Diagnostic (Washington and Kruzka) Flashcards
1
Q
What is a Rare orphan disease?
A
It is defined as a disease that affects fewer than 200,000 Americans at any given time
2
Q
What are cloning vectors used for?
A
DNA mapping and genotyping.
Method for fractioning complex starting DNA populations.
3
Q
Functional cloning
A
Gene function precedes identifying gene; requires knowledge about the function and structure of protein product
4
Q
What are examples of functional cloning?
A
Bioinformatics
Microarrays
5
Q
Positional cloning
A
gene mapping precedes gene identity (characterize the gene, its locus, and its allele) ; requires knowing the map position of a gene (physical and genomic)
6
Q
What are examples of positional cloning?
A
Genome/exome sequencing
Halotype maps
7
Q
What us Genomic mapping?
A
Describes the order of genes, markers, and the spacing between them
8
Q
Genetic map?
A
linkage association based on genetics recombination (statistical probability) in pedigree (linked to a marker)
- has immediate clinical application to provide information about a genes location
9
Q
Physical map
A
base pair sequence (sequencing/HGP)
10
Q
What are the two basic types of polymorphism that can be used for genotyping?
A
Restriction site polymorphism (RSP) - used to study disease
Variable number tandem repeats (VNTR) or restriction fragments polymorphism (RFLP)
11
Q
Single Nucleotide Polymorphism (SNPs)
A
exist as a base pair change in DNA
- subset of SNPs have been detected by loss of gain of a restriction site at the place of the change base = RSP
12
Q
VNTR
A
Repeat units ~9-65 bp long
arise from instability on an array of tandem repeats causing variable exchange in the repeat until
- looks at microsatalite and minisatallite region
13
Q
Linkage vs association
A
Linkage it the relationship between loci (Genetic). Provides powerful method for scanning the gene in 20-50Mb segments to locate a disease gene
Association is the statistical statement about co-occurrence of alleys and phenotypes
14
Q
Linkage Association
A
narrow down candidate gene region
15
Q
Non-syntenic vs systenic
A
Two genes are systemic if they are located on the same chromosome (both on chromosome 1)
Non-Syntenic - located on different chromosomes (one on chromosome 1 and the other on chromosome 2)
16
Q
Phase
A
a heterozygous genotype for each of two linked genes can have allele in either of two configurations - cis or trans
17
Q
trans configuration phase
A
also known as repulsion
mutant and marker alleles on opposite homologous chromosomes
18
Q
cis configuration phase
A
also know as coupling
both mutant and marker allele on the same homologous chromosome
19
Q
Chaismata in prophase results in what?
A
Recombinant allels - non parental chromosomes
20
Q
What does recombination fraction define?
A
Genetic distance (not physical distance
21
Q
Recombination equals to 0.5 tells you what?
A
That the allele/genes reside on the different chromosome or systemic alleles are very far apart
22
Q
Recombination equals to 0 tel you what?
A
That the allies reside close together, crossover rarely occurs and they are considered to be linked
23
Q
Linkage equilibrium (LE)
A
Two loci inheritance is an independent event = random assortment
24
Q
Linkage disequilibrium (LD)
A
two particular alleles at different loci are not segregating independently in a population (if you are not at 50% segregation)
This plays a fundamental role in gene mapping, fine mapping of complex disease genes and GWAS
25
When a new mutation occurs what does it create?
Linkage disequilibrium
26
quantify disequilibrium
difference between observed frequencies of two-locus calotype minus the frequencies it would be expected to show if the alleles are segregating at random.
D = P (AB) - P(A) x P(B)
Max - 0.5 (in linkage or in repulsion)
27
How does time and recombinational distance affect linkage distance?
