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What nucleotide is methylated?
Cytosine at the 5 position
All of the following syndromes are autosomal dominant except:
D) Wilson's disease
G) Hereditary retinoblastoma
D - Wilson's is AR
All of the following syndromes are autosomal recessive except:
A) MEN syndrome
B) Wilson's dz
D) Cystic Fibrosis
E) Hereditary hemochromatosis
A - MEN is autosomal dominant
All of the following are tumors found in Li-Fraumeni syndrome (P53 mutation) except:
A) Breast, stomach, colon CA
C) Clear cell RCC
E) Adrenocortical carcinoma
All of the following are X-linked dominant disorders except:
A) Incontinenta pigmenti
B) Fabry's disase
C) Fragile X
D) Vitamin D inherited rickets
Give examples of X-linked recessive disorders.
- Mnemonic: The Hunter Acquired HALF His Muscles from Mom (X-linked recessive)
- Alport syndrome
- Hemophilia A & B
- Muscular dystrophy
- Menke syndrome
What is the mutation for Fabry's dz? Clinical sxs?
Xq22 GLA gene → deficient α galactosidase → globotriasylceramide accumulation; pain in distal extremities, angiokeratomas, hypohydrosis, corneal & lenticular opacities, eventually die of renal failure, CVD or cerebrovascular dz
What may account for the clinical variability noted in mitochondrial diseases? What type of mitochondrial mutations present in childhood vs. later in life?
heteroplasmy (harboring dual population of nml and mutated mitochondria); nuclear DNA; mitochondrial DNA
What screening test is abnml in mitochondrial dz? What does a muscle bx show with trichrome & succinate dehydrogenase stain? What does EM show?
CSF or plasma lactic acid, ketones, acylcarnitines, urinary organic acids; ragged red fibers & stronger staining of vessels with succinate stain from abundant mitochondria; "parking lot" inclusions
What type of mutation causes Kearns-Sayre, Pearson, and progressive external ophthalmoplegia?
large mito DNA dels
What mito dz causes pigmentary retinopathy, sensorineural hearing loss, cardiomyopathy, cerebellar ataxia, hearing loss, myopathy, DM, and hypoPTH?
- *Note: these symptoms are seen in most of the mito dzs except for hypoPTH
What mito dz presents in childhood with lactic acidosis and stroke-like episodes with neurologic defects? What is the mutation? What is seen in the brain?
Mito encephalopathy w/ LA & stroke-like episodes (MELAS); MT-TL1 gene encoding tRNALEU due to single point mutation (A3243G)
What is the mutation in MERRF (myoclonic epilepsy with ragged red fibers)? What clinical finding is notable in addition to the regular mitochondrial-related manifestations?
A8344G, an MT-TK gene encoding tRNALYS; multiople lipomas
Which mito dz presents in young males with visual failure?
Leber hereditary optic neuropathy
What is notable about CpG regions of the DNA?
Hot spot where mutates to TpG at high rate
What are the three stop codons causing a nonsense mutation?
UAA, UAG, UGA (ex. β° thalassemia in Sardinia due to CAG to UAG at codon 24in )
What type of mutation is FAP? What is the mutation? Clinical manifestations?
truncation mutation; APC tumor suppressor gene on chrom 5q21 inhibits Wnt path & β catenin fxn so loss of this fxn → increased transcription of downstream genes like MYC and CCND1; colon, gastric (fundic), SI polyps, thyroid CA, desmoids
What happens in splice mutations?
GT/AG rule commonly present at end of introns so loss causes expression of introns → weird proteins
What is the gene mutation for Tay-Sachs?
HEXA gene on 15q from 4bp insertion (frameshift)
What 3 mechanisms underlie clinical symptoms of disease in female carriers of Hem A?
- 1) Asymmetric lyonization
- 2) Homozygosity
- 3) Hemizygosity (Turner syndrome)
What is the chromosomal mutation in mild Hemophilia B? Severe dz?
Xq27 with missense point mutations; frameshift, nonsense, microdel, and gross deletions
What populations can develop spontaneous F8 inhibitors? How long must the 1:1 mix be incubated? What is the prognosis?
Elderly and pregnant pts; 2 hrs; good, transient and disappears in a few mos
How is molecular diagnosis of Hemophilia A performed?
Southern blot analysis for inversion intron 22 or direct sequence analysis for other mutations like intron 1 inversion, or RFLP-based analysis (must have samples of two generations or more)
What chromosomal mutations are involved in Hemophilia A? What other dz should be considered if clinically looks like Hem A?
