockout, and knock-ins
- Would be transmitted to future generations
- If done during preimplantation dx or IVF ⇒ impacts all cells of embryo
- Currently ethically unacceptable
- Some mitochondrial-replacement techniques are used
Transgene
Piece of DNA or gene that usually has been created in a lab and incorporated into an organism’s genome through genetic engineering.
Promoter attached to the cDNA.
- Tissue specific promotors
- Promotors with high activity
- Drug sensitive promotors
Hardy-Weinberg
Overview
Coefficient of Inbreeding
Probability of inheriting two copies of the same allele from an ancestor that occurs on both sides of the pedigree.
Founder Effect
“Genetic bottleneck”
A small group breaks off from a larger population to form a new colony.
Leads to reduced genetic variation.
Non-random sample of genes ⇒ can significantly alter allelic frequency.
Achondroplasia
-
Inheritance & Pathogenesis:
- Autosomal dominant
-
Single gene mutation
- 80% de novo
- Only in paternal germline
- 80% de novo
- Constitutive activation of FGFR3 ⇒ dysplasia of cartilage to bone in growth plate
- Shows paternal age effect
-
Major Phenotypic Features:
- Rhizomelic short stature
- Megalencephaly
- Spinal cord compression
XYY Syndrome
-
Inheritance & Pathogenesis:
- Chromosomal
- Nondisjunction or anaphase lag in paternal gamete results in 2 copies of Y and one X
- Paternal age effect
-
Major Phenotypic Features:
- Normal at birth
- Subsequent tall stature
- Cognitive development normal but educational difficulties
- Radial-ulnar synostosis
Intrauterine Growth Restriction
-
Inheritance & Pathogenesis:
-
Multifactorial
- Spontaneous chromosomal deletion
- Uteroplacental insufficiency
- Teratogens
-
Multifactorial
-
Major Phenotypic Features:
- Poor growth
- Increased nuchal fold
- Dysmorphic facies
Holoprosencephaly
(Nonsyndromic Form)
-
Inheritance & Pathogenesis:
- Autosomal dominant
- Chromosomal
- Position-effect translocation of Sonic Hedgehog
- Loss of SHH ⇒ abnormal embryological developmental patterning
-
Major Phenotypic Features:
- Ventral forebrain maldevelopment
- Facial dysmorphism
- Developmental delay
Fragile X Syndrome
-
Inheritance & Pathogenesis:
- X-linked
-
Expansion of CGG repeat in 5’ UTR region of FMR1 gene
- > 200 repeats ⇒ hypermethylation and supression
-
Sex specific anticipation
- Only maternal transmission of premutation alleles
- Paternal transmission can actually dec. repeats
- FMRP protein chaperone some mRNAs for translation
-
Major Phenotypic Features:
- Intellectual disability
- Dysmorphic facies
- Male post-pubertal macroorchidism
Type II DM
-
Inheritance & Pathogenesis:
- Multifactoria
- Abnormal insulin secretion and insulin resistance
-
Major Phenotypic Features:
- Hyperglycemia
- Relative insulin deficiency
- Insulin resistance
- Obesity
- Acanthosis nigricans
CHARGE Syndrome
-
Inheritance & Pathogenesis:
- Autosomal dominant
- Single gene mutation of CHD7
- Abnormal gene product affects chromatin structure and gene expression during development
- Haploinsufficiency
-
Major Phenotypic Features:
- Coloboma of eye
- Heart defects
- Atresia of the choanae
- Retardation of growth/development
- Genital abnormalities
- Ear anomalies
- Facial palsy
- Cleft lip
- Tracheoesophageal fistula
Autism
(16p11.2 Deletion Syndrome)
-
Inheritance & Pathogenesis:
- Autosomal dominant or De Novo
- Contiguous gene deletion on 16p11.2
- 25 genes in region
- Pathogenesis unclear
- Can show copy number variant
-
Major Phenotypic Features:
- Varied intellectual disability
