Exam 1 Flashcards
1
Q
Mendel’s Laws
A
Independent assortment - Alleles of one gene segregate independently from alleles of another gene
Equal segregation - Alleles of a gene segregate into gametes at equal ratios
2
Q
Human Genome
A
20-30k genes (30%) Extragenic DNA (70%)
3
Q
Hardy-Weinberg Assumptions
A
- Population is infinitely large and effects of random genetic drift are negligible
- Mating is random with respect to genotypes
- No new mutation is introduced
- Natural selection does not affect geneotype frequency
4
Q
Hardy-Weinberg equation
A
p^2+2pq+q^2=1
5
Q
Founder Effect
A
A high frequency of a specific gene mutation in a population founded by a small ancestral group
6
Q
Population Bottleneck
A
Population is reduced, survivors with mutation spread the mutation resulting in a high mutation frequency in the newly burgeoning population.
7
Q
Jewish Related Diseases
A
Bloom syndrome Breast cancer Canavan disease Dysautonomia Factor IX deficiency Gaucher disease Idiopathic torsion dystonia Niemann-Pick disease Tay Sachs
8
Q
Patterns of Inheritance - Mendelian
A
Autosomal Dominant Autosomal recessive XLR XLD Y-linked
9
Q
Patterns of Inheritance - Non-Mendelian
A
Imprinting Mitochondrial Multifactorial Sporadic Conitguous gene syndromes
10
Q
Nucleotide vs Nuceloside
A
Nucleotide - sugar, nitrogenous base, phosphate
Nucleoside - sugar, nitrogenous base
11
Q
Pyrimidines - smaller
A
Thymine, Cystosine, Uracil
12
Q
Purines - larger
A
Adenine, Guanine
13
Q
Chargaff’s Rule
A
[A]=[T]
[C]=[G]
14
Q
RNA vs DNA
A
2’ OH on Ribose absent on Deoxyribose
15
Q
TATA Box
A
Promoter region
16
Q
5’ Cap and Poly A tail
A
Help in stability and transport of mature mRNA
17
Q
RNA Splicing
A
Donor site, acceptor site, branch site, lariat.
Lariat is donor plus branch end formed into a loop
Spliceosome splices out intron and carries it away
Splicesome contains small nuclear RNAs and small nuclear riboproteins
18
Q
Post translational protein modification
A
- Protein cleavage
- Ubiquitination - can mark a protein for degradation
- Addition of small chemical groups - phosphorylation, acetylation, methylation
- Addition of other types of chemical groups - Glycosylation, palmitoylation
19
Q
Point mutations
A
Transitions - A>G, G>A or T>C, C>T (more common)
Transversions - A>T, C>G, G>C, T>A
Deamination - C>U
20
Q
Pyrimidine Dimers
A
Ultraviolet light can create kinks in DNA, pyrimidines bond together which bends the DNA at that spot (kink)
Interferes with DNA replication and can lead to mutation
21
Q
Large scale del/dups
A
Often caused by errors in recombination and replication
22
Q
Insertions
A
Transposons can insert into stretches of DNA and disrupt transcription
-Alu elements most common transposable element in genome
23
Q
Repeat expansions
A
Short highly repetitive sequences are prone to slippage
-Increase in number of repeats leads to mutation
24
Q
Stop Codons
A
UAG
UGA
UAA
25
Mutation Nomenclature
Numbering begins at start codon ATG/AUG (1)
26
Analysis of complex DNA
PCR, Cloning - Selective replication of a segment of DNA
| Molecular hybridization - detection within a complex micture
27
Cloning - Formation of Recombinant DNA
1. Purification of DNA vector
2. Purification of complex DNA target
3. Digestion of both by restriction enzyme
4. Ligation of target DNA to vector
28
Cloning - Steps 2, 3, and 4
2. Transform recombinant DNA into host cells
3. Grow individual transformants to form colonies
4. Further expand clones and isolate recombinant DNA
29
PCR
- Need template DNA, oligo primers, DNTPs, thermostable DNA polymerase
- Can genotype repeat expansion diseases
- Allele-specific PCR is useful for detecting nucleotide variants
- 3 uses - SNP detection, large del/dups, repeat expansion diseases
30
Hybridization
In a complex mixture, can use a probe for a known nucleic acid or oligonucleotide. Hybridizes to sequence of interest.
- Southern Blot
- Microarray
31
Genotyping
ASOH - Allele specific oligonucleotide hybridization
- One oligonucleotide matches normal sequence, once matches mutant
- Used to find common mutations: CF, Achondroplasia, Sickle cell, Gaucher
32
Sequencing
Used when there is not a common mutation
| -DMD, Breast cancer, NF
33
Dideoxy sequencing (Sanger)
Use di-deoxy nucleotides, chain can't continue. Colors correspond to bases.