It cause Linkage disequilibrium to decrease
| Look at slide 17
28
Calculating Theta Recombination
The number of recombination/total
Convert fraction -> decimal -> cM -> bases apart
ex. Theta = 0.2 = 20% = 20cM = 2 x 10^7 bases apart
every 10% = 10cM = 1 x 10^6
Above 50% means that the genes are not close together
29
Lod Score
The ratio of the 2 likelihoods gives the odds of linkage
Most efficient statistic for evaluating pedigrees for linkage
Calculated for a range of theta values
Most likely recombinant is the value which the score is at its maximum
30
What does it mean when LOD (Z): >/= 3.0 (1000:1)
It means that it (linkage) is in favor
31
What does it mean when LOD (Z): = 2.0 (1:100)
It mens that it is against linkage
32
LOD (Z) equation
1/2 non recombination probability x 1/2 probability recombination / probability of no linkage
Non recombination fraction = 1-theta so the frequency with 2 parental types = 1-theta / 2
recombination fraction = theta so frequency with two parental yes is theta/2
frequency of no linkage with two parental types is 0.5/2
33
What is the phase for DMD marker alleles?
This is Duchane's muscular dystrophy and the D2 and L128 marker alleles are in coupling
34
Haploid Genotype
Also known as a halotype
A combination of alleles that are transmitted together on the same chromosome
- considered a unit
- no recombination
35
What is a HapMap?
tool that allows researcher to find genes and genetic variations that affect health and disease
36
What is GWS?
Genome Wide Association Studies?
Technology: Genomic DNA probes
Analysis: single nucleotide polymorphism ; estimate decrease in risk but you don't know which gene it is
37
HapMap and GWAS are generated by using?
By using single nucleotide polymorphism (SNPs) on a single chromatid that are statistically associated
38
What does mapping the relationships among SNPs tell you
How close these genes are (how related we are to others). By looking at SNPs you can determine what changes cause the disease to be more severe and what changes are helpful.
ex. CHF genetic SNP association decrease risk of AMD
In african americans for atherosclerosis and alzheimers having the ApoE2 mutation is better than having the ApoE4 mutation
39
fluorescence in situ hybridization (FISH)
Technology fluorescently labeled probes
Analysis: locate the position of specific DNA sequence on chromosome (del/dup. translocations)
This specifically looks at chromosome changes
40
Comparative Genomic Hybridization (CGH)
Technology: Genomic DNA Probes
Analysis: copy number variations, similarities and differences between the function, gene expression, and gene regulation
41
DNA microarray (DNA chip)
Technology: Small glass slide and DNA hybrid
Analysis: genetic variation at millions known genetic variants
42
How can you detect very small deletions (0.5-1X 10^7 basses), t to 10Mb?
by using FISH or Spectral Karyotyping (SKY)
Probes are derived from genomic libraries
43
Identification of Wolf Hirshorn syndrome (-4p) and Cri-du-chat syndrome (-5p) is done by?
FISH
| Chromosome staining occurs -> macro-changes to chromosome -> identification
44
Identification and diagnosis of DiGeorge and VCFS syndrome, Ph+CML (Clinical Cancer Vignette, trisomy 8), translocation of partial trisomy 21 46 XX +21 is done by what?
FISH
45
What is clinical cancer vignette?
Chromosome 9 - abl segment
Chromosome 22 - bar segment
Two segments break off and join -> hybrid chromosome -> Philly chromosome
Creates a different fluorescent color
46
What is Spectral Karyotyping (SKY) and multiplex-FISH (M-FISH)?
chromosome specific - labeled DNA fragments covering the length of each individual chromosome
detect inerchromosomal rearrangements and aneuploidy
used for hybridization experiments with metaphase chromosome spreads. Maternal and paternal copy of each chromosome labeled with same color
47
Deletion of the DMD gene can be detected by?
FISH
48
BRCA1 BrCa can be treated with?
olaparib 400mg twice daily
49
What only identifies variation in DNA that is common in the population?
Genome Wide Searches
50
Sequencing?
Determines every letter in DNA sequence, reveals rare mutations
51
Exam/transcriptome sequencing?
used to analyze rare mutations in expressed and coding regions
52
How are DNA microarrays used in GWAS?
Shows
1. Expression Analysis
2. Mutation analysis
3. Comparative analysis
53
What is a chip array?
cDNA copies correspond to a large number of different mRNAs = probes
54
What is expression analysis?
cDNA probe target cDNA transcriptome. Compares gene expression profile b/tw normal and diseased tissues.