- Impaired social and communication skills to frank autism spectrum d/o
- Minor dysmorphic features
Turner Syndrome
-
Inheritance & Pathogenesis:
- Chromosomal
-
Complete or partial monoploidy X
- Non-disjunction or loss of function
- Haploinsufficiency
-
Major Phenotypic Features:
- Short stature
- Ovarian dysgenesis
- Sexual immaturity
Thalassemia (α and β)
-
Inheritance & Pathogenesis:
- Autosomal recessive
- Single gene mutation
-
Locus heterogeneity
- Abnormal α-globulin or β-globulin of Hb
- Inadequate Hb production ⇒ hypochromia and microcytosis
- Unbalanced globulin accumulation ⇒ hemolytic anemia, ineffective erythropoiesis
-
Major Phenotypic Features:
- Hypochromic microcytic anemia
- Hepatosplenomegaly
- Extramedullary hematopoiesis
Sickle Cell Disease
-
Inheritance & Pathogenesis:
- Autosomal recessive
- Single gene disorder
- Missense mutation in hemoglobin β subunit gene
- Abnormal Hb w/ dec. solubility of deoxygenated Hb ⇒ polymers that distort RBCs
- Heterozygote advantage for malaria
-
Major Phenotypic Features:
- Anemia
- Infarction
- Asplenia
Sex Development Disorder
-
Inheritance & Pathogenesis:
-
Y-linked
- SRY mutation
-
Chromosomal
- Translocation of SRY region on Y chromosome
- Lack of functional SRY gene ⇒ 46,XY phenotypic female
-
Y-linked
-
Major Phenotypic Features:
-
Sterility
- Need two X chromosomes for oocyte maintenance
- Lack of azoospermic factors ⇒ abnormal sperm
- Reduced secondary sex features
- Unambiguous genitalia mismatched to chromosomal sex
-
Sterility
Polycystic Kidney Disease
-
Inheritance & Pathogenesis:
- Autosomal dominant
- Single gene mutation
- Genetic heterogeneity
- 85% w/ ADPKD1 mutation
- 15% w/ ADPKD2 mutation
- Some with neither
- Likely due to mislocalization of cell surface proteins
-
Major Phenotypic Features:
- Renal and hepatic cysts
- Progressive renal failure
- Intracranial saccular aneurysms
- Mitral valve prolapse
Progeria
-
Inheritance & Pathogenesis:
- Autosomal dominant
- Single gene mutation in LMNA gene
- Abnormal lamin A ⇒ nuclear instability ⇒ premature cell death
-
Major Phenotypic Features:
- Aged appearance in childhood
- Failure to thrive
- Alopecia
- Facial dysmorphia w/ beak nose
- Indurated skin with dyspigmentation
- Early cardiovascular disease / osteoporosis
Neurofibromatosis 1
-
Inheritance & Pathogenesis:
- Autosomal dominant
-
Single gene mutation in NF1
- Acts as a tumor suppressor gene
- Results in abnormal cellular proliferation
- Pleiotropy & variable expressivity
-
Major Phenotypic Features:
- Café au lait spots
- Lisch nodules
- Cutaneous and subcutaneous neurofibromas
- Increased risk for malignancies and learning disabilities
Huntington Disease
-
Inheritance & Pathogenesis:
- Autosomal dominant
- Expansion of CAG repeats in HD gene
- Mutant HD protein causes progressive degeneration of neurons, esp. in movement centers
- Neuronal dysfunction and death
- Sex-specific anticipation
-
Major Phenotypic Features:
-
Movement abnormalities
- Involuntary jerking or writhing movements (chorea)
- Cognitive abnormalities
- Psychiatric abnormalities
-
Movement abnormalities
Hemophilia A and B
-
Inheritance & Pathogenesis:
- X-linked recessive
-
Single gene mutation
- Locus heterogeneity
- Lack of Factor VIII or IX impairs clotting
-
Major Phenotypic Features:
- Bleeding
- Hemarthroses
- Hematomas
Glucose-6-Phosphase Dehydrogenase (G6PD)
Deficiency
-
Inheritance & Pathogenesis:
- X-lined recessive
- Single gene mutation
- Malfuntional G6PD ⇒ low [NADPH] ⇒ low [reduced glutathione] ⇒ oxidative stress ⇒ Heinz bodies ⇒ rigid RBC’s that hemolyze
