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
Gene
Nucleus
Cell Chromosomes

Protein

Adapted from Understanding Gene Testing, NIH, 1995

The DNA Double Helix
Sugar Base pair
phosphate
backbone Bases

Adenine (A) Cytosine (C)

Thymine (T) Guanine (G)
 DNA Transcription and
Translation
Growing
chain of
amino
acids

mRNA
Ribosome Protein

Nuclear
membrane Cell
DNA
membrane
Adapted from Understanding Gene Testing, NIH, 1995
 Gene Structure
RNA transcription Splice sites Stop site
start site

Promoter
Exon 1 Intron Exon 2 Intron Exon 3

5' end 3' end

Exon 1 Exon 2 Exon 3

mRNA
 MITOCHONDRIAL NUCLEAR
GENOME GENOME

10% GENE 90% EXTRAGENIC

10% 90% 80% 20%

EXON NON UNIQUE REPETITIVE
LOW COPY
CODING

TANDEM INTERSPERSED
PSEUDO INTRON UTR REPEAT SEQUENCE
GENE
Genetic Code
A codon is made of 3 base pairs
64 codons total

1 codon (AUG) encodes 61 codons encode 3 codons stop

methionine and starts 20 amino acids protein
translation of all proteins (redundant code) translation

A U G G C A U A A

Met Ala
Disease-Associated Mutations
Alter Protein Function

Functional protein Nonfunctional or

missing 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 testing
Direct mutational study
Linkage analysis
 RNA – based testing

 Protein – based testing

Preparing DNA for Analysis

Blood sample Centrifuge and DNA for analysis

extract DNA
from white blood
cells
 DNA
PELLET
DNA 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
DNA fragments loaded into wells

DNA fragments
separate by size
and charge
Voltage
Path of migration

+
RESTRICTION
ENZYME DIGESTION

ELECTROPHORESIS
PCR
Duchenne / Becker MD
SRY
 Allele Specific Oligonucleotide
(ASO) Hybridization
Patients
#1 #2 #3
Add radio-
labeled normal
DNA probes
#1 #2 #3
Add known
mutant DNA
Amplify DNA and probes
hybridize to membranes
Allele Specific Amplification
Thrombophilia
Single Strand Conformational
Polymorphism (SSCP)
Normal Mutated  DNA is denatured
DN into single strands
A
 Single strands fold;
shape is altered by
mutations
 Mobility of mutant
and normal strands
differ in gel
Gel mutation
SSCP
Cerebellar hemangioblastoma
Cerebellar hemangioblastoma
Retinal hemangioblastoma
Denaturing Gradient Gel
Electrophoresis (DGGE)
Normal Mutated  DNA denatured into single
strands
DNA
 Single strands reanneal
into normal and mutant
homoduplexes and
heteroduplexes
 Hetero- and homoduplexes
denature at different points
in gradient gel

Denaturing gradient gel

Heteroduplex Analysis (CSGE)

Amplify Cold
and Single-strand DNA
denature
DNA

Reannealed DNA

Mutated bands

Normal band
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

1, 2 3, 4 DNA markers near

gene of interest that
segregate with
1, 3 1, 4 2, 3 2, 4 disease
 Requires DNA
1
2 analysis of multiple
3 family members
4

Adapted from Offit K. Clinical Cancer Genetics: Risk Counseling & Management, 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

CTAGTGCATC TTTCTAGT TTTA

G A T C A C G T A G A A AG A T C A A A A T

CTAGTGCATC GTTCTAGT TTTA

G A T C A C G T A G C A AG A T C A A A A T

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 variant
risk factor for arterial and venous
thrombosis,NTD
associates with hyperhomocysteinemia
TT homozygotes have high Hcy
dietary manipulation with B6 is possible
potential risk for pre-eclampsia
Clinically useful polymorphism
 HFE hemochromatosis
C282Y and H63D polymorphisms
very common in caucasians
homozygote C282Y - HHC
compound 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
Prehybridization Posthybridization

Synthetic DNA probes Probes with

hybridized
DNA
Protein Truncation Assay
Normal Mutated  DNA transcribed to
DN mRNA
A
 RNA translated to
mRNA protein
 Protein run on gel
Protein
 Truncated protein
has different mobility
Gel in gel
mutation
Pregnancy and Prenatal diagnosis
Cyclops Cebocephaly
Holoprosencephaly sequence
spectrum
FRONTONASAL DYSPLASIA
Thanatophoric dysplasia
Use of Genetic Analysis
 Diagnostic use
Symptomatic
Presymptomatic
 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