34
Pyrosequencing
Luciferace and dNTPs - lights up when new base added
| -used for genotyping, short sequencing, and massively parallel sequencing
35
Massively Parallel Sequencing
Simulatneously sequence a panel of genes for genetic heterogenous disorders
36
Forward genetics
Study mutant phenotype, identify corresponding genotype
37
Reverse genetics
Known gene, generate mutant organism to study the gene's function in vivo
38
Gene knock-out
Removal of an essential part of, or the entire, gene
| -no active gene present
39
Gene knock-in
Insertion of a mutation into a gene
| -Both mutation and wt are active
40
Gene replacement
Only mutant gene is active
41
RNAi
miRNA (natural mechanism) and siRNA (a tool for experimental gene modification)
42
CRISPR/Cas9 genome editing
Removes mutation and replaces with a functional copy
43
Muller's morphs
+ = wt, m = mutant, Df = gene deletion, Dp = gene duplication, > = more severe than
44
Amorph
complete loss of gene function
45
Hyopmorph
Partial loss of gene function
46
Hypermorph
Increased gene function
47
Antimorph
Dominant mutation acting to oppose normal gene function
48
Neomorph
Dominant gain of function that is different than normal gene function
49
Morpholino knockdown
Morpholino hybridizes to mRNA, blocks translation
50
Transgenic organisms
1. Inject foreign DNA into one of the pronuclei of prior fertilized mouse egg
2. Transfer injected eggs into female mouse
3. 10-30% of offspring will exhibit foreign DNA, will be present in all tissues
4. Breed mice containing foreign DNA to continue mutation in germ line
51
Gene KO in mice
Mutant alleles introduced via injection of embryonic stem cells into developing embryo, injected embryo is placed in to "pseudo pregnant" mouse, creates chimera mice which can be bred
52
Conditional gene targeting
LoxP/Cre mice will only express Cre recombinase at a certain time or place in development
53
Garrod's Views
Concepts. What diseases are and why they exist. How to think about medicine.
54
Osler's Views
Facts. How to practice medicine. Treatment and management.
55
ACMG NBS criteria
- Clinical characteristics (incidence, burden of disease if untreated, phenotype in newborn)
- Analytical characteristics of the screening test (availability, platform)
- Diagnosis, treatment and management of disease in both acute and chronic form
56
Sensitivity
Proportion of people with the target disease who have a positive test
57
Specificity
Proportions of people without the target disease who have a negative test
58
Positive Predictive Value
Proportion of people with a positive test who have the disease
59
Negative Predictive Value
Proportion of people with a negative test who don't have the disease
60
Tandem Mass Spectrometry
Allows for focusing of mass spec for defined disorders, adaptable to most substrate disorders.
61
Uniform Screening Panel
- 29 core conditions (20 by mass spec)
- 25 secondary conditions (22 by mass spec)
- Later additions
62
Goals of NBS
- Sensitivity should be high
- Specificity and PPV vary by disorder
- Timing matters
- NBS is a SCREENING program to detect infants in need of a diagnostic test
63
ACT Sheets and Algorithms
Stepwise processes for NBS positives that help practitioners determine the next direction to take
64
PKU
Deficiency of levels of Phenylalanine Hydroxylase
AR inheritance
PAH works in liver, but liver is unaffected
Sometimes caused by BH4 mutations (biopterin, cofactor to PAH)
65
PKU Incidence
- 1/10,000 to 1/16,000
| - 300 new cases per year
66
PKU Phenotype
- Severe - serum Phe >1200 microMolar
- Moderate - serum Phe >600 to <600
- Intellectual disability and autistic behaviors
- Seizures
- White matter hyperintensities
- Genotype/Phenotype correlation
67
PKU Phenotype part 2
- Fair hair (tyrosine is a pigment precursor)
- Pale skin, blue eyes
- Developmental delays
- Microcephaly
- Eczema
- Musty order (phenylketones in urine)
68
Tyrosine
Precursor to pigment and neurotransmitters
69
PKU History
Folling - First description of the disease
Jervis - Showed that PKU was due to liver PAH deficiency
Bickel - Reported the reduced Phe diet
Guthrie - Newborn screening
70
Guthrie
Guthrie bacterial inhibition assay
- Blood spots from newborns plated with bacteria that requires Phe to grow
- Bacteria only grows on spots containing excess Phe
71
