Genetic Disorders
and Molecular Diagnosis
นายแพทย ดร. ณตพล ศุภณัฐเศรษฐกุล
ภาควิชาพยาธิวิทยา และนิติเวชศาสตร
คณะแพทยศาสตร มหาวิทยาลัยนเรศวร
Email : [email protected]
14 กันยายน พ.ศ. 2553
http://www.uga.edu/srel/kidsdoscience/images/genetics-puppets.gif
http://www.uwo.ca/biology/images/undergraduate/chromosome.jpg
Topics
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The Role of Genetics in Medicine
Genes and Chromosomes
Genetic disorders
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Single gene disorders
Chromosomal disorders
Polygenic or multifactorial diseases
Somatic cell genetic disorders
Topics
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Clinical genetics and Molecular Diagnosis
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Genetic testing for carrier detection
Prenatal diagnosis
Newborn screening
Molecular diagnosis
Genetic counseling
The Role of Genetics
in Medicine
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First report in human genetics : Jewish report
on hemophilia and gender transmission
In 1865, Mendel published his work
In 1956, the correct chromosome number
was reported
In January 1959, the chromosomal
abnormality associated with clinical disorders
was ever reported
Mapping of genes to chromosomes and
specific chromosome region, abnormal gene
functions
http://ghr.nlm.nih.gov/handbook/illustrations/chromosomallocation.jpg
Genes and Chromosomes
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Structure of DNA
Central dogma
Chromosomes
Gene
Cell cycle and cell divisions :
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mitosis
meiosis
Mutation
Structure of DNA
http://academic.brooklyn.cuny.edu/biology/bio4fv/page/molecular%20biology/16-05-doublehelix.jpg
“Central Dogma”
Transcription
DNA
Replication
Translation
RNA
Protein
Chromosomes
http://infohost.nmt.edu/~klathrop/DNA%20Coiling.jpg
Human Genome (chromosomes)
In Nucleus
In Mitochondria
3.1 x 109 bp
16,569 bp
10%
90%
Gene
<10%
Coding
Sequences
(Exon)
Non-gene (Extragenetic)
>90%
Non coding
Sequences
(Intron)
50%
Repetitive DNA
Tandem repeats
(satellite, minisatellite, microsatellite)
50%
Non repetitive DNA
Interspersed repeats
(SINES, LINES)
Cell cycle
http://ghs.gresham.k12.or.us/science/ps/sci/soph/cells/cycle/cycle.gif
http://www.cbp.pitt.edu/faculty/yong_wan/images/main_cell_cycle.jpg
Cell division
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Mitosis
Meiosis and gametogenesis
Crossing over
http://www.uhh.hawaii.edu/~ronald/392/Mitosis.JPG
http://www.mun.ca/biology/desmid/brian/BIOL2060/BIOL2060-20/2010.jpg
Mutation
หมายถึงกระบวนการที่กอใหเกิดการเปลี่ยนแปลง
ทุกรูปแบบ ที่เกิดขึน้ กับโมเลกุลของ DNA
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“Wild type” and “mutant allele”
Normal variation, polymorphism, inherited disease
Types of mutation
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Base substitution => silent mutation, missense, nonsense
Insertion => frameshift mutation
Deletion => frameshift mutation
Chromosomal abnormality
http://www.layevangelism.com/bastxbk/images/mutation.jpg
Genetic disorders
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Single gene disorders
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Mendelian inheritance
Non-Mendelian inheritance
Chromosomal disorders
Polygenic or multifactorial disorders
Somatic cell genetic disorders
I. Single gene disorders
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Terminology
Mechanism of mutation
Functional effects of mutation on protein
Mendelian inheritance of single gene
disorders
Terminology
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Genotype = genetic constitution of an individual
Phenotype = observed characteristic
Homozygote = identical alleles at a given locus
Heterozygote = two different alleles at a given locus
Autosomes = any chromosomes other than sex
chromosomes
Autosomal inheritance = involve any chromosomes
other than sex chromosomes
Pedigree charts = to illustrate the inheritance
http://naturalsciences.sdsu.edu/ta/classes/lab2.4/trait.jpg
Terminology
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Locus = position of DNA sequences, or gene
on a chromosome
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Allele = possible alternative form of a given
gene
http://www.geneticsandhealth.com/wp-content/allele.jpg
http://www.genome.gov/Pages/Hyperion/DIR/VIP/Glossary/Illustration/Images/autosome.gif
http://www.geneticsandhealth.com/wp-content/pedigree%20image_1.gif
http://www.geneticsandhealth.com/wp-content/pedigree%20image_2.gif
http://www.migeneticsconnection.org/genomics/Family%20History/pedigreeSymbols.jpg
Introduction
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Single gene disorders are caused by individual
mutant genes
