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IMPLICATIONS OF GENETIC
TESTING
Huong Le
Senior Hospital Scientist
Department of Molecular & Clinical Genetics
Royal Prince Alfred Hospital
Sydney , Australia
OUTLINE
Introduction
Types of Genetic test
Genetic testing of large genes
Familial Hypercholesterolemia (FH)
Hereditary Breast Cancer
Conclusions
INTRODUCTION
Gene Mutations
Disrupt protein function associated with
disease
DNA technology
Impact of Human Genome Project
GENE
http://www.ornl.gov/sci/techresources/Human_Genome/project/info.shtml
DNA
TECHNOLOGY
COMBIMATRIX
TYPES OF GENETIC TEST
Diagnostic
Confirm clinical diagnosis
Prenatal
Fetal prenatal diagnosis
Screening
Reproductive screening
Newborn screening
Community screening
Predictive
Huntington disease and cancer
TYPES OF GENETIC TEST
Current diagnosis focuses on single gene disorders eg.
CF, thalassaemias
The challenge ahead is to expand DNA diagnosis in
Disease involve with large gene / polygene
Multifactorial disorders
Genetic testing of
FAMILIAL HYPERCHOLESTEROLAEMIA
FH is an autosomal dominant disorder associated with
premature atherosclerosis increased risk for coronary artery
disease (CAD)
FH diagnosis criteria for index case
Cholesterol >7.5mmol/ or LDL > 4.9mmol/l in adult
Cholesterol > 6.7mmol/l or LDL > 4.0mmol/l if < 16 yrs
Plus family history of high cholesterol or MI (<55yrsM) or plus tendon
xanthoma
Definite FH & Probable FH
Genetic testing in FH patients-What genes ?
Adapted from S. Humphries UK
LDL Removed Most FH due to
from Blood LDLR mutations,
LDL RECEPTOR
5% by APOB, 2% PCSK9
LDL
Cholesterol carried
in “LDL” particle
BLOOD in Blood
Receptor
LIVER CELL Recycles
PCSK9 LDL broken down.
Diet and New-made
Chol Bile excreted
FH GENES
LDLR (Low Density Lipoprotein Receptor) gene
Location:19p13.2 ; Structure: 44,36 kb and 18 exons
Heterogeneity: 900 LDLR mutations report & 5% are
large deletion mutations
APOB (ApoB-100) gene
Location:2p24-p23 ; Structure: 42,65 kb and 29 exons
Arg3500Glu & Arg3500Trp have shown to cause
ligand-defective apoB-100 and associated with FH (5%)
Others
DIAGNOSTIC TECHNIQUES
ARMS (Amplification Refractory Mutation
System)
Commercial kit : allow for detection of LDLR, APOB
and PCSK9 mutations
S1 S2 S4 S5 S6 S7 S8… M1 M2 M3 M4 Bl
PCR +ve
PCR +ve
DIAGNOSTIC TECHNIQUES
DHPLC / WAVE system
A PCR product is injected onto a column of WAVE
system. DNA variant will be detected base on
heteroduplex formation
Use for screen for any sequence change in all
exons including promoter region
Positive profiles will be confirmed by sequencing
DIAGNOSTIC TECHNIQUES
DIAGNOSTIC TECHNIQUES
Sequencing
Direct sequencing technology
dye terminator
Fluorescent
AB DNA Analyzer 3730 with 96 capillaries
Sequencing analysis using SeqScape software
High throughput
Cheaper cost
Effective mutation detection
SEQUENCING analysis
DIAGNOSTIC TECHNIQUES
MLPA: Multiplex Ligation-dependent Probe
Amplification
Detection of aberrant copy number of 40
genomic DNA sequences in one PCR based
reaction. (Deletions, Duplications and Copy
Number Polymorphisms)
Applications: gene dosage and genotyping
MLPA
(Multiplex ligation dependent probe amplification)
MLPA results
Probe design
Schouten et al Nucleic Acids Res 30 e57 (2002)
MUTATION DETECTION RATE
The sensitivity of this molecular test depends
strongly on the certainty of the clinical diagnosis:
Definite FH cases have mutations detected in the
LDLR gene about 70% of the time.
Probable FH cases have mutations detected in the
LDLR gene about 40% of the time.