- Heterozygote advantage against malaria
-
Major Phenotypic Features:
- Hemolytic anemia
- Neonatal jaundice
Edwards Syndrome
Trisomy 18
-
Inheritance & Pathogenesis:
- Chromosomal
- Nondisjunction or anaphase lag (Chromosome 18)
- Translocation types rare
- Mosaicism with milder phenotypes
-
Major Phenotypic Features:
- Growth retardation
- Polyhydramnios
- Microcephaly
- Congenital malformations
- Clenched hand with overlapping fingers
- Hypotonia initially with hypertonia
- Severe intellectual disability
Polyploidy
-
Inheritance & Pathogenesis:
- Chromosomal
-
Abnormal chromosome # in multiples of 23
- Triploidy = 69 n
- Tetraploidy = 92 n
- Dispermy, Digyny, Diandry, Mosaicism
-
Major Phenotypic Features:
- Intrauterine growth retardation
- Placental molar changes
- Multiple congenital abnormalities
- Fetal mortality
Klinefelter Syndrome
XXY Males
-
Inheritance & Pathogenesis:
- Chromosomal
- Non-disjunction or anaphase lag in mom or dad
- 2 copies of X chromosome and 1 Y
- Paternal age effect
-
Major Phenotypic Features:
- Small testicles ⇒ sterility
- Tall stature
- Gynecomastia
- Specific learning disability
- Possible behavioral and emotional problems
Down Syndrome
-
Inheritance & Pathogenesis:
- Chromosomal
- Nondisjunction or anaphase lag (chromosome 21)
- Robertsonian translocation (14q21q) or (21q21q)
- Chromosomal
-
Major Phenotypic Features:
- Extra nuchal folds
- Delayed development
- Small size
- Hypotonia
- Single palmar crease
- Large/protruding tongue
- Congenital heart disease
Cri-du-chat Syndrome
-
Inheritance & Pathogenesis:
- Chromosomal
- Deletion (5p-)
- Continuous gene syndrome on the short arm of chromosome 5
-
Major Phenotypic Features:
- Cat-like cry
- Severe intellectual disability
- Microcephaly
- Low birth weight
- Round face
- Hypertelorism (wide set eyes)
- Low set ears
- Epicanthal folds
Spinal Muscular Atrophy
(SMA)
-
Inheritance & Pathogenesis:
- Autosomal recessive
-
Single gene mutation
- Deletion SMN1 gene (mostly
- Degeneration and loss of anterior horn cells of spinal cord and brain stem
- # of gene copies, # and type of mutations act together (epistasis)
-
Major Phenotypic Features:
- Progressive weakness and muscle atrophy
- Life expectancy < 2 years
Angelman Syndrome
-
Inheritance & Pathogenesis:
- Chromosomal
- Microdeletion (chromosome 15)
- Absence of maternally derived chromosome ⇒ uniparental disomy
-
Major Phenotypic Features:
- Severe intellectual disability
- Ataxic gait
- Seizures
- Inappropriate laughter
Myoclonic Epilepsy w/ Ragged-Red Fibers
(MERRF)
-
Inheritance & Pathogenesis:
- Matrilineal
- Mitochondrial
- >90% with point mutation in tRNAlys gene
- ↓ [tRNAlys]mito ⇒ ↓ translation and ↑ termination
- Complexes I and IV of ETC most effected
-
Major Phenotypic Features:
- Myopathy
- Dementia
- Myoclonic seizures
- Ataxia
- Deafness
Prader-Willi Syndrome
-
Inheritance & Pathogenesis:
- Chromosomal
- Microdeletion (chromosome 15)
- Absence of paternally derived chromosome ⇒ uniparental disomy
-
Major Phenotypic Features:
- Infantile feeding difficulties
- Childhood hyperphagia and obesity
- Hypotonia
- Cognitive impairment
- Short stature
- Dysmorphism
Patau Syndrome
Trisomy 13
-
Inheritance & Pathogenesis:
- Chromosomal
- Nondisjunction or anaphase lag (Chromosome 13)
- Robertsonian translocation
-
Major Phenotypic Features:
- Microcephaly
- Holoprosencephaly
- Cleft lip and cleft palate
- Polydactyly
- Profound intellectual disability
- Multiorgan abnormalities
