7/30/2019 LP 6 TesteMoleculareGeneticeDermatologie Part I
1/70
Disciplina de Dermatologie, Universitatea de Medicin Victor Babe Timioara
1
METODE DE DIAGNOSTIC MOLECULAR SI
GENETIC IN DERMATOLOGIE
7/30/2019 LP 6 TesteMoleculareGeneticeDermatologie Part I
2/70
The Central Dogma
The central dogma (due to
Francis Crick in 1958) states
that information flows are all
unidirectional:
The central dogma states that
once `information' haspassed into protein it cannot
get out again.
DNA RNA ProteinGenomeEvolution
Selection
Transcription Translation
7/30/2019 LP 6 TesteMoleculareGeneticeDermatologie Part I
3/70
7/30/2019 LP 6 TesteMoleculareGeneticeDermatologie Part I
4/70
Oncogenes: BAX, BCL2L1, CASP8, CDK4, ELK1,ETS1, HGF, JAK2, JUNB, JUND, KIT, KITLG, MCL1,MET, MOS, MYB, NFKBIA, NRAS, PIK3CA, PML,PRKCA, RAF1, RARA, REL, ROS1, RUNX1, SRC,STAT3, ZHX2.
Tumor Suppressor Genes: ATM, BRCA1, BRCA2,CDH1, CDKN2B, CDKN3, E2F1, FHIT, FOXD3, HIC1,IGF2R, MEN1, MGMT, MLH1, NF1, NF2, RASSF1,RUNX3, S100A4, SERPINB5, SMAD4, STK11, TP73,
TSC1, VHL, WT1, WWOX, XRCC1.
Oncogenic & Tumor Suppressor Properties: BCR,EGF, ERBB2, ESR1, FOS, HRAS, JUN, KRAS, MDM2,MYC, MYCN, NFKB1, PIK3C2A, RB1, RET, SH3PXD2A,TGFB1, TNF, TP53.
Transcription Factors: ABL1, BRCA1, BRCA2,CDKN2A, CTNNB1, E2F1, ELK1, ESR1, ETS1, FOS,FOXD3, HIC1, JUN, JUNB, JUND, MDM2, MEN1, MYB,MYC, MYCN, NF1, NFKB1, PML, RARA, RB1, REL,RUNX1, RUNX3, SMAD4, STAT3, TGFB1, TNF, TP53,
TP73, TSC1, VHL, WT1, ZHX2.
Epithelial-to-Mesenchymal Transition: BRCA2,CDKN2B, CTNNB1, ERBB2, HGF, JAK2, KIT, MCL1,NF1, RUNX3, S100A4, SMAD4, TGFB1, VHL.
Angiogenesis: AKT1, CTNNB1, EGF, ERBB2, NF1,PML, RUNX1, TGFB1.
Apoptosis: BAX, BCL2, BCL2L1, BRCA1, CASP8,E2F1, MCL1, MGMT, TNF, VHL.
Cell Adhesion: APC, CDH1, CDKN2A, CTNNB1,KITLG, NF1, NF2, TGFB1.
Cell Cycle: ATM, BRCA1, BRCA2, CCND1, CDK4,CDKN1A, CDKN2A, CDKN2B, CDKN3, E2F1, HGF,MEN1, STK11, TP53.
Chemotaxis, Cell Migration & Motility: HRAS, JAK2,MET, NF1, NF2, PRKCA, SERPINB5, STAT3.
DNA Damage & Repair: ABL1, APC, ATM, BRCA1,
BRCA2, CDKN1A, MEN1, MGMT, MLH1, PML, TP53,TP73, XRCC1.
7/30/2019 LP 6 TesteMoleculareGeneticeDermatologie Part I
5/70
Immunohistochemistry (IHC)
7/30/2019 LP 6 TesteMoleculareGeneticeDermatologie Part I
6/70
Nuclear markers
Cytoplasmic marker
Membranous Marker
7/30/2019 LP 6 TesteMoleculareGeneticeDermatologie Part I
7/70
Immunohistochemistry
Immunohistochemistry or IHC refers to the processof localizing antigens (e.g. proteins) in cells of atissue section exploiting the principle of antibodiesbinding specifically to antigens in biological tissues.
Immunohistochemical staining is widely used in thediagnosis of abnormal cells such as those found incancerous tumors.
Specific molecular markers are characteristic of
particular cellular events such as proliferation orcell death (apoptosis).
IHC is also widely used in basic research tounderstand the distribution and localization ofbiomarkers and differentially expressed proteins indifferent parts of a biological tissue.
Visualising an antibody-antigen interaction can beaccomplished in a number of ways. In the mostcommon instance, an antibody is conjugated to an
enzyme, such as peroxidase, that can catalyse acolour-producing reaction.
7/30/2019 LP 6 TesteMoleculareGeneticeDermatologie Part I
8/70
Surface Markers - CD CD markers, an abbreviation for human cluster of differentiation markers, are a classification
system for monoclonal antibodies against cell surface molecules on leukocytes and antigens from
other cells. Currently, more than 400 CD markers have been identified, although not all of them are of
diagnostic value.
Immunophenotyping can be used on paraffin-embedded samples, frozen sections, or with flowcytometry.
When faced with a possible cutaneous lympho-proliferative disorder, the dermatopathologistevaluates the overall histological architectural pattern of the biopsy.
Interpretation of CD marker staining on fixed tissue samples should be based on the cellular
distribution of staining (i.e., membranous, cytoplasmic, nuclear). Negative and positive controls are also used in the staining process to allow for comparison, toconfirm the specificity and sensitivity of the staining process, and to assist in determining theaffinity of a particular stain.
7/30/2019 LP 6 TesteMoleculareGeneticeDermatologie Part I
9/70
The first step in the immunophenotypic evaluation is determination if the dominant population ofcells are B-cells, T-cells, or neither.
Three markers are typically used for this initial classification: CD20, CD3, and CD45.
T-cell processes are typically CD3+, CD20-, CD45+.
B-cell processes are typically CD3-, CD20+, and CD45+.
CD markers are specific for a particular cell type or origin, but there can be overlap.
CD markers serve as an imperfect attempt to identify and classify some neoplastic cells. It is
probably more accurate and practical to state that the pattern of CD marker expression is stronglysuggestive of a certain cell type or lineage, but may not be definitive
7/30/2019 LP 6 TesteMoleculareGeneticeDermatologie Part I
10/70
Flow cytometric immunophenotyping
Some antibodies do not work with sectionscut from paraffin-embedded samples or withfrozen sections and necessitate flowcytometry. However, flow cytometry requiresthat cells being immunophenotyped beindividually suspended in liquid, an easy taskfor circulating cells in peripheral bloodsamples, but more complicated whendealing with skin samples.
It allows simultaneous multiparametricanalysis of the physical and/or chemicalcharacteristics of up to thousands ofparticles per second.
Flow cytometry uses the principles of lightscattering , light excitation, and emission of
fluorochrome molecules to generate specificmulti-parameter data from particles and cellsin the size range of 0.5um to 40um diameter.
http://www.sonyinsider.com/wp-content/uploads/2010/02/Flow-Cytometry-Diagram2.jpg7/30/2019 LP 6 TesteMoleculareGeneticeDermatologie Part I
11/70
Flow cytometric immunophenotyping In the flow cytometric evaluation of mature B-cell lymphoid neoplasms, it is useful to consider 4 broad groups as determined by their expression of CD5 and CD10.
For each group, additional flow cytometric data in combination with the morphology can narrow down the diagnostic.
