Genetic and Molecular อ.สู้

9/12/2011

1

Genetic Disorders and Molecular Diagnosis

นายแพทย ดร. ณตพล ศุภณัฐเศรษฐกุล

ภาควิชาพยาธิวิทยา และนิติเวชศาสตร

คณะแพทยศาสตร มหาวิทยาลัยนเรศวร

Email : [email protected]

http://www.uga.edu/srel/kidsdoscience/images/genetics-puppets.gif http://www.uwo.ca/biology/images/undergraduate/chromosome.jpg 13 กันยายน พ.ศ. 2554

Topics

The Role of Genetics in Medicine Genes and Chromosomes Genetic disorders

Single gene disorders Chromosomal disorders

Polygenic or multifactorial diseases Somatic cell genetic disorders

9/12/2011

2

Clinical genetics and Molecular Diagnosis Genetic testing for carrier detection Prenatal diagnosis Newborn screening Molecular diagnosis Genetic counseling

Topics

The Role of Genetics in Medicine

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

9/12/2011

3

http://ghr.nlm.nih.gov/handbook/illustrations/chromosomallocation.jpg

Genes and Chromosomes

Structure of DNA Central dogma Chromosomes Gene Cell cycle and cell divisions :

mitosis meiosis

Mutation

9/12/2011

4

Structure of DNA

http://academic.brooklyn.cuny.edu/biology/bio4fv/page/molecular%20biology/16-05-doublehelix.jpg

“Central Dogma”

DNA RNA Protein

Translation Transcription

Replication

9/12/2011

5

Chromosomes

http://infohost.nmt.edu/~klathrop/DNA%20Coiling.jpg

Human Genome (chromosomes)

In Nucleus 3.1 x 109 bp

In Mitochondria 16,569 bp

Gene

Coding Sequences

(Exon)

Non coding Sequences

(Intron)

Non-gene (Extragenetic)

Repetitive DNA

Tandem repeats (satellite, minisatellite, microsatellite)

Interspersed repeats (SINES, LINES)

Non repetitive DNA

10% 90%

<10% >90% 50% 50%

9/12/2011

6

Cell cycle

http://www.cbp.pitt.edu/faculty/yong_wan/images/main_cell_cycle.jpg

http://ghs.gresham.k12.or.us/science/ps/sci/soph/cells/cycle/cycle.gif

Cell division

Mitosis Meiosis and gametogenesis Crossing over

9/12/2011

7

http://www.uhh.hawaii.edu/~ronald/392/Mitosis.JPG

http://www.mun.ca/biology/desmid/brian/BIOL2060/BIOL2060-20/2010.jpg

9/12/2011

8

Mutation หมายถึงกระบวนการที่กอใหเกิดการเปลี่ยนแปลง

ทุกรูปแบบ ที่เกิดขึ้นกับโมเลกุลของ DNA

“Wild type” and “mutant allele” Normal variation, polymorphism, inherited disease Types of mutation

Base substitution => silent mutation, missense, nonsense Insertion => frameshift mutation Deletion => frameshift mutation Chromosomal abnormality

http://www.layevangelism.com/bastxbk/images/mutation.jpg

9/12/2011

9

Genetic disorders

Single gene disorders

Mendelian inheritance Non-Mendelian inheritance

Chromosomal disorders Polygenic or multifactorial disorders Somatic cell genetic disorders

I. Single gene disorders

Terminology Mechanism of mutation Functional effects of mutation on protein Mendelian inheritance of single gene disorders

9/12/2011

10

Terminology 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

9/12/2011

11

Terminology Locus = position of DNA sequences, or gene on a chromosome Allele = possible alternative form of a given gene

http://www.geneticsandhealth.com/wp-content/allele.jpg

9/12/2011

12

http://www.genome.gov/Pages/Hyperion/DIR/VIP/Glossary/Illustration/Images/autosome.gif

http://www.geneticsandhealth.com/wp-content/pedigree%20image_1.gif

9/12/2011

13

http://www.geneticsandhealth.com/wp-content/pedigree%20image_2.gif

http://www.migeneticsconnection.org/genomics/Family%20History/pedigreeSymbols.jpg

9/12/2011

14

Introduction

Single gene disorders are caused by individual mutant genes There are approximately 11,000 single gene disorders, affecting 1% of the populations Follow Mendelian patterns of inheritance

