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