Lecture 3 Genetic Diagnosis In Clinical And Forensic Medicine

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  

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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 Cell

Nucleus Chromosomes

Protein Adapted from Understanding Gene Testing, NIH, 1995

The DNA Double Helix Sugar phosphate backbone

Base pair Bases

Adenine (A)

Cytosine (C)

Thymine (T)

Guanine (G)

DNA Transcription and Translation Growing chain of amino acids mRNA Ribosome

DNA

Nuclear membrane

Adapted from Understanding Gene Testing, NIH, 1995

Protein

Cell membrane

Gene Structure RNA transcription start site

Splice sites

Stop site

Promoter Exon 1

Intron

Exon 2

Intron

5' end

Exon 3 3' end

Exon 1 Exon 2

mRNA

Exon 3

MITOCHONDRIAL GENOME 10% 10% EXON

NUCLEAR GENOME

GENE

90% 90%

80% UNIQUE LOW COPY

NON CODING

PSEUDO GENE

INTRON

EXTRAGENIC

UTR

TANDEM REPEAT

20% REPETITIVE

INTERSPERSED SEQUENCE

Genetic Code A codon is made of 3 base pairs 64 codons total 1 codon (AUG) encodes methionine and starts translation of all proteins

61 codons encode 20 amino acids (redundant code)

3 codons stop protein 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, Xlinked, Y-linked  Multifactorial (complex) disorders : NTD, DM, HTN, hyperlipidemia 

Techniques in Genetics 

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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 

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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 extract DNA from white blood cells

DNA for analysis

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 radiolabeled normal DNA probes

Amplify DNA and hybridize to membranes

Add known mutant DNA probes

#1

#2

#3

Allele Specific Amplification

Thrombophilia

Single Strand Conformational Polymorphism (SSCP) Normal DN A

Gel

Mutated

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DNA is denatured into single strands

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Single strands fold; shape is altered by mutations

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Mobility of mutant and normal strands differ in gel

mutation

SSCP

Cerebellar hemangioblastoma

Cerebellar hemangioblastoma

Retinal hemangioblastoma

Denaturing Gradient Gel Electrophoresis (DGGE) Normal

Mutated



DNA denatured into single strands

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Single strands reanneal into normal and mutant homoduplexes and heteroduplexes

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Hetero- and homoduplexes denature at different points in gradient gel

DNA

Denaturing gradient gel

Heteroduplex Analysis (CSGE) Amplify and denature DNA

Cold Single-strand DNA

Reannealed DNA Mutated bands Normal band

SEQUENCING

Linkage Analysis 





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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

1, 3 1, 4 2, 3 2, 4 1 2 3 4

DNA markers near gene of interest that segregate with disease

 Requires DNA

analysis of multiple family members

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   

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

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 DN A

Mutated



DNA transcribed to mRNA



RNA translated to protein



Protein run on gel

mRNA Protein

Truncated protein has different mobility in gel mutation 

Gel

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

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Informed decision making

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Non-directiveness

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Confidentiality

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Empathy and support

GENETIC DATABASES 

ATLASES (dysmorphology, radiology)

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CATALOGS (chromosome, cancer cytogenetic)

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PUBMED / MEDLINE

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OMIM (www.ncbi.nlm.nih,gov/omim)

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GENETICISTS