Maternal PKU
- Structural birth defects - cardiac defect, esophageal atresia
- Microcephaly, intellectual disability
- Craniofacial effects - elongated philtrum, medial epicanthal folds, thin upper lip
72
PAH
Chromosome 12q
73
PKU Treatment/Therapies
- Low phe diet
- Vitamin supplementation/amino acid supplementation
- Tyrosine supplementation
- Prevent fasting/catabolism
- 225-450 mg Phe/day (normal person consumes 4000 mg)
74
PKU New Therapies
- Kuvan - large doses of biopterin - may stabilize poorly functioning PAH
- LNAA therapy - bombard blood brain barrier with other amino acids to prevent Phe uptake
75
PKU Barriers to compliance
```
Peer pressure
Costs of specialty food
Insurance companies
Intolerance to supplements
Limited variety
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76
Structural proteins of the ECM
- Form a large percentage of bulk body protein
- Large multi-domain homo/heteropolymers
- Extensively modified (oxidation, crosslinking, glycosylation)
- Directly influence cell behavior via integrin receptors
- Regulate the activity of and are regulated by growth factors
77
Inheritance of connective tissue diseases
Mostly dominant
- Haploinsufficiency - amorph/hypomorph (Marfan, SVAS)
- Dominant negative - antimorph (Osteogenesis imperfecta)
- Gain of function - hypermorph (Achondroplasia)
78
Marfan Syndrome
- 2-3/10,000 incidence
- 1/4 to 1/3 are de novo
- Skeletal, ocular, cardiovascular, pulmonary, skin and integument, dura
79
Ocular features of Marfan
Major - ectopia lentis
Minor - Abnormally flat cornea, myopia, increased globe length, hypoplastic iris
Other - open angle glaucoma, nuclear cataract
80
Cardiovascular features of Marfan
Major - dilation of ascending aorta with/without aortic regurgitation
Minor - Mitral valve prolapse, dilation of pulmonary artery before age 40, calcification of the mitral valve before age 40, dilation of descending thoracic or abdominal aorta before the age of 50
Other - arrhythmia, dilated cardiomyopathy, diastolic dysfunction
81
Management of Marfan
Skeletal
- Surgical stabilization of scoliosis
- Surgical correction of pectus excavatum
- Hormonal treatment with estrogen to reduce growth
Visual
- Corrective lenses to prevent amblyopia
- Ectopia lentis: surgical removal and artificial lens replacement
- Laser eye surgery
Cardiovascular
- Annual echo
- B-andrenergic blockade to reduce rate of dilation/dissection
- Avoid exertion
- Replacement of aortic root when moderately dilated (>50mm)
- Mitral valve repair
82
Pleiotropy
A gene or mutation in a gene causes phenotypic changes in multiple organs or tissues
Mechanisms:
-Toxic metabolite with multiple target tissues
-Gene product required for homeostasis of multiple organs
-Gene is necessary for a function of a developmental precursor of multiple organs
83
FBN1 mutations and Marfan
Chromosome 15
- Fibrillin microfibrils in ECM
- Most missense mutations located in calcium-binding EGF-like domains
- Some genotype phenotype correlation
84
Disease mechanisms in Marfan
Dominant negative
- overexpression of mutant protein in cells results in abnormal microfibrils
- mutant cells do not assemble microfibrils
Haploinsufficiency
85
Marfan and TGFBeta
TGFbeta antagonism rescues Marfan syndrome in mice
| Losartan antagonizes TGFbeta to rescue MFS in mice - possible medicine for Marfan
86
Diseases related to Marfan
Some FBN1 mutations cause isolated Marfan symptoms
Weill-Marchesani Syndrome
- AD
- Short stature, muscular build, brachycephaly, severe myopia, ectopia lentis, joint stiffness
Stiff skin syndrome
-Mutations in Arg-Gly-Asp of FBN1
FBN2
-congenital contractural arachnodactyly
87
Homocystinuria
AR
- CBS deficiency
- tall stature, long limbs, arachnodactyly, limited joint mobility, pectus deformity, osteoporosis
- highly arched palates, crowded teeth
- ectopia lentis, myopia
- mitral valve prolapse, MI
- fine, brittle hair, hernia
- intellectual disability, seizures
- fatty liver disease, pancreatitis
88
Treatment homocystinuria
- 50% of patients are responsive to pyrixodine (vitamin B6), usually they are mildly affected
- Pyrixodine nonresponders - low methionine, cystein supplementation, betaine treatment
89
Elastin characteristics
```
Secreted as soluble monomer tropoelastin
Lacks internal Met
Contains 2 cysteines at C-terminus