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There are approximately 11,000 single gene
disorders, affecting 1% of the populations
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Follow Mendelian patterns of inheritance
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Autosomal dominant inheritance
Autosomal recessive inheritance
X-linked recessive inheritance
X-linked dominant inheritance
Y-linked (Holandic) inheritance
http://www.uic.edu/classes/bms/bms655/gfx/pedigree1.gif
Autosomal Dominant
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Phenotypically expressed in homozygotes and
heterozygotes for that gene
There is vertical inheritance (affected child usually
has an affected parent)
Unaffected family members usually have unaffected
partners, and they produce normal children
Affected family members, usually have unaffected
partners, and they produce a 1:1 ratio of normal and
affected children
Usually both sexes are equally affected, and they
are equally likely to pass on the disease
Examples
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Achondroplasia
Acute intermittent porphyria
Familial hypercholesterolemia
Marfan syndrome
Von Willebrand disease
Achondroplasia
http://www.sciencemuseum.org.uk/on-line/genes/images/1-3-5-1-4-2-1-1-1-3-0.jpg
http://www.moondragon.org/obgyn/graphics/achondroplasiacomparison.jpg
http://www.commonwealthadoption.org/images/johnston.jpg
Marfan syndrome
http://www.nhlbi.nih.gov/health/dci/images/Marfan_lowres.gif
http://www.homoeopathiker.de/homoeopathik/syphilis/bilder/marfan3.jpg
http://www.uic.edu/classes/bms/bms655/lesson5.html
Autosomal Recessive
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Expressed only in homozygotes, otherwise
can be trait
There is horizontal inheritance (normal parents
often have more than one affected child)
Affected individuals have phenotypically
normal parents
Affected individuals usually have unaffected
partners and all their children will be carriers
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If a carrier has an unaffected partner, there is a 50%
chance of the children being carriers
Only mating between heterozygotes will produce
affected individuals, with an expected frequency of
1 in 4
There is an association with consanguinity due to
sharing of genes in families (rare recessive genetic
disorders are more likely to arise through
consanguinity)
Both sexes are equally affected and equally likely to
pass the mutation to the next generation
http://upload.wikimedia.org/wikipedia/commons/thumb/3/3e/Autorecessive.svg/300px-Autorecessive.svg.png
Example
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Alpha thalassemia
Beta thalassemia
Congenital adrenal hyperplasia
Cystic fibrosis
Hemochromatosis
Phenylketonuria
Sickle cell disease
Tay-Sachs disease
http://healthnet.md.chula.ac.th/text/forum1/thalassemia2/picture/thalassemia.jpg
http://home.kku.ac.th/acamed/kanchana/p7.jpg
http://www.microscopyu.com/galleries/pathology/images/sicklecell/sicklecellanemia40x02small.jpg
http://www.irondisorders.org/News/SickleCell.jpg
http://www.uic.edu/classes/bms/bms655/gfx/pedigree5.gif
X-linked Dominant
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Rare, and difficult to distinguish from AD
except that affected males have normal sons,
but all daughters are affected
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Example : X-linked hypophosphatemic
rickets, Rett syndrome
http://www.uic.edu/classes/bms/bms655/lesson7.html#RECESSIVE
X-linked Recessive
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Many more males than females show the recessive
phenotype
The disease is transmitted by a carrier female, who
is usually asymptomatic
If a mother is a carrier, her son have a 50% chance
of being affected and her daughters a 50% chance
of being carriers
An affected male will usually have no affected
offspring, but all his daughters will be carriers and,
in turn, 50% of their sons will be affected
No sons of the affected male will inherit the gene
(there is no male-to-male transmission)
Example
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Duchenne muscular dystrophy
Becker muscular dystrophy
Glucose-6-phosphate dehydrogenase
(G6PD) deficiency
Hemophilia A
Hemophilia B
Lesch-Nyhan syndrome
Gower’s sign
Duchenne muscular dystrophy
http://www.neurologyarticles.com/sitebuilder/images/Scan10106-176x315.jpg
http://medicalimages.allrefer.com/large/x-linked-recessive-genetic-defects.jpg
Y-linked (Holandic)
http://cas.bellarmine.edu/tietjen/HumanBioogy/bills_developmental_abnormalities.htm
http://cas.bellarmine.edu/tietjen/HumanBioogy/Finished%20Images/gen17.gif
II. Chromosomal Abnormalities
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Terminology
Introduction
Numerical Chromosome abn.