7/30/2019 LP 6 TesteMoleculareGeneticeDermatologie Part I
12/70
Flow cytometric immunophenotyping Among mature lymphoid neoplasms with a T-cell phenotype, expression of CD4 and CD8 can be used to formulate a list of diagnostic
possibilities and determine what additional information is required for further classification
7/30/2019 LP 6 TesteMoleculareGeneticeDermatologie Part I
13/70
Clonality Diagnosis
7/30/2019 LP 6 TesteMoleculareGeneticeDermatologie Part I
14/70
T-Cell Receptor and Immunoglobulin Gene
Rearrangements in Diagnosing Skin Disease
The most important advance in the molecular immunological features of lymphomas has been the
recognition that each normal T and B cell bears a unique antigen receptor on its cell surface that
serves as a specific marker for that cell and all of its clonal progeny.
If the cell should undergo malignant transformation, then this same structure becomes a tumor-
specific marker, as well.
For B cells, this marker is the immunoglobulin (Ig) molecule.
For T cells, it is the T-cell receptor (TCR).
7/30/2019 LP 6 TesteMoleculareGeneticeDermatologie Part I
15/70
B-Cell Receptor B cell development occurs through several stages, each stage representing
a change in the genome content at the antibody loci.
An antibody is composed of two identical light (L) and two identical heavy (H)chains, and the genes specifying them are found in the 'V' (Variable) regionand the 'C' (Constant) region.
In the heavy-chain 'V' region there are three segments; V, D and J, whichrecombine randomly, in a process called VDJ recombination, to produce aunique variable domain in the immunoglobulin of each individual B cell.
Similar rearrangements occur for light-chain 'V' region except there are onlytwo segments involved.
Stage Heavy chain Light chain
Progenitor (or pre-pro) B cells germline germline
Early Pro (or pre-pre)-B cells undergoes D-J rearrangement germline
Late Pro (or pre-pre)-B cells undergoes V-DJ rearrangement germline
Large Pre-B cells is VDJ rearranged germline
Small Pre-B cells is VDJ rearranged undergoes V-J rearrangement
Immature B cells is VDJ rearranged VJ rearranged
Mature B cells is VDJ rearranged VJ rearranged
The B-cell receptor is atransmembrane receptor proteinlocated on the outer surface of B-cells.
When a B-cell is activated by itsfirst encounter with an antigen thatbinds to its receptor (its "cognateantigen"), the cell proliferates anddifferentiates to generate apopulation of antibody-secretingplasma B cells and memory B cells.
http://8e.devbio.com/image.php?id=1187/30/2019 LP 6 TesteMoleculareGeneticeDermatologie Part I
16/70
T-Cell Receptor The TCR, which is anchored in the cell membrane,
consists of two halves which form a pair (or dimer) ofprotein chains. The halves are called the alpha () andbeta () fragments (in / T cells, the halves are gamma() and delta () fragments).
Each fragment is divided in turn into a constant (C) andvariable (V) region. The constant region has an end whichis anchored in the cell membrane.
The variable region faces outward and binds to the HLAmolecule and the antigen it presents. On the chain, thevariable region is called V and the constant region iscalled C; on the chain they are called V and Crespectively.
Processes for TCR formation are similar to thosedescribed for B cell antigen receptors
The TCR alpha chain is generated by VJ recombination,whereas the beta chain is generated by V(D)Jrecombination (both involve a somewhat random joining ofgene segments to generate the complete TCR chain).
Similarly, generation of the TCR gamma chain involves VJrecombination, whereas generation of the TCR delta chain
occurs by V(D)J recombination.
7/30/2019 LP 6 TesteMoleculareGeneticeDermatologie Part I
17/70
7/30/2019 LP 6 TesteMoleculareGeneticeDermatologie Part I
18/70
Clonality
Mycosis fungoides can arise from a background of chronic inflammation via the gradual selection
of one dominant T-cell clone that becomes increasingly malignant over time, probably as a result
of sequential somatic mutations.
Cutaneous patches containing superficial T-cell infiltrates with deletion of certain antigens and the
presence of dominant clonality are often diagnosed as mycosis fungoides, even when the
histopathological features are not fully diagnostic.
Clinically nodular skin lesions composed of atypical lymphoid infiltrates that exhibit abnormal
patterns of antigen expression and contain molecular evidence of dominant clonality are usually
regarded as lymphomas, even when this diagnosis cannot be made on morphological grounds
alone.
Thus, the principle has emerged that cutaneous lymphomas do not necessarily arise de novo but
can instead develop gradually from different types of chronic inflammatory processes.
7/30/2019 LP 6 TesteMoleculareGeneticeDermatologie Part I
19/70
Clonality
Once a diagnosis of lymphoma has been established, TCR or IgH gene rearrangement assays
can also be used to determine the disease stage of patients and to monitor their response to
therapy.
Occult involvement of lymph nodes by mycosis fungoides is prognostically relevant.
Because of their enhanced sensitivity relative to routine histological testing, molecular assays can
more accurately define remission and detect early relapse.
Patients who stop treatment when representative skin biopsy specimens are nonspecifichistologically but still positive by molecular analysis tend to relapse rapidly.
7/30/2019 LP 6 TesteMoleculareGeneticeDermatologie Part I
20/70
7/30/2019 LP 6 TesteMoleculareGeneticeDermatologie Part I
21/70
Polymerase Chain Reaction (PCR)
andSingle Nucleotide Polymorphism
Wh I h H G ?
7/30/2019 LP 6 TesteMoleculareGeneticeDermatologie Part I
22/70
What Is the Human Genome?
Human Cell
Nucleus
Chromosomes
DNA d Ch St t
7/30/2019 LP 6 TesteMoleculareGeneticeDermatologie Part I
23/70
DNA and Chromosome Structure
DNA molecule(chromosome)
Chemicalbases
A
T
G
C
Th G C t i G
7/30/2019 LP 6 TesteMoleculareGeneticeDermatologie Part I
24/70
The Genome Contains Genes
Gene 2 Coding region Protein 2
Protein 1
Noncoding region
Noncoding region
Gene 1 Coding region
V i ti i th H G
7/30/2019 LP 6 TesteMoleculareGeneticeDermatologie Part I
25/70
Variation in the Human Genome
Person 1 Person 2
= Variations in DNA
Wh t I V i ti i th G ?
7/30/2019 LP 6 TesteMoleculareGeneticeDermatologie Part I
26/70
What Is Variation in the Genome?
Common Sequence
Variations
Polymorphism
Deletions
Translocations
Insertions
Chromosome
V i ti C i N Ch
7/30/2019 LP 6 TesteMoleculareGeneticeDermatologie Part I
27/70
Variations Causing No Changes
= Variations in DNA that cause no changes
V i ti C i H l Ch
7/30/2019 LP 6 TesteMoleculareGeneticeDermatologie Part I
28/70
Variations Causing Harmless Changes
= Variations in DNA that cause harmless changes
V i ti C i L t t Ch
7/30/2019 LP 6 TesteMoleculareGeneticeDermatologie Part I
29/70
Variations Causing Latent Changes
Many years laterMany years later
= Variations in DNA that cause latent effects
SNP A th M t C
7/30/2019 LP 6 TesteMoleculareGeneticeDermatologie Part I
30/70
SNPs Are the Most CommonType of Variation
At least 1 percent
of the populationMost of the population
Commonsequence
G to C
SNPsite
Variantsequence
Wh A SNP Si ifi t?
7/30/2019 LP 6 TesteMoleculareGeneticeDermatologie Part I
31/70
Why Are SNPs Significant?