FH GENETIC TEST AT
RPAH, SYDNEY
FH GENETIC TEST AT RPAH
FLOW CHART
FH
PROBAND
* Step5:
Step1: Step2: Step3: * Step4:
SEQUENCING
LDLR sequencing LDLR – DHPLC APOB MLPA
promoter & all
Exon4 & Exon12 (5% large deletions)
exons
NEGATIVE POSITIVE NEGATIVE POSITIVE POSITIVE NEGATIVE POSITIVE NEGATIVE
POSITIVE NEGATIVE
result profile profile result result result result result
result result *
Go to step2 Go to step3
Confirm by
Issue report Issue report Issue report Issue report FH cannot
sequencing
be excluded
Issue report
GENETIC TESTING OF
BREAST CANCER
Multifactorial disorder
BRCA1 and BRCA2 are two genes associated with
hereditary breast cancer and ovarian cancer
DNA testing for BRCA cancer predisposition mutations
perform for
Proband
At risk relatives
Risk of developing cancer which is associated with
BRCA mutations appears to be variable
GENETIC TESTING OF
BREAST CANCER
BRCA1 (hereditary breast /ovarian cancer)
Location: 17q12-21
Structure: 81,16 kb and 23 exons & encoded a
protein of 1863 amino acids
~ 500 mutations have been reported
BRCA2 (hereditary breast /ovarian cancer)
Location: 13q12-3
Structure: 84,19 kb and 28 exons & encoded a
protein of 3418 amino acids
~ 300 mutations have been reported
BRCA-Associated Cancers:
Lifetime Risk
BRCA1 BRCA2
GENETIC TESTING OF
BREAST CANCER
Features that indicate increased likelihood of having
BRCA mutations
Multiple cases of early onset breast cancer within
one family
Ovarian cancer (with family history of breast or
ovarian cancer)
Breast and ovarian cancer in the same woman
Bilateral breast cancer
Ashkenazi Jewish heritage
Male breast cancer
GENETIC TESTING OF
BREAST CANCER
In familial breast cancer, germ line mutations in the
BRCA1 or BRCA2 genes are followed by somatic
inactivation of the wild-type allele
BRCA1 and BRCA2 are thus thought to be tumour
suppressors since loss of heterozygosity at either
locus can lead to retention of the mutant allele in
tumours from heterozygous carriers.
BRCA mutation detection
strategy at Prince of Wales
Hospital, Sydney
Ashkenasi Jewish heritage
ARMS: c.185delAG,c. 5382insC, c. 617delT
Sequencing of BRCA1 followed by BRCA2 using
M13 labelled primers in a 96 well 20μl format with
stepped annealing temperatures.
SeqScape analysis software
MLPA analysis
GENETIC TESTING OF
BREAST CANCER
Adapt from Jennifer E. Axilbund, Cancer Risk Assessment Program
The Johns Hopkins Hospital
RESULT INTERPRETATION
Proband:
Mutation present: conferring high risk of
cancer for the family members
Mutation absent:
Limited / low sensitive techniques
Mutation in other regions / genes
Other factors
Inconclusive result:
Important in clinical problem when
A variant with unknown clinical significant
VARIANT ANALYSIS
Classification variants as deleterious or neutral
based on:
Frequency of variant in cases and controls
Segregation of sequence variant with disease in family
Co-occurrence of variant with a known deleterious mutation in
one or more tested individuals
Nature position of amino acid substitution plus degree of
conservation among different species, protein modeling
Result of functional assay
RESULT INTERPRETATION
At risk relative
Mutation present: increase risk of cancer
Mutation absent: similar risk as in general
population having cancer predisposing
BRCA1/2 mutations
MUTATION DETECTION RATE
Only 1/3 cases have mutations in
BRCA1/2 genes
Low detection rate of BRCA1/2
mutations
Limited / low sensitive techniques
Mutation in other regions / other genes
Other factors
GENETIC COUNSELING
Genetic counseling is required to address issues that arising
from the results of advance in genetic testing in order to assure
appropriate genetic testing can be translated into clinical care
Information from genetic test can affect the lifestyles of
individual and their family members
The challenge focuses on issues including privacy, informed consent,
risk assessment, decision making, disease intervention, insurance
and employment.
FUTURE DIRECTION OF
GENETIC TESTING
Microarray analysis
Facilitate analysis of all known mutations in a gene
at same time or all mutations in many genes can be
assayed in a single chip.
Applications have expanded in disease diagnosis,
drug discovery and toxicological research (R. Uma
& T. Rajkumar 2007)
Genome-wide approaches capable of analysing
thousands of genes and gene products and provide
a better biological understanding of breast cancer
and other cancers
FUTURE DIRECTION OF
GENETIC TESTING
Identify breast cancer profile (e.g. known subtype)
has potential to improve prognosis and predict the
best individual therapeutic scheme
Whole genome sequencing
Aim at US $ 1000 personal genome project within
10 years
454 from Roche company can do 20 MB in 4 hours
MOLECULAR BIOMARKER USEFUL FOR
TREATMENT RESPONSE
(James et al Oncologist 2007;12;142-150)
CONCLUSIONS
Identify family specific mutation recommendation
for predictive testing can be used to screen for at
risk relatives
provide opportunities for earlier disease management
thus reduce morbidity rate and enhance effectiveness
of prevention
However the complexities of all possible genetic
test results are still the challenge ahead in term of
result interpretation / translation into clinical care
Issues arise from genetic testing also need to be
addressed
The future model of genetic services
(Trent et al Encyclopedia of Diagnostic Genomics and Proteomics
2004; p676-681).