Structural chromosome abn.
Chromosome instability synd.
http://images.wikio.com/images/p/51ca/the-world-s-worst-genetic-mutation-in-a-dog.jpg
Terminology
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Karyotype = the chromosome constitution of an individual,
normal human karyotype is 46,XY (male) and 46,XX (female)
Polyploidy = the number of hapoloid chromosome sets is
greater than two (2n), triploidy, tetraploidy
Aneuploidy = chromosome number is not an exact multiple of
haploid number, monosomies, trisomies
Trisomy = three representatives of a given chromosome, trisomy
21 in Down dyndrome
Monosomy = one member of a chromosome pair is missing,
Turner syndrome (45,X)
Translocation = the transfer of one segment of a chromosome
to another
http://www.asklenore.info/miscarriage/bick/images/fig3.jpg
http://www.ucl.ac.uk/~ucbhjow/bmsi/lec7_images/47_xx_21.gif
http://www.myoops.org/cocw/tufts/courses/20/content/D293242/C293223.jpg
Spectral Karyotyping (SKY) Analysis
http://www.nature.com/onc/journal/v24/n33/images/1208711f1.jpg
Introduction
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Maybe numerical or structural
Nomenclature
Numerical disorders :
[47, XY,+21],
[45,X]
Structural disorders :
[46,XY,t(14;21)(q11,p10)],
[46,XY,dup(5) (q20-q30)],
[46,XY,del(15)(q11-q13)],
(break point, margin, or region)
Examples
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Autosomal disorders :
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Sex chromosome disorders :
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Trisomy 21 (Down syndrome)
Trisomy 18 (Edwards syndrome)
Trisomy 13 (Patau syndrome)
Klinefelter syndrome (47,XXY)
Turner syndrome (45,X)
XXX = female with an extra X chromosome
XYY = male with an extra Y chromosome
XX male = Y sequences are transferred to the X
chromosome, look like male with Klinefelter
syndrome
http://cas.bellarmine.edu/tietjen/HumanBioogy/Finished%20Images/gen30.gif
http://cas.bellarmine.edu/tietjen/HumanBioogy/bills_developmental_abnormalities.htm
http://www.virtualsciencefair.org/2004/mcgo4s0/public_html/t2/dwn_chromo.gif
http://www.robbinspathology.com/content/figure.cfm?ImageSeqNo=108200&screenWidth=1280&screenHeight=800
http://cas.bellarmine.edu/tietjen/HumanBioogy/bills_developmental_abnormalities.htm
http://www.robbinspathology.com/content/figure.cfm?ImageSeqNo=109400&screenWidth=1280&screenHeight=800
http://cas.bellarmine.edu/tietjen/HumanBioogy/bills_developmental_abnormalities.htm
The XYY Man is Spider Scott (Stephen
Yardley), a burglar with an extra "Y"
chromosome - which makes him
predisposed towards crime. So when he's
tapped up fresh out of prison by British
intelligence to do a big job for a big price,
he goes for it - which leads to a chain of
events that threaten his life - and indeed
other people's lives.
It's one of those 70s plots that involves
various secret agents from several
different countries - South Africa,
Rhodesia (it still existed) and China in
particular.