Person 1 Person 2
= SNP variations in DNA
SNP marks Gene A
Gene BGene A
SNP may cause Gene Bto make altered protein
Amino Acids
7/30/2019 LP 6 TesteMoleculareGeneticeDermatologie Part I
32/70
Amino Acids
Lysine side chain
20 Different Amino Acids
Basic Structureof an Amino Acid
Graphic Representationof an Amino Acid
Lysine
Carboxyl group
Amino group
Genes to Proteins I
7/30/2019 LP 6 TesteMoleculareGeneticeDermatologie Part I
33/70
Genes to Proteins I
DNA
T
AC
G
C
A
A
T
A
TG
C
A
T
T
A
U
G
C
G
U
U
A
U
AC
G
U
A
A
mRNA
Genes to Proteins II
7/30/2019 LP 6 TesteMoleculareGeneticeDermatologie Part I
34/70
Genes to Proteins II
Genes to Proteins III
7/30/2019 LP 6 TesteMoleculareGeneticeDermatologie Part I
35/70
Genes to Proteins III
Ribosome
mRNA
tRNA
A
Codons:AUG=Methionine=StartCGU=ArginineUAU=TyrosineACG=Threonine
UAA=Stop
Methionine
Arginine
Threonine
Tyrosine
U G C G U U A U A C U A AG
StopTyrosineMethionine
ThreonineArginine
Protein Folding and Function
7/30/2019 LP 6 TesteMoleculareGeneticeDermatologie Part I
36/70
Protein Folding and Function
Amino acid chain grows
and folds
into a 3-D structure.
SNPs in Coding Regions
7/30/2019 LP 6 TesteMoleculareGeneticeDermatologie Part I
37/70
SNPs in Coding RegionsNo Changes in Protein
DNA SNP C to G
RNA CodonCUG to CUC
ProteinLeucine to Leucine
No change in shape
Leucine Leucine
mRNA
G A C
C U G C U C
CUG CUC
G A G
SNPs in Coding Regions
7/30/2019 LP 6 TesteMoleculareGeneticeDermatologie Part I
38/70
SNPs in Coding RegionsSubtle, Harmless Changes in Protein
DNA SNP A to C
RNA CodonGAU to GAG
ProteinAspartic acid
to Glutamic acid
Slight change in shape
Aspartic acid Glutamic acid
mRNA
C T A
G A U G A G
GAU GAG
C T C
SNPs in Coding Regions
7/30/2019 LP 6 TesteMoleculareGeneticeDermatologie Part I
39/70
SNPs in Coding RegionsHarmful Changes in Protein Mutations
DNA SNP T to A
RNA CodonGAU to GUU
ProteinAspartic acid
to Valine
Change in shape
Aspartic acid Valine
mRNA
C T
G A U G U U
GAU GUU
C AA A
7/30/2019 LP 6 TesteMoleculareGeneticeDermatologie Part I
40/70
PCR Requirements
Magnesium chloride: .5-
2.5mM
Buffer: pH 8.3-8.8
dNTPs: 20-200M
Primers: 0.1-0.5M
DNA Polymerase: 1-2.5
units
Target DNA: 1 g
7/30/2019 LP 6 TesteMoleculareGeneticeDermatologie Part I
41/70
7/30/2019 LP 6 TesteMoleculareGeneticeDermatologie Part I
42/70
Gel electrophoresis
Heterozygous = having two
different alleles for a single
trait.
Wild type
Mutant
Homozygous = having identical
alleles for a single trait.
SNPs in Coding Regions
7/30/2019 LP 6 TesteMoleculareGeneticeDermatologie Part I
43/70
SNPs in Coding RegionsSubtle Changes in Proteins
That Only Switch on Under Certain ConditionsSmoking
Switched-ongenes
Pattern AMany years later
= SNPs causing latent effects
Pattern BMany years later
SNP Profiles and Response to
7/30/2019 LP 6 TesteMoleculareGeneticeDermatologie Part I
44/70
SNP Profiles and Response toDrug Therapy
Does Not Respond to Standard Drug Treatment
Breast Cancer Patients
Individual SNP Profiles Are Sorted
SNP profile A SNP profile B
SNP profile D
SNP profile E SNP profile C
Responds to Standard Drug Treatment
7/30/2019 LP 6 TesteMoleculareGeneticeDermatologie Part I
45/70
Gene SNP
TNFa Chromosome: 6; Location: 6p21.3 rs2228088, rs3179060, rs35131721, rs4645843, rs1800620, rs1800618, rs11574936,
IL-1a Chromosome: 2; Location: 2q14 rs3783588, rs55910084, rs1801715, rs3783581, rs17562, rs17561, rs61538608, rs20540, rs3783531,
IL-2 Chromosome: 4; Location: 4q26-q27 rs1051753, rs2069763, rs3087209,
IL-4 Chromosome: 5; Location: 5q31.1 rs4986964, rs56279116, rs55743996, rs35648164, rs71645915,
IL-6 Chromosome: 7; Location: 7p21 rs34280821, rs2069830, rs11544633, rs56383910, rs34012176, rs71708959,
rs2069860, rs13306435, rs34709428, rs2069849,
IL-8 Chromosome: 4; Location: 4q13-q21 rs1803205, rs71745371,
IL-12 Chromosome: 5; Location: 5q31.1-q33.1 rs34012639, rs55780930, rs2230052, rs56272177, rs35990253, rs55691228, rs56043315, rs1042154,
rs1042155,
IL-13 Chromosome: 5; Location: 5q31 rs55733734, rs56035208, rs34255686, rs34654684, rs20541, rs56258826,
IL-17 Chromosome: 6; Location: 6p12 rs17880588, rs17878530,
IL-22 Chromosome: 12; Location: 12q15 rs2227507,
IL-23 Chromosome: 12; Location: 12q13.3 rs61937689, rs11465746, rs11171806, rs71772333,
VEGF Chromosome: 6; Location: 6p12 rs25648, rs45533131, rs62401172,
Stanford University
http://www.stanford.edu/http://www.pharmgkb.org/index.jsphttp://www.stanford.edu/http://www.stanford.edu/http://www.stanford.edu/7/30/2019 LP 6 TesteMoleculareGeneticeDermatologie Part I
46/70
Adalimumab, Etanercept
rs983332 at chr1:87904968This variant is significantly associated with the efficacy of anti-TNF (Adjusted P-value: 0.000005; OR: 10.2 (2.6, 59.2)). The study is a genome-wide associationstudy using the Illumina HapMap300 SNP chip on 89 RA patients prospectively followed after beginning of anti-TNF therapy.
rs928655 at chr1:89622162 in GBP6This variant is significantly associated with the efficacy of anti-TNF (Adjusted P-value: 0.00003; OR: 5.5 (1.8, 20.2)). The study is a genome-wide association study
using the Illumina HapMap300 SNP chip on 89 RA patients prospectively followed after beginning of anti-TNF therapy. rs13393173 at chr2:169097337 in LASS6This variant is significantly associated with the efficacy of anti-TNF (Adjusted P-value: 0.000004; OR: 6.8 (1.7, 40.3)). The study is a genome-wide association studyusing the Illumina HapMap300 SNP chip on 89 RA patients prospectively followed after beginning of anti-TNF therapy.
rs437943 at chr4:35048493This variant is significantly associated with the efficacy of anti-TNF (Adjusted P-value: 0.000004; OR: 4.6 (1.8, 12.3)). The study is a genome-wide association studyusing the Illumina HapMap300 SNP chip on 89 RA patients prospectively followed after beginning of anti-TNF therapy.
rs1800629 at chr6:31651010 in LTA, TNFThe TNF:(-308)G>A polymorphism is a weak marker for response to anti-TNF treatment, with A-allele carriers being significantly less l ikely to respond than patientswith the GG genotype.
rs10945919 at chr6:164106667This variant is significantly associated with the efficacy of anti-TNF (Adjusted P-value: 0.0000003; OR: 4.6 (1.8, 12.3)). The study is a genome-wide associationstudy using the Illumina HapMap300 SNP chip on 89 RA patients prospectively followed after beginning of anti-TNF therapy.
