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Biochem Notes:

[purple = step 1 drill questions]

Collagen synthesis, structure, and disorders:

1. Steps of Collagen synthesis?


a. Synthesis
b. Hydroxylation
c. Glycosylation
d. Exocytosis
e. Proteolytic processing
f. Cross-linking
2. What is the structure of collagen?
a. Triple helix of amino acid chains of repeating Gly-X-Y amino acids
b. Hydroxylated proline and lysine typically in X position
3. Hydroxylation of collagen fibers proline and lysine require what?
a. Vitamin C
b. Deficiency of vit C? Scurvy
i. Leads to ↓ collagen hydroxylation
ii. Symptoms:
1. Petechiae surrounding hair follicles
2. Loose teeth
3. Bleeding gums
4. Poor wound healing
5. Poor bone development
4. After hydroxylation, the collagen fibers are glycosylated, and then formed into triple
helices (aka procollagen). In what condition is this defective?
a. Osteogenesis Imperfecta (OI)
i. Genetic bone disorder, MC form = AD = ↓ prod of collagen type 1
ii. Symptoms:
1. Multiple fractures with minimal trauma (brittle bones)
2. Blue sclerae
3. Skeletal deformities
4. Hearing loss
5. Dental issues (lack of dentin)
5. Once procollagen is formed and secreted, propeptides are cleaved into insoluble
tropocollagen. How is tropocollagen then stabilized?
a. Cross-linkage of lysine-hydroxylysine occurs via copper containing lysyl oxidase
b. Defective in Ehler-Danlos and Menkes
6. What genes are implicated in Ehler-Danlos?
a. Lysine hydroxylase and collagen genes  defects in crosslinking collagen fibers
b. Classic EDS = mutation in type 5 collagen fibers
c. Symptoms:
i. Hyperextensible joints
ii. Fragile skin
iii. Hypermobile joints
iv. Dislocations
v. Varicose veins
vi. Ecchymoses
7. Menkes disease is an XR disorder d/t deficiency in what?
a. Copper (d/t defective menkes protein)
b. Leads to ↓ in lysine oxidase activity (as a copper is needed co-factor)
c. Symptoms:
i. Brittle, kinky hair
ii. Growth retardation
iii. Hypotonia
8. What tissues have type 1 collagen?
a. Bone, dentin, skin, tendons, fascia
b. Ex. Decreased production in OI type 1
9. What tissues have type 2 collagen?
a. Cartilage, hyaline, vitreous body, nucleus pulposus
10. What tissues have type 3 collagen?
a. Reticulin = skin, blood vasculature, uterus, fetal tissue, granulation tissue
b. Ex: Vascular type Ehler-Danlos = defect in type 3 collagen
11. What tissues have type 4 collagen?
a. Basement membrane (basal lamina), Lens
b. Ex. Defective in Alport syndrome; targeted by autoantibodies in Goodpasture
syndrome

Genetic Lab techniques:

1. 3 steps involved in PCR?


a. Denaturation (of DNA)
b. Annealing = cooling  DNA primers annealing to specific sections of DNA to be
amplified
c. Elongation = heat stable DNA polymerase replicates the DNA sequence following
the primer
2. What does a southern blot measure?
a. DNA that has been enzymatically cleaved and electrophoresis onto a gel
b. Then moved onto a filter, denatured, and exposed to a radiolabeled DNA probe
c. Then binds and is seen on film
3. What does a northern blot show?
a. RNA that has been cleaved “SNoW DRoP”
b. Gives reflection of gene expression
4. What does a western blot show? Southern = DNA
a. Sample proteins onto a gel and labeled with ELISA Northern = RNA
b. Used for HIV testing (o) (o)
5. What does a southwestern blot show? Western = Protein
a. ID DNA binding proteins
6. ELISA? (Southwestern = DNA + Protein)
a. ID specific antigens in a blood sample. Uses
labeled antibodies bound to enzymes that
produce a signal (Direct ELISA)
OR
b. Uses 2nd antibody that’s bound to an enzyme that produces a signal
7. FISH?
a. Fluorescent DNA or RNA used to probe specific sites of gene of interest on a
chromosome
b. Can ID locations of genes and visualize anomalies such as microdeletions
8. Karyotyping?
a. Metaphase chromosomes stained and visualized, ordered and counted to dx any
chromosomal abnormalities such as trisomies or sex chromosome disorders
9. What is recombinant DNA?
a. Insertion of a gene into a plasmid vector containing antibiotic resistance genes
b. Can be used for generation of genomic libraries or make recombinant proteins
for gene therapy
10. What is restriction endonuclease?
a. Recognizes palindrome sequences in dsDNA and cuts it, leaving sticky ends
b. Used to make restriction maps of DNA, produce fragments
11. What does reverse transcriptase do?
a. Makes a complimentary DNA (NO introns!) strand from mRNA
b. Used to make recombinant DNA or used in retroviruses like HIV
12. What are some methods of modifying gene expression?
a. Transgenic strategies = involve random gene insertion into mouse genome
b. Targeted insertion/deletion = via recombination
13. What is the Cre-lox system?
a. Inducibly manipulate genes to specific developmental points
b. Study a gene whose deletion induces embryonic death
14. What is RNA interference?
a. dsRNA synthesized that’s complimentary to mRNA of interest  then trans-
infected into human cells  dsRNA separating and binding to the mRNA  ↓
gene expression  KNOCK OUT‼