http://modculture.typepad.com/photos/uncategorized/2007/03/14/xyy.jpg
http://www.biology.iupui.edu/biocourses/n100/images/XYYMale.gif
Structural Chromosome
Abnormalities
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Result from breakage and limitations of
DNA repair systems
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Mechanisms
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Translocation
Inversion
Duplication
Deletion and ring chromosome
Isochromosome
http://www.robbinspathology.com/content/figure.cfm?ImageSeqNo=107050&screenWidth=1280&screenHeight=800
Cri du chat syndrome
mental retardation, low birth weight, low set ears, cat-like cry
http://cas.bellarmine.edu/tietjen/HumanBioogy/Finished%20Images/gen30.gif
http://www.cityofhope.org/PublishingImages/Research/Shared%20Resources%20%20Equipment/Cytogenetics%20Core%20Laboratory/abnormal_karyotype_Large.jpg
III. Multifactorial Disorders
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Terminology
Multifactorial disorders
Threshold model of multifactorial disorders
Examples of multifactorial disorders
Terminology
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Polygenic inheritance : the inheritance of
traits that are influenced by many genes at
different loci
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Multifactorial disorder : disorder in which
both environmental and genetic factors are
important
Multifactorial Inheritance
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Normal human characteristics : Blood
pressure, height, finger ridges, and intelligence
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Congenital malformations : neural tube
defects, non-syndromic cleft lip and palate, and
congenital heart disease
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Common disorders of adult life : DM,
Hypertension, peptic ulcer, and schizophrenia
Heritability and Environmental
factors
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Heritability : percentage denoting that the
genetic contribution of a given disease
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If heritability is high, there is a high correlation in
relatives
Environmental factors : can be manipulated
to reduce an individual’s susceptibility below
than the threshold
Threshold model for
Multifactorial disorders
(Tendency)
http://www.uic.edu/classes/bms/bms655/gfx/figure18.gif
Example
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Diabetes mellitus, type I (Insulin-dependent)
Essential hypertension (62% heritability)
Atherosclerosis (65% heritability)
Peptic ulcer (37% heritability)
Schizophrenia (85% heritability)
Asthma (80% heritability)
Alzheimer disease
The age of expression of the major types of genetic disease
Clinical genetics
and Molecular Diagnosis
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Genetic testing for carrier detection
Prenatal diagnosis
Newborn screening
Molecular diagnosis
Genetic counseling
Genetic Testing for Carrier Detection
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To identify asymptomatic heterozygotes for
AR traits, or AD disorders that have limited
penetrance or late onset
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Confined to small ethnic populations in which
there is an anomalously high incidence of a
particular disease
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Examples : Thalassemia, Cystic fibrosis,
ADPKD, Cascade screening
Prenatal diagnosis
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To assure of having unaffected children when the risk of
having an affected child is unacceptably high
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NOT equivalent to the assurance of having normal
children, It does not address all possible birth defects
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Noninvasive techniques : ultrasonography, maternal
serum screening
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Invasive technique : amniocentesis, chorionic villus
sampling, cordocentesis
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DNA analysis (PCR, Southern blot), Cytogenetic analysis
http://www.nlm.nih.gov/medlineplus/ency/images/ency/fullsize/1062.jpg
Cordocentesis
http://www.pennhealth.com/health_info/pregnancy/graphics/images/en/19177.jpg
http://www.pennhealth.com/health_info/pregnancy/graphics/images/en/19175.jpg
Chorionic villi sampling
http://www.nlm.nih.gov/medlineplus/ency/images/ency/fullsize/19163.jpg
http://www.pennhealth.com/health_info/pregnancy/graphics/images/en/19166.jpg
Newborn screening
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To identify treatable genetic disorders such as
phenylketonuria, galactosemia, maple syrup urine
disease, and congenital adrenal hyperplasia in
newborn infants
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Dietary management is usually an early intervention
to eliminate clinical symptoms that would otherwise
lead to severe disability, mental impairment, or dealth
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Prevalence, severity of a condition, availability,
effectiveness of a treatment, and cost will determine
which disorder will be screened
http://www.health.state.mn.us/divs/fh/mcshn/ncfu/images/pkupath.gif
Molecular techniques
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DNA , RNA Extraction
Gel Electrophoresis
Polymerase Chain Reaction (PCR)
Direct Hybridization Method
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Southern blot analysis
Northern blot analysis
Fluorescence In situ Hybridization (FISH)
DNA Microarrays
Cytogenetics
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Karyotyping
Spectral karyotyping (SKY analysis)
DNA
Extraction
http://www.favorgen.us/sitebuilder/images/Blood_Genomic_DNA_Mini_chart-437x463.jpg
DNA Gel Electrophoresis
http://oceanexplorer.noaa.gov/explorations/03bio/background/molecular/media/gel_plate_600.jpg
http://universe-review.ca/I11-50-gelectrophoresis.jpg
Polymerase Chain
Reaction (PCR)
Taq DNA
Polymerase
http://www.bio.miami.edu/~cmallery/150/gene/c7.20.7.pcr.jpg
http://www.obstbau-kompetenzzentrum.de/arbeitsbereiche/Folder.2006-11-07.1358/Thermocycler_.jpg
http://users.ugent.be/~avierstr/principles/pcrcopies.gif
http://images.google.co.th/imgres?imgurl=http://users.ugent.be/~avierstr/principles/pcrcopies.gif&imgrefurl=http://users.ugent.b
e/~avierstr/principles/pcr.html&usg=__kCFieOdf4YXbyVmdNjY2uR9bquI=&h=306&w=598&sz=14&hl=th&start=9&um=1&tbnid
=3Kupvmv7CJ2gM:&tbnh=69&tbnw=135&prev=/images%3Fq%3Dpcr%26ndsp%3D18%26hl%3Dth%26sa%3DN%26um%3D1
Southern Blot Analysis
http://www.mmi.mcgill.ca/mmimediasampler2002/images/5no15/5no15overview.gif
Blot Detection Method
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Southern blot = DNA detection (DNA probe)
Edwin Southern, Professor of Biochemistry, 1975
Northern blot = RNA detection (RNA probe)
Western blot = Protein detection (Antibody)
Southwestern blot = DNA binding protein (DNA
probe)
Eastern blot = Protein post translation modification
(probe detect CHO, lipid, etc.)