rs854547 at chr7:94761792 in PPP1R9AThis variant is significantly associated with the efficacy of anti-TNF (Adjusted P-value: 0.000006; OR: 3.6 (1.5, 9.3)). The study is a genome-wide association studyusing the Illumina HapMap300 SNP chip on 89 RA patients prospectively followed after beginning of anti-TNF therapy.
rs854548 at chr7:94763756 in PON1, PPP1R9AThis variant is significantly associated with the efficacy of anti-TNF (Adjusted P-value: 0.000003; OR: 8.5 (2.6, 36.5)). The study is a genome-wide association studyusing the Illumina HapMap300 SNP chip on 89 RA patients prospectively followed after beginning of anti-TNF therapy.
rs854555 at chr7:94768327 in PON1This variant is significantly associated with the efficacy of anti-TNF (Adjusted P-value: 0.000002; OR: 4.6 (1.8, 12.3)). The study is a genome-wide association studyusing the Illumina HapMap300 SNP chip on 89 RA patients prospectively followed after beginning of anti-TNF therapy.
rs868856 at chr9:27479251 in MOBKL2BThis variant is significantly associated with the efficacy of anti-TNF (Adjusted P-value: 0.0000005; OR: 4.9 (1.8, 14.0)). The study is a genome-wide associationstudy using the Illumina HapMap300 SNP chip on 89 RA patients prospectively followed after beginning of anti-TNF therapy.
rs7046653 at chr9:27480967 in MOBKL2BThis variant is significantly associated with the efficacy of anti-TNF (Adjusted P-value: 0.0000005; OR: 4.9 (1.8, 14.0)). The study is a genome-wide associationstudy using the Illumina HapMap300 SNP chip on 89 RA patients prospectively followed after beginning of anti-TNF therapy.
rs2814707 at chr9:27526397 in MOBKL2BThis variant is significantly associated with the efficacy of anti-TNF (Adjusted P-value: 0.000002; OR: 5.2 (1.8, 16.7)). The study is a genome-wide association studyusing the Illumina HapMap300 SNP chip on 89 RA patients prospectively followed after beginning of anti-TNF therapy.
rs3849942 at chr9:27533281This variant is significantly associated with the efficacy of anti-TNF (Adjusted P-value: 0.000005; OR: 5.0 (1.7, 15.8)). The study is a genome-wide association studyusing the Illumina HapMap300 SNP chip on 89 RA patients prospectively followed after beginning of anti-TNF therapy.
rs774359 at chr9:27551049 in C9orf72This variant is significantly associated with the efficacy of anti-TNF (Adjusted P-value: 0.0000006; OR: 5.4 (1.9, 17.3)). The study is a genome-wide associationstudy using the Illumina HapMap300 SNP chip on 89 RA patients prospectively followed after beginning of anti-TNF therapy.
rs6138150 at chr20:23795009This variant is significantly associated with the efficacy of anti-TNF (Adjusted P-value: 0.000003; OR: 11.1 (2.5, 103.3)). The study is a genome-wide associationstudy using the Illumina HapMap300 SNP chip on 89 RA patients prospectively followed after beginning of anti-TNF therapy.
rs6028945 at chr20:38254219This variant is significantly associated with the efficacy of anti-TNF (Adjusted P-value: 0.0000002; OR: 11.2 (2.3, 108.1)). The study is a genome-wide associationstudy using the Illumina HapMap300 SNP chip on 89 RA patients prospectively followed after beginning of anti-TNF therapy.
rs6071980 at chr20:38301990This variant is significantly associated with the efficacy of anti-TNF (Adjusted P-value: 0.000003; OR: 7.6 (1.9, 44.6)). The study is a genome-wide association studyusing the Illumina HapMap300 SNP chip on 89 RA patients prospectively followed after beginning of anti-TNF therapy.
Stanford University
http://www.pharmgkb.org/do/serve?objId=PA134964409&objCls=Genehttp://www.pharmgkb.org/do/serve?objId=PA134925480&objCls=Genehttp://www.pharmgkb.org/do/serve?objId=PA30474&objCls=Genehttp://www.pharmgkb.org/do/serve?objId=PA435&objCls=Genehttp://www.pharmgkb.org/do/serve?objId=PA33661&objCls=Genehttp://www.pharmgkb.org/do/serve?objId=PA33529&objCls=Genehttp://www.pharmgkb.org/do/serve?objId=PA33661&objCls=Genehttp://www.pharmgkb.org/do/serve?objId=PA33529&objCls=Genehttp://www.pharmgkb.org/do/serve?objId=PA134886513&objCls=Genehttp://www.pharmgkb.org/do/serve?objId=PA134886513&objCls=Genehttp://www.pharmgkb.org/do/serve?objId=PA134886513&objCls=Genehttp://www.pharmgkb.org/do/serve?objId=PA134908144&objCls=Genehttp://www.pharmgkb.org/index.jsphttp://www.pharmgkb.org/do/serve?objId=PA134908144&objCls=Genehttp://www.pharmgkb.org/do/serve?objId=PA134886513&objCls=Genehttp://www.pharmgkb.org/do/serve?objId=PA134886513&objCls=Genehttp://www.pharmgkb.org/do/serve?objId=PA134886513&objCls=Genehttp://www.pharmgkb.org/do/serve?objId=PA33529&objCls=Genehttp://www.pharmgkb.org/do/serve?objId=PA33661&objCls=Genehttp://www.pharmgkb.org/do/serve?objId=PA33529&objCls=Genehttp://www.pharmgkb.org/do/serve?objId=PA33661&objCls=Genehttp://www.pharmgkb.org/do/serve?objId=PA435&objCls=Genehttp://www.pharmgkb.org/do/serve?objId=PA30474&objCls=Genehttp://www.pharmgkb.org/do/serve?objId=PA134925480&objCls=Genehttp://www.pharmgkb.org/do/serve?objId=PA134964409&objCls=Genehttp://www.stanford.edu/http://www.stanford.edu/http://www.stanford.edu/http://www.stanford.edu/http://www.pharmgkb.org/index.jsp7/30/2019 LP 6 TesteMoleculareGeneticeDermatologie Part I
47/70
Infliximab
rs1061622 at chr1:12175542 in TNFRSF1BFor this SNP in the TNFRSF1B gene a significant correlation was found between 196R allele carriers and low response to infliximab therapy.
rs983332 at chr1:87904968This variant is significantly associated with the efficacy of anti-TNF treatment (Adjusted P-value: 0.000005; OR: 10.2 (2.6, 59.2)). The study is a genome-wideassociation study using the Illumina HapMap300 SNP chip on 89 RA patients prospectively followed after beginning of anti-TNF therapy.
rs928655 at chr1:89622162 in GBP6This variant is significantly associated with the efficacy of anti-TNF treatment (Adjusted P-value: 0.00003; OR: 5.5 (1.8, 20.2)). The study is a genome-wideassociation study using the Illumina HapMap300 SNP chip on 89 RA patients prospectively followed after beginning of anti-TNF therapy.
rs396991 at chr1:159781166 in FCGR3AThis variant may be a useful marker to predict response to infliximab in Japanese patients with rheumatoid arthritis.
rs13393173 at chr2:169097337 in LASS6This variant is significantly associated with the efficacy of anti-TNF treatment (Adjusted P-value: 0.000004; OR: 6.8 (1.7, 40.3)). The study is a genome-wideassociation study using the Illumina HapMap300 SNP chip on 89 RA patients prospectively followed after beginning of anti-TNF therapy.