Xq28 with 50% intron 22 inversion in Caucasians, which predicts likelihood of developing anti-FVIII Abs (*look for the X and #8 for FVIII!), 5% with intron 1 inversion; vWD type IIN (inability of vWF to bind F8 →↓ F8 levels)
What is the prevalence of cystic fibrosis? Most common disease-causing mutation? MC mutation in Ashkenazi jews?
1/2000 live births in Caucasians with gene frequency of 1:20; ΔF508 mutation with lack phenylalanine (F) residue → rapidly degraded protein → Na and Cl transport mutations → hyperviscous secretions; W1282X truncating mutation
How is CF diagnosed?
- 1) molecular testing for CFTR 7q31.2 mutation
- 2) 2 consecutive abnml pilocarpine iontophoresis sweat chloride tests (>60mEq/L dx of CF)
- 3) Abnml transepithelial nasal potential difference showing increased voltage potential diff across nasal epithelium
What is the newborn screening test for CF?
immunorx trypsinogen assay (IRT) with elevated IRT levels in CF, used within first 2 mos of life
What three pulmonary infections are common in CF?
S. aureus, B. cepacia and P. aeruginosa (mucoid Pseudomonas is diagnostic of CF)
What type of mutation is in BRCA1? What are the BRCA genes? Lifetime risk of breast cancer with the mutation? What are the common mutations in Ashkenazi jews?
insertion of cytosine in exon 20 → frameshift & premature stop codon (75% of cases have protein truncation); tumor suppressor genes mediate DNA repair; 70%; 1) two-pair del in codon 23 (185delAG) of BRCA1, 2) 5382insC mut in BRCA1, and 3) 6174delT mut in BRCA2
What are the trinucleotide repeats in the following diseases:
1) Fragile X
2) Myotonic dystrophy
3) Spinocerebellar ataxia, type 1
4) Friedrich ataxia
5) Huntington Dz
- 1) CGG, 5'UT
- 2) CTG, 3'UT
- 3) CAG-polyglutamine
- 4) GAA, intron 1
- 5) CAG
What is the gene involved in Huntington's? How many repeats in a normal person is capable of having affected offspring?
HD gene at 4p16; >36 CAG repeats
How does restriction fragment length polymorphisms identify disease?
Use restriction endonucleases to digest DNA into fragments, visualized by Southern blot. DNA polymorphisms result in varying lengths of DNA from person to person. Look for differences, ex. loss of restriction sites, fragments "informative markers" that are missing in family members w/out dz
What is nested PCR and what is a major disadvantage?
Initial amp product is subjected to a 2nd round of PCR using a new set of primers; high rate contamination
What is competitive PCR used for?
Quantification using two templates: starting amount is known for one template and the unknown in the other, then compare the products of both to quantify (used in HIV-1 and HCV RNA)
What protects DNA during the annealing process of PCR?
Annealing buffer: NaCl or KCl, Tris-HCl, Triton X-100
What is real-time PCR?
Detection of rxn products simultaneously with amplification, used for qualitative or quantitative results
What are allele-specific oligonucleotides?
15-18 base length frags complimentary to the sequence of interest that can hybridize to the DNA sample (ex. reaction product of PCR) if the sequence is present
How can Mg affect PCR? How does heme affect PCR?
↑ binding but ↓specific amplification; heme is an inhibitor
What RT-PCR primer is used for the mRNA poly-A tail?
In DNA sequencing, what is used to terminate the chain?
dideoxynucleotides (ddNTPs) lack 3' OH → chain termination at varying lengths → sequence by automation or gel
All of the following methods are used to detect unknown mutations except:
A) single strand conformation polymorphism
C) heteroduplex formation
D) chemical cleavage of mismatch
E) RNase A: cuts a pyrimidines
- *In heteroduplex, wt DNA mixes with mutated DNA, and the wt-mut complexes migrate slower
What is the size of an avg FISH probe? What mutations are not detected by FISH?
30kb+; small mutations (best detected by PCR)
Name 4 types of probes used in FISH.
- 1) locus-specific ID probe (ex. BCR-ABL)
- 2) Chromosome enumeration probes
- 3) Whole chromosome painting probe for complex structural rearrangements
- 4) Subtelomeric DNA probes for gene-rich area to detect hidden dels or translocs
Name 3 causes of artifacts in FISH.
