Genetic Diagnosis in Clinical and Forensic Medicine
Chanin Limwongse, MD
Division of Medical Genetics, Department of Medicine,Faculty of Medicine Siriraj Hospital
Uses of Genetic Diagnostic Techniques
Forensic investigation Diagnosis of genetic and inherited
diseases Diagnosis of infectious diseases Pharmacogenetic uses
Diagnosis of diseases
Dysmorphic individual Spontaneous abortion Bone tumor Recurrent DVT Intractable epistaxis Childhood muscular weakness Malignant hyperthermia
Chromosomes, DNA, and Genes
CellCellNucleusNucleus
ChromosomesChromosomes
Gene
ProteinProtein
Adapted from Adapted from Understanding Gene TestingUnderstanding Gene Testing, NIH, 1995, NIH, 1995
The DNA Double Helix
Adenine (A)Adenine (A)
Thymine (T)Thymine (T)
Cytosine (C)Cytosine (C)
Guanine (G)Guanine (G)
BasesBases
Sugar Sugar phosphate phosphate backbonebackbone
Base pairBase pair
DNA Transcription and Translation
mRNAmRNA
RibosomeRibosome
Growing Growing chain of chain of amino amino acidsacids
ProteinProtein
Nuclear Nuclear membranemembrane Cell Cell
membranemembraneDNADNA
Adapted from Adapted from Understanding Gene TestingUnderstanding Gene Testing, NIH, 1995, NIH, 1995
5' end 5' end
PromoterPromoter
RNA transcription RNA transcription start sitestart site
3' end 3' end
Gene Structure
Stop siteStop site
Intron Exon 2 IntronExon 1 Exon 3
Splice sitesSplice sites
Exon 2Exon 1 Exon 3
mRNAmRNA
NUCLEAR GENOME
MITOCHONDRIAL GENOME
GENE
EXON
EXTRAGENIC
NON
CODING
PSEUDOGENE
INTRON UTR
UNIQUE LOW COPY
REPETITIVE
TANDEM REPEAT
INTERSPERSED SEQUENCE
10% 90%
10% 90% 80% 20%
Genetic Code
A codon is made of 3 base pairs
64 codons total
1 codon (AUG) encodes 1 codon (AUG) encodes methionine methionine andand starts starts
translation of all proteinstranslation of all proteins
3 codons stop 3 codons stop protein protein
translationtranslation
61 codons encode 61 codons encode 20 amino acids20 amino acids
(redundant code)(redundant code)
U A A
A U G
Met
G C A
Ala
Disease-Associated Mutations Alter Protein Function
Functional proteinFunctional protein Nonfunctional or Nonfunctional or missing proteinmissing protein
Classification of Genetic Disorders
Chromosomal aberrations : trisomy 21, Turner, Klinefelter
Mendelian disorders : AD, AR, X-linked, Y-linked
Multifactorial (complex) disorders : NTD, DM, HTN, hyperlipidemia
Techniques in Genetics
Cytogenetic technique conventional chromosome analysis FISH, M-FISH, SKY
comparative genomic hybridization
Molecular genetic technique DNA, RNA, and protein testing
Biochemical genetic testing blood and urine metabolite analysis
Clinical specialty testing U/S, CVS, cordocentesis, fetoscopy fetal cell in maternal blood
METAPHASE SPREAD
Karyotype
Chromosome Analysis (Karyotyping)
Suspect a chromosomal aberration MR of unknown cause Multiple congenital anomalies (>= 3 major
anomalies) Habitual abortion , neonatal death / stillbirth Severe manifestation of X-linked disease in
female Cancer tissue Specific symptom-based indication : ambiguous
genitalia, SS Family history of a carrier of chromosomal
aberration
Single copy probe FISH
Interphase FISH
Spectral Karyotyping (SKY)
Fiber FISH
FISH
CHD- Di George / VCF 22q deletion syndrome
Floppy baby - PWS Neuropathy - CMT 17p11 duplication CML - BCR/Abl fusion Sex chromosome abnormalities Post bone marrow transplantation
Comparative Genomic Hybridization
Comparative Genomic Hybridization
Molecular Genetic Testing
DNA – based testingDirect mutational studyLinkage analysis
RNA – based testingProtein – based testing
Preparing DNA for Analysis
Blood sampleBlood sample Centrifuge and Centrifuge and extract DNA extract DNA
from white blood from white blood cellscells
DNA for analysisDNA for analysis
DNA PELLET
DNA Diagnostic TechniquesDNA Diagnostic Techniques
Direct mutation analysis Membrane hybridization techniques Restriction digestion technique PCR-based techniques Electrophoretic mobility-based techniques Combination of the four above Denaturing HPLC DNA Sequencing Microarray-based techniques
Electrophoresis of DNA
VoltageVoltage
++
DNA fragments loaded into wellsDNA fragments loaded into wells
Path of migrationPath of migration
DNA fragments DNA fragments separate by size separate by size
and chargeand charge
RESTRICTION ENZYME DIGESTION
ELECTROPHORESIS
PCR
Duchenne / Becker MD
SRY
Add radio-Add radio-labeled normal labeled normal DNA probesDNA probes
Amplify DNA and Amplify DNA and hybridize to membraneshybridize to membranes
Allele Specific Oligonucleotide(ASO) Hybridization
Add known Add known mutant DNA mutant DNA
probesprobes
PatientsPatients
#1#1 #2#2 #3#3
#1#1 #2#2 #3#3
Allele Specific Amplification
Thrombophilia
Single Strand Conformational Polymorphism (SSCP)
DNDNAA
GelGel