rs437943 at chr4:35048493This variant is significantly associated with the efficacy of anti-TNF treatment (Adjusted P-value: 0.000004; OR: 4.6 (1.8, 12.3)). The study is a genome-wideassociation study using the Illumina HapMap300 SNP chip on 89 RA patients prospectively followed after beginning of anti-TNF therapy.
rs1800629 at chr6:31651010 in LTA, TNFThe TNF:(-308)G>A polymorphism is a weak marker for response to anti-TNF treatment, with A-allele carriers being significantly less l ikely to respond than patientswith the GG genotype.
rs10945919 at chr6:164106667
This variant is significantly associated with the efficacy of anti-TNF treatment (Adjusted P-value: 0.0000003; OR: 4.6 (1.8, 12.3)). The study is a genome-wideassociation study using the Illumina HapMap300 SNP chip on 89 RA patients prospectively followed after beginning of anti-TNF therapy.
rs854547 at chr7:94761792 in PPP1R9AThis variant is significantly associated with the efficacy of anti-TNF treatment (Adjusted P-value: 0.000006; OR: 3.6 (1.5, 9.3)). The study is a genome-wideassociation study using the Illumina HapMap300 SNP chip on 89 RA patients prospectively followed after beginning of anti-TNF therapy.
rs854548 at chr7:94763756 in PON1, PPP1R9AThis variant is significantly associated with the efficacy of anti-TNF treatment (Adjusted P-value: 0.000003; OR: 8.5 (2.6, 36.5)). The study is a genome-wideassociation study using the Illumina HapMap300 SNP chip on 89 RA patients prospectively followed after beginning of anti-TNF therapy.
rs854555 at chr7:94768327 in PON1This variant is significantly associated with the efficacy of anti-TNF treatment (Adjusted P-value: 0.000002; OR: 4.6 (1.8, 12.3)). The study is a genome-wideassociation study using the Illumina HapMap300 SNP chip on 89 RA patients prospectively followed after beginning of anti-TNF therapy.
rs868856 at chr9:27479251 in MOBKL2BThis variant is significantly associated with the efficacy of anti-TNF treatment (Adjusted P-value: 0.0000005; OR: 4.9 (1.8, 14.0)). The study is a genome-wideassociation study using the Illumina HapMap300 SNP chip on 89 RA patients prospectively followed after beginning of anti-TNF therapy.
rs7046653 at chr9:27480967 in MOBKL2BThis variant is significantly associated with the efficacy of anti-TNF treatment (Adjusted P-value: 0.0000005; OR: 4.9 (1.8, 14.0)). The study is a genome-wideassociation study using the Illumina HapMap300 SNP chip on 89 RA patients prospectively followed after beginning of anti-TNF therapy.
rs2814707 at chr9:27526397 in MOBKL2BThis variant is significantly associated with the efficacy of anti-TNF treatment (Adjusted P-value: 0.000002; OR: 5.2 (1.8, 16.7)). The study is a genome-wideassociation study using the Illumina HapMap300 SNP chip on 89 RA patients prospectively followed after beginning of anti-TNF therapy.
rs3849942 at chr9:27533281This variant is significantly associated with the efficacy of anti-TNF treatment (Adjusted P-value: 0.000005; OR: 5.0 (1.7, 15.8)). The study is a genome-wideassociation study using the Illumina HapMap300 SNP chip on 89 RA patients prospectively followed after beginning of anti-TNF therapy.
rs774359 at chr9:27551049 in C9orf72This variant is significantly associated with the efficacy of anti-TNF treatment (Adjusted P-value: 0.0000006; OR: 5.4 (1.9, 17.3)). The study is a genome-wideassociation study using the Illumina HapMap300 SNP chip on 89 RA patients prospectively followed after beginning of anti-TNF therapy.
rs6138150 at chr20:23795009This variant is significantly associated with the efficacy of anti-TNF treatment (Adjusted P-value: 0.000003; OR: 11.1 (2.5, 103.3)). The study is a genome-wideassociation study using the Illumina HapMap300 SNP chip on 89 RA patients prospectively followed after beginning of anti-TNF therapy.
rs6028945 at chr20:38254219This variant is significantly associated with the efficacy of anti-TNF treatment (Adjusted P-value: 0.0000002; OR: 11.2 (2.3, 108.1)). The study is a genome-wide
association study using the Illumina HapMap300 SNP chip on 89 RA patients prospectively followed after beginning of anti-TNF therapy.
Stanford University
http://www.pharmgkb.org/do/serve?objId=PA36610&objCls=Genehttp://www.pharmgkb.org/do/serve?objId=PA134964409&objCls=Genehttp://www.pharmgkb.org/do/serve?objId=PA28065&objCls=Genehttp://www.pharmgkb.org/do/serve?objId=PA134925480&objCls=Genehttp://www.pharmgkb.org/do/serve?objId=PA30474&objCls=Genehttp://www.pharmgkb.org/do/serve?objId=PA435&objCls=Genehttp://www.pharmgkb.org/do/serve?objId=PA33661&objCls=Genehttp://www.pharmgkb.org/do/serve?objId=PA33529&objCls=Genehttp://www.pharmgkb.org/do/serve?objId=PA33661&objCls=Genehttp://www.pharmgkb.org/do/serve?objId=PA33529&objCls=Genehttp://www.pharmgkb.org/do/serve?objId=PA134886513&objCls=Genehttp://www.pharmgkb.org/do/serve?objId=PA134886513&objCls=Genehttp://www.pharmgkb.org/do/serve?objId=PA134886513&objCls=Genehttp://www.pharmgkb.org/do/serve?objId=PA134908144&objCls=Genehttp://www.pharmgkb.org/index.jsphttp://www.pharmgkb.org/do/serve?objId=PA134908144&objCls=Genehttp://www.pharmgkb.org/do/serve?objId=PA134886513&objCls=Genehttp://www.pharmgkb.org/do/serve?objId=PA134886513&objCls=Genehttp://www.pharmgkb.org/do/serve?objId=PA134886513&objCls=Genehttp://www.pharmgkb.org/do/serve?objId=PA33529&objCls=Genehttp://www.pharmgkb.org/do/serve?objId=PA33661&objCls=Genehttp://www.pharmgkb.org/do/serve?objId=PA33529&objCls=Genehttp://www.pharmgkb.org/do/serve?objId=PA33661&objCls=Genehttp://www.pharmgkb.org/do/serve?objId=PA435&objCls=Genehttp://www.pharmgkb.org/do/serve?objId=PA30474&objCls=Genehttp://www.pharmgkb.org/do/serve?objId=PA134925480&objCls=Genehttp://www.pharmgkb.org/do/serve?objId=PA28065&objCls=Genehttp://www.pharmgkb.org/do/serve?objId=PA134964409&objCls=Genehttp://www.pharmgkb.org/do/serve?objId=PA36610&objCls=Genehttp://www.stanford.edu/http://www.stanford.edu/http://www.stanford.edu/http://www.stanford.edu/http://www.pharmgkb.org/index.jsp7/30/2019 LP 6 TesteMoleculareGeneticeDermatologie Part I
48/70
Methotrexate rs4846051 at chr1:11777044 in MTHFR
The C allele of this variant was associated with increased risk of toxicity in African American Rheumatoid Arthritis patients receiving methotrexate.