- 1) Truncation artifact -section thru nucleus so lose some chrom material → false neg
- 2) Aneuploidy and polyploidy in neoplastic cells → apparent duplications
- 3) Autofluorescence from endogenous enzymatic activity
What types of mutations are detected with spectral karyotyping?
aneuploidy and translocations
In TCR gene rearrangement assays, what is the gene detected in Southern blot? PCR? What chains are rearranged first in T devt?
PCR - TCRγ, Southern TCRδ; γ and δ chains
What is the most common inherited defect among northern europeans? What is the prevalence?
Hereditary hemochromatosis (HH); 1:200-1:300 in US
What is the most common mutation in hereditary hemochromatosis (HH)? What mutations more commonly lead to dz? What type of protein is involved in the HFE mutation? Clinical sxs?
C282Y (>80%) and H63D (~5%); homozygous C282Y highest disease likelihood but many don't have clinically signif dz; HLA Class I protein associates with β2-microglobulin, which may regulate transferrin binding to receptor; liver damage, bronze DM, cardiomyopathy, pituitary gland
Which of the following are tumors found in Cowden syndrome (more than one may apply):
B) Dysplastic gangliocytoma
C) Colorectal CA
D) Follicular carcinoma
E) Breast CA
F) Uterine leiomyomas
All of them - in the colon it's usually hamartomatous polyps but they're at risk for colon CA
What is the pathognomonic lesion for the following PTEN related disorders:
2) Proteus syndrome
3) Bannayan-Riley Ruvalcaba
- 1) cerebellar dysplastic gangliocytoma
- 2) connective tissue nevi
- 3) None but it has weird stuff like pigmented macules of the glans penis and lipomas
What does PTEN encode?
Phosphatidylinositol 3,4,5 triphosphate 3-phosphatase, it's a phosphatase that acts in PI3 Kinase/Act cell cycle arrest and apoptotic pathway in lipids & proteins
All of the following tumors are found in Beckwith-Wiedemann syndrome except:
B) Wilms tumor
C) Adrenal cortical carcinomas
What is the mutation in Beckwith Wiedemann?
Abnml transcription of 11p15.5 → translocation of ZNF215, methylation of KCNQ1 and/or IGF1, etc.
What is the most common mutation in hereditary Rb? What type of mutation is typically involved? What other tumors are they at risk for?
chrom 13q14, a tumor suppressor gene; point mutations in ~70% of cases; pineal gland tumors, PNET/Ewings, bone tumors (osteosarcomas, chondrosarcomas), fibrosarcomas, leukemias, lymphomas
What non-gyn malignancies are asociated with BRCA 1 mutations?
Prostate, pancreas, colon
Which BRCA is more often associated with male breast ca? What non-gyn tumors do they get?
BRCA2; gallbladder, prostate, melanoma, pancreas, stomach
What is the mutation in familial medullary thryoid CA (FMTC)? what is the mutation in MEN2A? MEN2B?
RET gene (10q) mutations on cysteine residues (609, 611, 618, 620); RET mutation affecting exons 10-11 affecting cysteine residue 634; RET mutation affecting exon 16 from single point mutation (M918T)
Men 1 mutation?
Menin on 11q13
What is the mutation for combined FV and FVIII deficiency?
Chrom 18q on LMAN1 gene encoding ERGIC-53, which is a chaperone of rough ER and Golgi for intracellular transport of FV and VIII
Chromosomes for all the following factors:
1) FVII & FX
5) Combined FV & FVIII
- 1) FVII & FX - chrom 13q
- 2) FVIII - Xq28
- 3) FIX - Xq27
- 4) FV - 1q21-25
- 5) Combined FV & FVIII - chrom 18q
- 6) FXIII - chroms 6p (A subunit) and 1q (B subunit)
What chromosomal translocation is particularly unsuitable for PCR detection?
t(11;14) MCL b/c bcl-1 gene is too big to amplify
What are telomeres? How does it relate to apoptosis?
Molecules that are reverse transcriptases with RNA subunits that add TTAGGG to the ends of chromosomes to prevent loss of chromosomal cmpts and prevent fusion; they shorten with each cell division and when too short, undergo apoptosis
What is the prognosis for AML with CEBPA or KIT mutations?
- CEBPA - good prognosis (associated with CD7+, AML M1 and M2)
- KIT - bad
What specific FLT mutation is tied to a poor prognosis?
Internal tandem duplication (not tyrosine kinase domain mutations)