55
What is mutation analysis?
gDNA probe targets gDNA variation - SNPs
56
What is comparative Genomic hybridization?
Identification of an increase or decrease chromosomal fragments harboring genes involved in a disease
57
comparative genomic hybridization (CGH)
also used in GWAS
also known as chromosomal microarray analysis (CMA) - analysis of copy number changes (gains/loses in the DNA content) and can only detect unbalanced chromosomal changes (not balance or inversions)
used in diagnosis of tumor cells
Abnormalities (< 5 Mb( detected with banding - microscopic
58
How do you analyze Array CGH?
Fluorescent signals are typically presented as rations - tells yo intensity of hybridization
Ratio = 1 = single equivalent to a control sample
Ratio = 1.5 = Trisomy for an autosome (case to control ratio of 3:2)
Ratio 0.5 = Monosomy for an autosome (case to control ratio of 1:2)
Samples are usually hybridized with control of sex
59
Who is the father of Genomics?
Frederick Sanger
deduced the complete sequence of insulin
developed
- methods for determine small sequence of RNA
- using the dideoxycytidine technique
- adaptation of efficient cloning methods for whole-genome shotgun
60
What does everyone respond differently to medicines?
Variations in our SNPs and cytochrome P45
61
Pharmacogenetics?
study of monogenic traits involving individual variations in drug metabolism
62
Pharmacogenomics?
study of pathways encoding proteins that influence pharmacokinetics and pharmacodynamics
63
What is a major cause in inter-indivual variation of drug affect?
allele polymorphism
64
What are the four basic stages in drug metabolism?
```
Absorption
Distribution
Metabolism
Interaction
Excretion
```
ADME
65
What are the pharmocokinetic factors ?
absorption, distribution, excretion = METABOLISM/CATABOLISM
66
What are the Pharmacodynamic factors?
target interaction, signaling cascade of downstream target = THE EFFECT
67
What are two classical enzymatic examples of RxGenetics?
gutyrlcholinesterase (BCHE) - hydrolysis of succinylcholine, short acting muscle relaxant (neurotransmitter antagonist)
N-acetyle transferase (NAT) - acetylation of isoniazid), anti-tuberculosis drug
68
What is the monogenic variation of BCHE?
Some treated with short-acting succinycholine -> prolonged muscle paralysis -> lethal adverse response
To determine if they metabolize choline well you give the dibucaine -> tells you if you are a fast or slow metabolizer
Atypical form - missence - SNPG290A -> affects active site -> reduces catalytic hydrolysis of succinylcholine -> reduce resistance to inhibition by dibucaine
69
What is NAT?
It is a isoniazid (prodrug) that reached therapeutic concentrations in serum but must be processed first
metabolism via acetylation where isonicotinic acyl is coupled with NADH by bacteria enzyme leads to inhibition of synthesis of mycobacterial call wall
70
What is the monogenetic variation of NAT2?
Slow acytlaters: 10-20% reduction of NAT2 in liver cytosol - affect messenger RNA -> phenotypes by caffeine catabolism
71
Where are the NAT genes located?
NAT1, NAT2, AND NATp are located on chromosome 8
72
What are the genes for NAT2?
G191A, C282T, T341C
73
What are the side effects for NAT2?
```
severe and sometimes fatal hepatitis
headache
poor concentration
weight gain
poor memory
depression
```
increased risk of autoimmune disorders post drug exposure
74
NAT2 polymorphism: T431C/C481T is more prevalent in?
White and African - 28% (unlike asians 7%)
75
NAT2 polymorphism: G191A is more prevalent in?
Asians - 10-18% (unlike where and africans <5%)
76
What is CYP2D6?
A type of Cytochrome P450 that has deletions, critical SNPs, duplications (highly polymorphic).
They are important in drug metabolism
77
CYP2D6 Homozygous deletion, frameshift, and stop codon
Slow metabolizers
78
CYP2D6 heterozygous deletion, frameshift, and stop codon
Intermediate metabolizers
79
CYP2D6 duplicated, multiplied
fast metabolizers
| Don't give them as much as the drug
80
Cytochrome P450 2C9
absent in 1% caucasians and africans -Americans
Primary metabolism of
- NSAIDS (cOX-2)
S-Warfarin (active form)
- Phenytoin
Inhibited by Fluconazole
81
No CYP2D enzyme and CYP2D6 duplications is more common in?