rs1801131 at chr1:11777063 in MTHFRAt 6 months methotrexate and folic acid therapy, of early rheumatoid arthritis patients with the MTHFR 1298AA genotype showed good improvement relative tocombined CA and AA genotypes (OR 2.3), while 1298C allele carriers developed more adverse drug events (OR 2.5) (e.g. pneumonitis, gastrointestinal ADEs, skin
and mucosal ADEs, and elevated liver rs1801133 at chr1:11778965 in CLCN6, MTHFRThis variant is associated with methotrexated-induced mucositis, thrombocytopenia and hepatic toxicity
rs1801133 at chr1:11778965 in CLCN6, MTHFRIn 330 patients who completed 3 months methotrexate treatment for psoriasis, no significant genotypic associations were found between clinical outcome (e.g.efficacy, toxicity) and 50 SNPs in pathway genes for methotrexate metabolism (ATIC, FPGS, GGH, MTHFR), including 47 common ( >5% minor allele frequency)haplotype-tagging SNPs (r(2) > 0.8) plus 3
rs1801133 at chr1:11778965 in CLCN6, MTHFRMTHFR rs1801133, 667CT or 667TT genotypes were associated with an increased risk of methotrexate treatment discontinuation due to adverse events (relativerisk 2.01), mostly as a result of increased risk of elevated levels of liver enzyme alanine aminotransferase (relative risk 2.38) in rheumatoid arthritis patients.
rs1801133 at chr1:11778965 in CLCN6, MTHFRIn a retrospective analysis of 61 Italian patients experiencing methotrexate toxicity during treatment for acute lymphoblastic leukemia or acute promyelocyticleukemia, carriers of the MTHFR 677TT genotype (60%) showed significantly greater drug-induced toxicity (p=0.03) compared to CC and CT genotypes.
rs1801133 at chr1:11778965 in CLCN6, MTHFRRisk or phenotype-associated allele: CT and TT genotypes. Phenotype: The 677 CT or TT genotypes were associated with greater incidence of discontinuation ofmethorexate treatment because of adverse events, mainly due to elevation of liver enzymes. Study size: 236. Study population/ethnicity: Patients who startedmethorexate treatment with (n = 157) or without (n = 79) folic or folinic acid supplementation for rheumatoid arthritis. Significance metric(s): RR = 2.01 Type ofassociation: CO, GN.
rs13120400 at chr4:89252551 inABCG2SNP is associated with clinical reponse to methotrexate in patients with psoriasis.
rs17731538 at chr4:89274403 inABCG2SNP is associated with clinical reponse to methotrexate in patients with psoriasis.
rs11545078 at chr8:64101318 in GGHGGH 452C>T has been associated with decreased catalytic activity and higher accumulation of long-chain methotrexate-polyglutamate.
rs1800909 at chr8:64113866 in GGHThis study found that only patients with the GGH 16C-allele and one or no copies of the GGH 452C-16T haplotype were associated with good clinical improvementat 3 months upon treatment with methotrexate.
rs4149081 at chr12:21269288 in SLCO1B1Risk or phenotype-associated allele: G allele, with additive genotypic effect. Phenotype: Genome-wide analysis of 398,699 germline SNPs showed association ofthe rs4149081 G allele with increased methotrexate (MTX) plasma clearance, with an additive effect per G allele (increase of 12.7 mL/min/m(2) per allele in 434subjects), after adjusting for age, race, sex, and MTX regimen. Variants rs11045879 and rs4149081 were in linkage disequilibrium (r(2) = 1). The G allele was
associated with increased risk of gastrointestinal toxicity (mucositis) (OR = 15.3, p = 0.03). Pharmacokinetics differed by e thnicity (MTX clearance:African>Caucasian). Study size: 434 (discovery cohort), 206 (independent validation cohort), 640 (combined cohort). Study population/ethnicity: Multiethnic children(5.92 median age , 1.02-18.85 range) with ALL given 3,014 courses of methotrexate at 2-5 g/m(2) enrolled in Tennessee. Significance metric(s): increased MTXclearance: p = 1.7 x 10(-9) (n = 434), p = 0.017 (n = 206), p = 6.7 x 10(-10) (n = 640); increased GI toxicity: OR = 15.3, p = 0.03. Type of association: CO; GN; PK;ADR; TOX
http://www.pharmgkb.org/do/serve?objId=PA245&objCls=Genehttp://www.pharmgkb.org/do/serve?objId=PA245&objCls=Genehttp://www.pharmgkb.org/do/serve?objId=PA26551&objCls=Genehttp://www.pharmgkb.org/do/serve?objId=PA245&objCls=Genehttp://www.pharmgkb.org/do/serve?objId=PA26551&objCls=Genehttp://www.pharmgkb.org/do/serve?objId=PA245&objCls=Genehttp://www.pharmgkb.org/do/serve?objId=PA26551&objCls=Genehttp://www.pharmgkb.org/do/serve?objId=PA245&objCls=Genehttp://www.pharmgkb.org/do/serve?objId=PA26551&objCls=Genehttp://www.pharmgkb.org/do/serve?objId=PA245&objCls=Genehttp://www.pharmgkb.org/do/serve?objId=PA26551&objCls=Genehttp://www.pharmgkb.org/do/serve?objId=PA245&objCls=Genehttp://www.pharmgkb.org/do/serve?objId=PA390&objCls=Genehttp://www.pharmgkb.org/do/serve?objId=PA390&objCls=Genehttp://www.pharmgkb.org/do/serve?objId=PA432&objCls=Genehttp://www.pharmgkb.org/do/serve?objId=PA432&objCls=Genehttp://www.pharmgkb.org/do/serve?objId=PA134865839&objCls=Genehttp://www.pharmgkb.org/index.jsphttp://www.pharmgkb.org/do/serve?objId=PA134865839&objCls=Genehttp://www.pharmgkb.org/do/serve?objId=PA432&objCls=Genehttp://www.pharmgkb.org/do/serve?objId=PA432&objCls=Genehttp://www.pharmgkb.org/do/serve?objId=PA390&objCls=Genehttp://www.pharmgkb.org/do/serve?objId=PA390&objCls=Genehttp://www.pharmgkb.org/do/serve?objId=PA245&objCls=Genehttp://www.pharmgkb.org/do/serve?objId=PA26551&objCls=Genehttp://www.pharmgkb.org/do/serve?objId=PA245&objCls=Genehttp://www.pharmgkb.org/do/serve?objId=PA26551&objCls=Genehttp://www.pharmgkb.org/do/serve?objId=PA245&objCls=Genehttp://www.pharmgkb.org/do/serve?objId=PA26551&objCls=Genehttp://www.pharmgkb.org/do/serve?objId=PA245&objCls=Genehttp://www.pharmgkb.org/do/serve?objId=PA26551&objCls=Genehttp://www.pharmgkb.org/do/serve?objId=PA245&objCls=Genehttp://www.pharmgkb.org/do/serve?objId=PA26551&objCls=Genehttp://www.pharmgkb.org/do/serve?objId=PA245&objCls=Genehttp://www.pharmgkb.org/do/serve?objId=PA245&objCls=Gene7/30/2019 LP 6 TesteMoleculareGeneticeDermatologie Part I
49/70
7/30/2019 LP 6 TesteMoleculareGeneticeDermatologie Part I
50/70
Fluorescence in Situ Hybridisation (FISH)
7/30/2019 LP 6 TesteMoleculareGeneticeDermatologie Part I
51/70
Molecular Cytogenetic Analysis of Chromosomal Translocation
Translocations generate novelchromosomes. In a translocation, asegment from one chromosome istransferred to a nonhomologouschromosome or to a new site on the samechromosome.
The genomes of closely related species,they can see that translocations haveoccurred many times during the course ofevolution.
Translocations that give an organism anadaptive advantage are very rare.
Translocations are more often associatedwith negative consequences like cancer.
In many cases, are considered to be theprimary cause of various cancers.
Nonreciprocal translocations are one-way translocations in which a
chromosomal segment is transferred to a nonhomologous chromosome. a)
An idiogram of a reciprocal translocation between chromosomes 12 and
17. b) An ideogram of a Robertsonian translocation between
chromosomes 14 and 21.