Dot blot (Slot blot) = Nucleotide or Protein
Fluorescent In situ
Hybridization
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Localized specific DNA sequences on
chromosomes using fluorescent probes
Fluorescence microscopy
Localized specific mRNA = gene expression
http://micro.magnet.fsu.edu/primer/techniques/fluorescence/anatomy/images/fluoromicrofigure1.jpg
http://en.wikipedia.org/wiki/File:FISH_(Fluorescent_In_Situ_Hybridization).jpg
DNA Microarrays
http://img.medscape.com/fullsize/migrated/430/330/cc430330.fig2.gif
http://en.wikipedia.org/wiki/File:NA_hybrid.svg
http://www.fao.org/DOCREP/003/X6884E/x6884e00.jpg
http://neurophilosophy.files.wordpress.com/2006/08/cdna-array.jpg
http://farm3.static.flickr.com/2077/2034113679_ac5a5c688d_m.jpg
Cytogenetics : Karyotyping
http://www.virtualsciencefair.org/2004/mcgo4s0/public_html/t2/dwn_chromo.gif
Spectral Karyotyping (SKY) Analysis
http://www.cityofhope.org/PublishingImages/Research/Shared%
20Resources%20%20Equipment/Cytogenetics%20Core%20Laboratory/HumanSK
Y_Large.jpg
Genetic Counseling
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Indications for genetic counseling
Information conveyed in genetic counseling
http://www.gen.vcu.edu/images/counseling.jpg
Indications for
Genetic Counseling
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Known or suspected hereditary disease
in a patient or family
Birth defects
Mental retardation
Advanced maternal age
Family history of early onset cancer
Recurrent pregnancy loss
Teratogen exposure
Consanguinity
Information Conveyed in
Genetic Counseling
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The magnitude of the risk of occurrence
or recurrence
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The impact of the disease on the patient
and the family
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The possibility of modification of either
the impact or the risk
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Anticipated future development
References
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คัมภิรานนท, อมรา., “พันธุศาสตรมนุษย (Human Genetics), พิมพครั้งที่ 2.” เท็กซ แอนด
เจอรนัล พับลิเคชั่น, กรุงเทพมหานคร, 2546.
บุญแสง, วิชัย., และคณะ., “ลายพิมพดีเอ็นเอ..จากสารพันธุกรรมสูเ ทคโนโลยีพิสจู นบุคคล,
พิมพครั้งที่ 2.” สํานักงานพัฒนาวิทยาศาสตร และเทคโนโลยีแหงชาติ, ปทุมธานี, 2545.
Gelehrter, T.D., Collins, F.S., Ginsburg, D., “Principles of Medical Genetics, 2nd edition.”
Williams & Wilkins, Maryland, 1998.
Lamb, N.E., “Crash course : Cell Biology and Genetics.” Mosby-Elsevier, Inc.,
Philadelphia, 2007.
Kumar, V., et al., “Robbins and Cotran Pathologic Basis of Disease, 7th edition.” Elsevier
Inc., Philadelphia, 2005.
Rimoin, D. L., editors, et al., “Emery and Rimoin’s Principles and Practice of Medical
Genetics, 4th edition.” Churchill Livingstone, London, 2002, volume 1.