Insoluble, highly stable crosslinked polymer
Produced early in life
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90
Supravalvular Aortic Stenosis
Narrowing of the ascending aorta
Mortality - Sudden Death (severe biventricular obstruction)
Can be non-syndromic
Associated with Williams-Beuren syndrome
91
Familial supravalvular aortic stenosis
AD
Arterial stenosis
1/200,000
92
Williams-Beuren Syndrome
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Rare AD
1/20,000
Sporadic
Characteristic facial features
Intellectual disability, developmental delay
Gregarious personality
Hypercalcemia
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93
Mechanism of ELN mutations in SVAS
Less elastin
Increased vascular smooth muscle cell proliferation
Leads to SVAS
94
Cutis Laxa
AD
- emphysema
- hernias
- pulmonary artery stenosis
- aortic aneurysms
- 30% mutations are de novo
95
Mechanism of ELN mutations in ADCL
Mutant tropoelastin has impaired association with fibrilin microfibrils, increased aggregations with wildtype tropoelastin
- unfolded protein response and increased apoptosis
- Increased TGFbeta signaling
96
Collagens
Most abundant protein in the body
- makes up 70% of bone protein
- There are 28 types of collagen encoded over 45 genes
- long chains with glycine-proline-hydroxyproline
- often form a triple helix
97
Collagen I
Encoded by COL1A1 and COL1A2 genes
| Exists as trimer
98
Proline Hydroxylation
Proline hydroxylation requires vitamin C
Proline hydroxylation stabilizes collagen triple helix
Scurvy results from vitamin C deficiency (James Lind)
99
Osteogenesis Imperfecta
```
1/20,000
Bone fragility, blue sclerae, short stature
Collagen Type I mutations
AR
"Brittle bone syndrome"
```
100
Mechanisms of OI
- most are missense replacements of Gly
| - Loss of function mutations result in AD OI
101
Treatment of OI
Bisphosphonates to increase bone mineral content
Rodding
Bone marrow transplantation (experimental)
Animal studies point to TGFbeta antagonism
102
Ehlers Danlos Syndrome
Skin hyperextensibility and joint hypermobility
Easy bruising
Abnormal
Cigarette paper scarring
103
FGFR3 mutations
Achondroplasia
| Hypochondroplasia
104
Mechanisms of FGFR3 mutations
Too much differentation and too little cell division of growth plate
105
Causes of Hypertyrosinemia
1. Transient tyrosinemia of newborn
2. Severe hepatocellular dysfunction
3. Inborn errors of tyrosine catabolism
4. Scurvy
5. NTBC Treatment
6. Hyperthyroidism
7. Postprandial state
106
Hereditary Tyrosinemia Type I
Rare AR
Life threatening
Phenotypes
- Acute with liver failure at infancy
- Subacute/chronic with a protracted course
- Renal tubular dysfunction
- Rickets (liver failure leads to lack of vitamin D activation)
- 80-90% mortality in childhood
107
HTT1 Cause
Block at the conversion of fumarylacetoacetate, causes it to be converted to succinyl acetone instead
Succinylacetone is a product inhibitor of the enzyme that converts Tyrosine to 4-OH Phenylpyruvate, resulting in excess tyrosine
108
Untreated prognosis of HTT1
- Liver disease
- Renal Tubular dysfunction
- Neurological crises similar to pts with porphyria
- Liver cancer
109
Conventional treatments of HTT1
Diet
| Liver transplant
110
NTBC
Initially an herbicide
Caused eye lesions in rats due to excess of tyrosine
Causes a block in the chain that prevents conversion of 4-OH phenylpyruvate but ALLOWS conversion of tyrosine
Made into a medicine for HTT1 patients called Orfadin
111
NTBC effect on HTT1
- Reverse acute symptoms of liver failure
- Allows for normal growth
- Reverse/prevent renal dysfunction
- Prevents rickets
- Prevents neurological crises
- Prevent or delay need for liver transplant
112
4-hydroxphenylpyruvate dioxygenase (4PPD)
Also called tyrosinemia III
AR
Leads to hypertyrosinemia
113
Tyrosine aminotransferase (TAT) deficiency
Characteristic: Keratosis palmoplantaris
-hyperkaratosis of palms and feet
Ocular and cutaneous symptoms
Half of pts have intellectual disability
Treatment: Dietary restriction of Phe and Tyr
114
Alkaptonuria
```
Rare AR
Homogentisic acid cannot be broken down
(Defects in homogentisic acid oxidase)
Ochronosis (blackening of joints)
Arthritis
Spinal narrowing leading to back pain
Blackened stool/urine
Possible can be treated by NTBC
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