Europe
82
For SNP what does the minor allele frequency have to be?
>1%
83
What date did the human genome project complete?
14 April 2003
84
```
Case 1
12 y.o girl
Short stature
Normal development
Pulmonary stenosis
```
Noonan syndrome
85
What are the symptoms of Noonan syndrome?
Eyes wides, ears low
Congenital heart disease
Characteristic craniofacial findings
Developmental delay (variable)
86
What is Noonan syndrome caused by?
Caused by mutations in the RAS-MAPK pathway (gain function), with PTPN11 being the most common mutated gene. (single base pair change)
87
What test would you use to detect Noonan Syndrome?
Sanger Sequencing Based on selective incorporation of chain-terminating dideoxynucleotides by DNA polymerase during in vitro DNA replication
(most widely used for 25 years but is now being replaced by Next Generation Sequencing)
88
```
Case 2
10 year old boy from Lagos Nigeria
Being seen today for tetralogy of Fallot (heart tubes mixed up)
Nasal voice
Developmental delay
```
22q11.2 deletion syndrome - diGeorge Syndrome (Velocardioficial syndrome; Shprintzen)
89
What are the symptoms of 22q11.2 deletion syndrome?
Heart malformations conotruncal malformations (tetralogy of Fallot, interrupted aortic arch, and truncus arteriosus)
Palatal abnormatlities (velopharyngeal incompetence)
Distinctive facial characteristics
Immune system anomalies
Developmental delay
Increased risk for mental illness
90
How do you test for 22q11.2 deletion?
This condition contains copy number variant which Array CGH would be used to detect (Probe hybridization)
Microarray is based on hydrogen bonding
91
What is copy number variant (CNV)?
alternative structure in genomic DNA that typically includes deletions (reduced copy number) and duplications (more copies) in adjacent segments of DNA.
CNVs may be disease causing OR may vary innocently among individuals, thus we can also consider them to be a type of polymorphism
92
What are the steps for Array CGH?
1-3: Patient and control DNA are labeled with fluorescent dyes and applied to the microarray
4: Patient and control DNA compete to attach, or hybridize to the microarray
5: microarray scanner measure the fluorescent signal
6: computer software analyzes the data and generates a plot
You that where the allele is missing there will be some amplification. Everything else will look normal and have the same ratio.
93
Case 3
| How can genomic technology be used in a case control study to evaluate a common disease such as autism?
You would use a SNP chip with GWAS
SNPs of disease population will be compared to SNPs of normal population - looking for common SNP
It allows you to find association of genes.
You will not know exact specifics but will know that certain SNPs are more prevalent in diseased population
94
What are some Mendelian diseases caused by rare alleles?
Achondroplasia
Duchenne Muscular Dystrophy
Huntington Disease
95
What are some common disease caused by common variants? Detected by GSA
Type II Diabetes
Coronary artery disease
PTSD
Autism (common genetic variants on 5p14.1)
96
Case 4
Multifactoral
Difficult case
Patient has multiple symptoms that don't line up with on particular spectrum
What do you do when you end up in this situation?
Next Generation sequencing = Massive parallel sequencing
Chop up chromosome and align it with a reference sequence (from Human genome project) -> this will allow you to detect heterozygosity and nucleotides that have been changed -> PCR is used to amplify sequence
97
What is the scope and limitations of next generation sequencing?
5 – 10% of the genome cannot be confidently mapped/aligned to reference genome
SNV ~ 90% of total burden of genetic variation with a sensitivity of > 95%
Insertions and deletions (~ 10%) with sensitivities of 50-80%
98
Whole Exome Sequencing (protein coating)
Condenses genome to 1.5 billion -> obtain fragments -> hybridize -> Bate with beads -> capture DNA -> sequence
99
What can cause rare disorders that occurred in case 4? Where there was no specific disease that was able to be diagnosed.
It could have been a possibility of gremlin mosaicism
As in the the Zebrafish