NormalNormal MutatedMutated
mutationmutation
DNA is denatured into single strands
Single strands fold; shape is altered by mutations
Mobility of mutant and normal strands differ in gel
SSCP
Cerebellar hemangioblastoma
Cerebellar hemangioblastoma
Retinal hemangioblastoma
Denaturing Gradient Gel Electrophoresis (DGGE)
DNA denatured into single strands
Single strands reanneal into normal and mutant homoduplexes and heteroduplexes
Hetero- and homoduplexes denature at different points in gradient gel
DNADNA
Denaturing gradient gelDenaturing gradient gel
NormalNormal MutatedMutated
Heteroduplex Analysis (CSGE)
Normal bandNormal band
Mutated bandsMutated bands
Single-strand DNASingle-strand DNAColdCold
Reannealed DNAReannealed DNA
Amplify Amplify and and
denature denature DNADNA
SEQUENCING
Linkage Analysis
Attempt to predict the inheritance of mutant allele by using close-by polymorphic DNA markers
PRO: only need to know gene position, use standard molecular technique
CON: requires multiple samples, not definitive, expensive
Methods: PCR of polymorphic DNA markers
Linkage Analysis
Looks for pattern of Looks for pattern of DNA markers near DNA markers near gene of interest that gene of interest that segregate with segregate with diseasedisease
Requires DNA Requires DNA analysis of multiple analysis of multiple family membersfamily members
1, 21, 2 3, 43, 4
1, 31, 3 1, 41, 4 2, 32, 3 2, 42, 4
11223344
Adapted from Offit K. Adapted from Offit K. Clinical Cancer Genetics: Risk Counseling & Management,Clinical Cancer Genetics: Risk Counseling & Management, 1998 1998 ASCO
Linkage Analysis Probability (of crossing over
dependent upon distance of markers) Need to have multiple members Need to know mode of inheritance Doesn’t need to know type of
mutation
ADPKD Linkage
DMD Linkage
Polymorphism
C T A G T G C A T C T T T C T A G T T T T A
G A T C A C G T A G A A AG A T C A A A A T
G A T C A C G T A G C A AG A T C A A A A T
C T A G T G C A T C G T T C T A G T T T T A
T to G polymorphism
VNTR and Satellites
VNTR 50-200 bp size repeat
Minisatellite 6-20 bp size repeat
Microsatellite 2-4 bp size repeat
CAG, CTG, CCG
SNPs
The smallest polymorphism in the genome Biallelic, triallelic or rarely tetra-allelic Occur approximately every 100 bp of human
DNA Most are intronic SNPs and do not result in
change in protein structure Very useful as map markers for gene tracking
Clinically useful polymorphism
MTHFR gene C667T variantrisk factor for arterial and venous
thrombosis,NTDassociates with
hyperhomocysteinemia TT homozygotes have high Hcy dietary manipulation with B6 is possiblepotential risk for pre-eclampsia
Clinically useful polymorphism
HFE hemochromatosisC282Y and H63D polymorphismsvery common in caucasianshomozygote C282Y - HHCcompound heterozygote C282Y/H63D
homozygote H63D - normal Useful clinical test leading to surveillance
APOE polymorphism
Differ at residue 112 (site A) and 158 (site B) Different electrophoretic mobility due to charge E2 --- cysteine/cysteine E3 --- cysteine/arginine --- wild type allele and
most common
E4 --- arginine/arginine --- Alzheimer’s association E2/E2 --risk for familial dysbetalipoproteinemia
Principle of Microarray (Chip) Assay
Synthetic DNA probesSynthetic DNA probes
PrehybridizationPrehybridization PosthybridizationPosthybridization
Probes with Probes with hybridized hybridized
DNADNA
Protein Truncation Assay DNA transcribed to
mRNA
RNA translated to protein
Protein run on gel
Truncated protein has different mobility in gel
DNDNAA
mRNAmRNA
ProteinProtein
GelGel
NormalNormal MutatedMutated
mutationmutation
Pregnancy and Prenatal diagnosis
Cyclops Cebocephaly
Holoprosencephaly sequence spectrum
FRONTONASAL DYSPLASIA
Thanatophoric dysplasia
Use of Genetic Analysis
Diagnostic useSymptomaticPresymptomatic
Prognostic Use Carrier detection Family planning
Premarital, preimplantation, prenatal Paternity testing and identification
Genetic Counseling
Genetic counseling is a process in which counselor facilitate the understanding of genetic information pertinent to the counselee’s personal and family history, and assist the counselee in a non-biased manner to make an informed decision based on his/her own judgement regarding his/her reproduction and future.
Content of Genetic Counseling
Diagnosis Disease burden including treatment Inheritance mode Recurrence risk Reproductive options Each members’ (couple , pt, other family
members) viewpoint of disease
Principles of Genetic Counseling
Autonomy
Informed decision making
Non-directiveness
Confidentiality
Empathy and support
GENETIC DATABASES
ATLASES (dysmorphology, radiology)
CATALOGS (chromosome, cancer cytogenetic)
PUBMED / MEDLINE
OMIM (www.ncbi.nlm.nih,gov/omim)
GENETICISTS