7/30/2019 LP 6 TesteMoleculareGeneticeDermatologie Part I
52/70
Translocations Can Produce Oncogenes
The translocation places the coding sequence of one gene (Gene B) in proximity to the regulatorysequence for a different gene (Gene A).
The translocation involving chromosomes 8 and 14 places the MYC proto-oncogene fromchromosome 8 under the control of the powerful immunoglobin heavy chain gene (IGH) promoteron chromosome 14.
The MYC protein normally signals for cell proliferation, and the translocation causes high levels ofMYC overexpression in lymphoid cells, where the IGH promoter is normally active.
Aberrant oncogene expression from chromosomal translocation frequently leads to cellular
immortalization and clonal expansion.
Translocations Mb ( j b k i t i 150 b )
Mb ( j b k i t i 150 b )
7/30/2019 LP 6 TesteMoleculareGeneticeDermatologie Part I
53/70
Translocations
Can Produce
Oncogenes
A rearrangement of the bcl-2proto-oncogene onchromosome 18 with theimmunoglobulin heavy chainregion on chromosome 14,leads to deregulated BCL-2
production. Bcl-2 has been shown to
prevent programmed celldeath (apoptosis) thusimmortalizing the cell.
The t(14;18) translocation ischaracteristic of B-celllymphomas, occurring in up to90% of follicular lymphomas.
It is also found in 20% to 30%of diffuse large B-celllymphomas, where it is anindicator of poor prognosis.
Bcl2 Chromosome 18
Mbr (major breakpoint region, 150 bp)
JH
C
Double strand DNA break by RAG1/2
Chromosome 14
Bcl2 C t(14;18) translocation
bcl2 CE C 3E
Unregulation of Bcl2 expression by IgH enhancers
Translocation takes place in B cell precursors.
Transformation takes place
during B cell activation in GC.
Bcl2 Chromosome 18
Mbr (major breakpoint region, 150 bp)
JH
C
Double strand DNA break by RAG1/2
Chromosome 14
Bcl2 C t(14;18) translocation
bcl2 CE C 3E
Unregulation of Bcl2 expression by IgH enhancers
Translocation takes place in B cell precursors.
Transformation takes place
during B cell activation in GC.
activation
Germinal center Germinal center
apoptosis
IgH-Bcl2
activation
Germinal center Germinal center
Plasma cells
Memory cells
follicular lymphoma
Apoptosis inhibitedMost follicular lymphoma Ig V regions containsomatic hypermutation.
activation
Germinal center Germinal center
apoptosis
IgH-Bcl2
activation
Germinal center Germinal center
Plasma cells
Memory cells
follicular lymphoma
Apoptosis inhibitedMost follicular lymphoma Ig V regions containsomatic hypermutation.
7/30/2019 LP 6 TesteMoleculareGeneticeDermatologie Part I
54/70
Depending on probe design (eg, the
distance between the regionsrecognized) and the state of thegenomic DNA at the time of fixation,a fused signal may appear either asa colocalized red and green signal oras a single yellow signal.
When using break-apart probes,red/green signal pairs will
occasionally appear to be slightlyseparated because of the secondarystructure of the target DNA.
7/30/2019 LP 6 TesteMoleculareGeneticeDermatologie Part I
55/70
(A) Interphase nuclei hybridized with the LSI IGH break apart probe (Vysis).The two nuclei at the top display a significant dissociation of the red and
green signals (arrows) indicating the presence of a translocation affectingthe IGH.
(B) Interphase nucleus with the LSI MYC/IGH double fusion probe (Vysis).The presence of two fused red and green signals (arrows) indicates that atranslocation t(8;14)(q24;q32) juxtaposing the MYC and IGH loci has takenplace. The isolated red and green signals point to the unrearranged MYCand IGH alleles, respectively.
7/30/2019 LP 6 TesteMoleculareGeneticeDermatologie Part I
56/70
Deregulation of BCL6 either by juxtaposition next to an IG locus or by promotor substitution due toa chromosomal translocation can be detected in about 30% of systemic diffuse-large B-celllymphomas (DLCBL).
t(14;18) cytogenetically identical to that occurring in FL can lead to activation of the MALT1oncogene. This gene is also targeted by a recurrent t(11;18)(q21;q21) present in approximately30% of systemic marginal zone lymphomas of MALT type, which leads to fusion of the MALT1gene with the apoptosis inhibitor-2 (API2) gene in 18q21.
7/30/2019 LP 6 TesteMoleculareGeneticeDermatologie Part I
57/70
Molecular Cytogenetic Analysis of Chromosomal Translocation in
Primary Cutaneous B-cell Lymphomas
Overall, translocations affecting one IG locus are estimated to be present in at leasthalf of the nodal B-cell non-Hodgkin lymphomas. In contrast to systemic B-cell
lymphomas only few data exist on the presence of recurrent translocations in primary
cutaneous B-cell lymphomas (PCBCL).
The t(14;18) translocation does not occur in PCBCL, which suggests the involvement
of different pathogenetic mechanisms compared with their nodal counterparts.
The detection of a t(14;18) translocation in cutaneous B-cell lymphoma should
suggest the presence of systemic disease, which underlies the need for exhaustive
staging procedures.
7/30/2019 LP 6 TesteMoleculareGeneticeDermatologie Part I
58/70
Gene Microarray Expression Technology
Oncogenes: BAX, BCL2L1, CASP8, CDK4, ELK1,ETS1, HGF, JAK2, JUNB, JUND, KIT, KITLG, MCL1,
7/30/2019 LP 6 TesteMoleculareGeneticeDermatologie Part I
59/70
ETS1, HGF, JAK2, JUNB, JUND, KIT, KITLG, MCL1,MET, MOS, MYB, NFKBIA, NRAS, PIK3CA, PML,PRKCA, RAF1, RARA, REL, ROS1, RUNX1, SRC,STAT3, ZHX2.
Tumor Suppressor Genes: ATM, BRCA1, BRCA2,CDH1, CDKN2B, CDKN3, E2F1, FHIT, FOXD3, HIC1,IGF2R, MEN1, MGMT, MLH1, NF1, NF2, RASSF1,RUNX3, S100A4, SERPINB5, SMAD4, STK11, TP73,TSC1, VHL, WT1, WWOX, XRCC1.
Oncogenic & Tumor Suppressor Properties: BCR,EGF, ERBB2, ESR1, FOS, HRAS, JUN, KRAS, MDM2,MYC, MYCN, NFKB1, PIK3C2A, RB1, RET, SH3PXD2A,TGFB1, TNF, TP53.
Transcription Factors: ABL1, BRCA1, BRCA2,CDKN2A, CTNNB1, E2F1, ELK1, ESR1, ETS1, FOS,FOXD3, HIC1, JUN, JUNB, JUND, MDM2, MEN1, MYB,MYC, MYCN, NF1, NFKB1, PML, RARA, RB1, REL,RUNX1, RUNX3, SMAD4, STAT3, TGFB1, TNF, TP53,TP73, TSC1, VHL, WT1, ZHX2.
Epithelial-to-Mesenchymal Transition: BRCA2,CDKN2B, CTNNB1, ERBB2, HGF, JAK2, KIT, MCL1,NF1, RUNX3, S100A4, SMAD4, TGFB1, VHL.
Angiogenesis: AKT1, CTNNB1, EGF, ERBB2, NF1,PML, RUNX1, TGFB1.
Apoptosis: BAX, BCL2, BCL2L1, BRCA1, CASP8,E2F1, MCL1, MGMT, TNF, VHL.
Cell Adhesion: APC, CDH1, CDKN2A, CTNNB1,KITLG, NF1, NF2, TGFB1.