Patterns of inheritance:
1. Type of inheritance pattern in familial hypercholesterolemia, Huntington’s,
Neurofibromatosis, and Marfans?
a. AD = affects structural genes usually
b. Familial hypercholesterolemia = ↑ LDL and absent LDL receptors
i. Severe atherosclerotic disease with corneal arcus and tendon xanthomas
c. Huntington disease:
i. Symptoms = depression, early onset dementia, choreiform movements,
caudate atrophy
ii. d/t trinucleotide repeats of CAG in chromosome 4
d. NF type 1:
i. Symptoms = café au lait spots, cutaneous neurofibromas, optic gliomas,
pheochromocytomas, lisch nodules
ii. 100% penetrance
e. NF type 2:
i. Chromosome 22
ii. Symptoms = B/L acoustic schwannomas, juvenile cataracts, meningiomas
and ependymomas
2. Describe AD inheritance.
a. Affected individuals have an affected parent
b. Either sex is affected
c. Variable to late onset usually
3. PKD: what mode of inheritance and what mutations?
a. AD
b. Chromosome 16 = PKD 1
c. Chromosome 4 = PKD 2
4. FAP: mode of inheritance and mutations?
a. AD
b. 5q APC gene
5. Hereditary spherocytosis: mode of inheritance and deficiency in what?
a. AD
b. Spectrin/Ankyrin deficiency causing hemolytic anemia
6. Li Fraumeni: mode of inheritance and gene defect?
a. AD
b. TSG p53  multiple malignancies: sarcoma, breast, leukemia, adrenal gland
7. Marfan syndrome: mode of inheritance and defect in what protein and gene?
a. AD
b. FBN1 (fibrillin) on chromosome 15  defective elastin binding
8. MEN syndromes associated with what and what mode of inheritance?
a. AD
b. Tumors of endocrine system (pancreas, parathyroid, pituitary, thyroid, adrenal
medulla)
9. Tuberous sclerosis: mode of inheritance?
a. AD
b. Incomplete penetrance + variable expression
c. Symptoms = “HAMARTOMAS”
10. VHL: mode of inheritance and defect?
a. AD
b. TSG VHL gene deletion on Chromosome 3
c. Numerous benign and malignant tumors
11. Type of inheritance pattern in Sickle cell, CF, PKU, and Kartegener’s?
a. AR = affects genes encoding for enzymes usually
12. Describe AR inheritance.
a. Unaffected parents
b. Either sex affected
c. Early/uniform onset
d. Often catalytic proteins
e. Sometimes occurs in consanguinity (incest)
13. What do fragile X and hypophosphatemic rickets (vit D resistant rickets) have in
common?
a. Both are XD!
14. XD inheritance:
a. Males cannot pass onto sons
b. Mothers transmit to 50% of offspring
c. Fathers to ALL daughters
15. Type of inheritance in Duchenne, G6PD def, Lesch-Nyhan, Ornithine transcarbamylase
def, Hemophilia A/B?
a. XR‼
16. What is a sign of mitochondrial inheritance?
a. Transmitted though mothers
b. Variable expression
c. Mitochondrial myopathies = ragged red fibers on muscle biopsy
17. Define codominance:
a. Both alleles contribute to phenotype
b. Ex: blood groups: A, B, AB; α1 antitrypsin deficiency
18. What is variable expressivity?
a. Disease may present differently in different individuals
b. Ex: NF type 1
19. What is incomplete penetrance?
a. Mutant genotype may not produce a mutant phenotype
b. Ex: BRCA 1 gene for breast cancer ↑ chances of getting breast cancer to 60%
(not 100%)

Trisomy disorders:

1. Down syndrome is a disorder of what chromosome?


a. Chromosome 21
b. Signs:
i. Flat facies
ii. Prominent epicanthal folds
iii. Single palmer crease
iv. Duodenal atresia
v. Endocardial cushion defects
c. ↑ risk of:
i. early onset Alzheimer’s dementia
ii. ALL (> AML)
d. At what stage of meiosis is Down syndrome a/w?
i. Meiotic nondisjunction (usually in Meiosis I, sometimes Meiosis II)
e. Detection:
i. 1st trimester:
1. U/S = ↑ nuchal translucency + hypoplastic nasal bone
2. ↓ Serum PAPP-A
3. ↑ β-hCG
nd
ii. 2 trimester:
1. Quad screen = ↑ AFP, ↑ β-hCG, ↓ estriol, ↑ Inhibin-A
2. Edwards syndrome:
a. Trisomy 18
b. Findings:
i. Severe intellectual disability
ii. Rocker bottom feet
iii. Micrognathia (small jaw)
iv. Low set ears
v. Clenched hands with overlapping fingers
vi. Prominent occiput
vii. Congenital heart disease
viii. Usually, death within 1 year
c. Detection:
i. 1st trimester = ↓ PAPP-A and free β-hCG
ii. 2nd trimester = Quad = ↓ AFP, ↓ β-hCG, ↓ estriol, ↓/nL Inhibin-A
3. Patau syndrome:
a. Trisomy 13
b. Findings:
i. Severe intellectual disability
ii. Rocker bottom feet
iii. Microphthalmia
iv. Microcephaly/Holoproscencephaly
v. Cleft lip/palate
vi. Polydactyly
vii. Congenital heart disease
viii. Death within 1 year
c. Detection:
i. 1st trimester = ↓ β-hCG, ↓ PAPP-A, ↑ nuchal translucency
4. Cri-du-chat:
a. Deletion in chromosome 5 short arm (p)
b. Findings:
i. Microcephaly
ii. Mod to severe intellectual disability
iii. High pitched crying/mewing
iv. Epicanthal folds
v. Cardiac abnormalities (VSD)
5. Williams syndrome:
a. Deletion of long arm (q) of chromosome 7 – region of elastin gene
b. Findings:
i. Elfin facies
ii. Intellectual disability
iii. Hypercalcemia
iv. Overly Friendly
v. CVS problems

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