Cell Cycle: ATM, BRCA1, BRCA2, CCND1, CDK4,CDKN1A, CDKN2A, CDKN2B, CDKN3, E2F1, HGF,MEN1, STK11, TP53.
Chemotaxis, Cell Migration & Motility: HRAS, JAK2,MET, NF1, NF2, PRKCA, SERPINB5, STAT3.
DNA Damage & Repair: ABL1, APC, ATM, BRCA1,BRCA2, CDKN1A, MEN1, MGMT, MLH1, PML, TP53,
TP73, XRCC1.
7/30/2019 LP 6 TesteMoleculareGeneticeDermatologie Part I
60/70
Non-Hodgkin lymphoma (NHL) Non-Hodgkin lymphoma (NHL) is a heterogeneous, complex, and progressive clonal expansion of
B-, T-lymphocytes and rarely NK-cells or their precursors.
Our taxonomy of lymphomas, which is based mostly on histopathology and immunophenotyping,
includes about 30 distinct entities arising from diverse cells types. The genetic complexity of lymphomas probably explains the clinical diversity with traditional
methods and genomic expression analysis.
Microarrays technique is effective in deciphering this clinical diversity.
A number of published studies identify gene expression signatures for major non-Hodgkinlymphoma types and subtypes, and uncover gene expression patterns that correlate with variouscharacteristics of non-Hodgkin lymphoma.
Mature T-cell and NK-cell neoplasms Mycosis fungoides (MF)
Variants of MF Pagetoid reticulosis (localized disease)
Folliculotropic, syringotropic, granulomatous variants
Subtype of MF
Granulomatous slack skin
Sezary syndrome
CD30+ T-cell lymphoproliferative disorders of the skin
Lymphomatoid papulosis
Primary cutaneous anaplastic large cell lymphoma
Subcutaneous panniculitis-like T-cell lymphoma Primary cutaneous peripheral T-Cell lymphoma (PTL),unspecified
Subtypes of PTL
Primary cutaneous aggressive epidermotropic CD8+T-cell lymphoma (provisional)
Cutaneous gamma/delta-positive T-cell lymphoma(provisional)
Primary cutaneous CD4+ small/medium-sizedpleomorphic T-cell lymphoma (provisional)
Extranodal NK/T-cell lymphoma, nasal type
Hydroa vacciniforme-like lymphoma (variant)
Adult T-cell leukemia/lymphoma
Angioimmunoblastic T-cell lymphoma
Mature B-cell neoplasms Cutaneous marginal zone B-cell lymphoma (MALT-type)
Primary cutaneous follicle center lymphoma
Growth patterns
Follicular
Follicular and diffuse
Diffuse
Cutaneous diffuse large B-cell lymphoma, leg type
Cutaneous diffuse large B-cell lymphoma, others
Intravascular large B-cell lymphoma
Lymphomatoid granulomatosis Chronic lymphocytic leukemia
Mantle cell lymphoma
Burkitt lymphoma
Immature hematopoietic malignancies
Blastic NK-cell lymphoma CD4+/CD56+ hematodermicneoplasm
Precursor lymphoblastic leukemia/lymphoma
T-lymphoblastic lymphoma
B-lymphoblastic lymphoma
Myeloid and monocytic leukemias
Hodgkin lymphoma
7/30/2019 LP 6 TesteMoleculareGeneticeDermatologie Part I
61/70
Current Microarray Technology
Tissue Lysis mRNA cDNA
OpticalDetection
Scanning
Image Analysis
Data Analysis
Amplification
Fluorescent Labeling
7/30/2019 LP 6 TesteMoleculareGeneticeDermatologie Part I
62/70
In standard microarrays, the probes are attached via surface engineering to a solid surface by acovalent bond to a chemical matrix (via epoxy-silane, amino-silane, lysine, polyacrylamide orothers).
The solid surface can be glass or a silicon chip, in which case they are colloquially known as anAffy chip when an Affymetrix chip is used.
The core principle behind microarrays is hybridization between two DNA strands, the property ofcomplementary nucleic acid sequences to specifically pair with each other by forming hydrogenbonds between complementary nucleotide base pairs.
A high number of complementary base pairs in a nucleotide sequence means tighter non-covalentbonding between the two strands.
After washing off of non-specific bonding sequences, only strongly paired strands will remainhybridized.
So fluorescently labelled target sequences that bind to a probe sequence generate a signal thatdepends on the strength of the hybridization determined by the number of paired bases, thehybridization conditions (such as temperature), and washing after hybridization.
7/30/2019 LP 6 TesteMoleculareGeneticeDermatologie Part I
63/70
Two-color microarrays or two-channel microarrays are typicallyhybridized with cDNA preparedfrom two samples to be compared(e.g. diseased tissue versushealthy tissue) and that are labeledwith two different fluorophores.
Fluorescent dyescommonly used for cDN
A
7/30/2019 LP 6 TesteMoleculareGeneticeDermatologie Part I
64/70
Microarray analysis
commonly used for cDNAlabeling include Cy3, whichhas a fluorescenceemission wavelength of 570nm (corresponding to thegreen part of the lightspectrum), and Cy5 with afluorescence emissionwavelength of 670 nm(corresponding to the redpart of the light spectrum).
The two Cy-labeled cDNAsamples are mixed andhybridized to a singlemicroarray that is thenscanned in a microarrayscanner to visualizefluorescence of the twofluorophores after excitationwith a laser beam of a
defined wavelength. Relative intensities of each
fluorophore may then beused in ratio-basedanalysis to identify up-regulated and down-regulated genes.
7/30/2019 LP 6 TesteMoleculareGeneticeDermatologie Part I
65/70
Repression Induction
7/30/2019 LP 6 TesteMoleculareGeneticeDermatologie Part I
66/70
7/30/2019 LP 6 TesteMoleculareGeneticeDermatologie Part I
67/70
Components of DNA Microarray image analysis are
(1) Grid Alignment Problem,
(2) Foreground Separation,
(3) Quality Assurance,
(4) Quantification and
(5) Normalization.
7/30/2019 LP 6 TesteMoleculareGeneticeDermatologie Part I
68/70
Variations in microarray image Data processing:
grid geometry,
foreground and background intensity,
spot morphology
Right image shows variations of spots; a regular
spot, an inverse spot or a ghost shape, a
spatially deviating spot inside of a grid cell, a
spot radius deviation, a tapering spot or a comet
shape, spot with a hole or a doughnut shape, a
partially missing spot and a scratched spot.
Examples of accurate (top) and inaccurate
(bottom) foreground separation
7/30/2019 LP 6 TesteMoleculareGeneticeDermatologie Part I
69/70
Hierarchical clustering Microarray data sets are commonly very large, and analytical precision is influenced
by a number of variables. Statistical challenges include taking into account effects of
background noise and appropriate normalization of the data. For statistical analysis and visualization of gene expression data a large number of
commercial and non-commercial software tools have been developed (e.g., Gene
Spring, Gene Cluster, Cluster, and Treevoew, SAM and dCHIP).
Hierarchical clustering output as dendogram or tree attached to a heatmap
representation of the clustered matrix
Clustering aims at grouping objects, such as genes, together, according to somemeasure of similarity, so that objects within one group or cluster are more similar to
each other than to objects in other groups. It is a mean to visualize patterns of gene
expression in the data.
7/30/2019 LP 6 TesteMoleculareGeneticeDermatologie Part I
70/70
Hierarchical clustering The final, computational form, of
the Pearson correlation coefficien:
To return to the context ofhierarchical clustering, a Pearsoncorrelation coefficient must becomputed for every possible genecomparison.
When clustering an entire genome
of 6,000 or more genes this canmean a considerable number ofcomparisons must be performed,yet the results can providevaluable generalizations about thegenes' relationships