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

9/12/2011

15

Autosomal Dominant

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

Achondroplasia Acute intermittent porphyria Familial hypercholesterolemia Marfan syndrome Von Willebrand disease

9/12/2011

16

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

Achondroplasia

http://www.nhlbi.nih.gov/health/dci/images/Marfan_lowres.gif

http://www.homoeopathiker.de/homoeopathik/syphilis/bilder/marfan3.jpg

Marfan syndrome

9/12/2011

17

http://www.uic.edu/classes/bms/bms655/lesson5.html

Autosomal Recessive

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

9/12/2011

18

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

9/12/2011

19

Example

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

9/12/2011

20

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

9/12/2011

21

http://www.uic.edu/classes/bms/bms655/gfx/pedigree5.gif

X-linked Dominant

Rare, and difficult to distinguish from AD except that affected males have normal sons, but all daughters are affected Example : X-linked hypophosphatemic rickets, Rett syndrome

9/12/2011

22

http://www.uic.edu/classes/bms/bms655/lesson7.html#RECESSIVE

X-linked Recessive

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)

9/12/2011

23

Example

Duchenne muscular dystrophy Becker muscular dystrophy Glucose-6-phosphate dehydrogenase (G6PD) deficiency Hemophilia A Hemophilia B Lesch-Nyhan syndrome

http://www.neurologyarticles.com/sitebuilder/images/Scan10106-176x315.jpg

Gower’s sign

http://medicalimages.allrefer.com/large/x-linked-recessive-genetic-defects.jpg

Duchenne muscular dystrophy

9/12/2011

24

Y-linked (Holandic)

http://cas.bellarmine.edu/tietjen/HumanBioogy/Finished%20Images/gen17.gif http://cas.bellarmine.edu/tietjen/HumanBioogy/bills_developmental_abnormalities.htm

II. Chromosomal Abnormalities

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

9/12/2011

25

Terminology 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

9/12/2011

26

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

9/12/2011

27

http://www.nature.com/onc/journal/v24/n33/images/1208711f1.jpg

Spectral Karyotyping (SKY) Analysis

Introduction 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)

9/12/2011

28

Examples Autosomal disorders :

Trisomy 21 (Down syndrome) Trisomy 18 (Edwards syndrome) Trisomy 13 (Patau syndrome)

Sex chromosome disorders : 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

9/12/2011

29

http://cas.bellarmine.edu/tietjen/HumanBioogy/bills_developmental_abnormalities.htm

http://www.virtualsciencefair.org/2004/mcgo4s0/public_html/t2/dwn_chromo.gif

9/12/2011

30

http://www.robbinspathology.com/content/figure.cfm?ImageSeqNo=108200&screenWidth=1280&screenHeight=800


9/12/2011

31



9/12/2011

32

http://www.biology.iupui.edu/biocourses/n100/images/XYYMale.gif

http://modculture.typepad.com/photos/uncategorized/2007/03/14/xyy.jpg

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.

Structural Chromosome Abnormalities

Result from breakage and limitations of DNA repair systems Mechanisms

Translocation Inversion Duplication Deletion and ring chromosome Isochromosome

9/12/2011

33


http://cas.bellarmine.edu/tietjen/HumanBioogy/Finished%20Images/gen30.gif

mental retardation, low birth weight, low set ears, cat-like cry

Cri du chat syndrome

9/12/2011

34

http://www.cityofhope.org/PublishingImages/Research/Shared%20Resources%20-%20Equipment/Cytogenetics%20Core%20Laboratory/abnormal_karyotype_Large.jpg

III. Multifactorial Disorders

Terminology Multifactorial disorders Threshold model of multifactorial disorders Examples of multifactorial disorders

9/12/2011

35

Terminology

Polygenic inheritance : the inheritance of traits that are influenced by many genes at different loci Multifactorial disorder : disorder in which both environmental and genetic factors are important

Multifactorial Inheritance

Normal human characteristics : Blood pressure, height, finger ridges, and intelligence Congenital malformations : neural tube defects, cleft lip and palate, and congenital heart disease Common disorders of adult life : DM, Hypertension, peptic ulcer, and schizophrenia

9/12/2011

36

Heritability and Environmental factors

Heritability : percentage denoting that the genetic contribution of a given disease

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

http://www.uic.edu/classes/bms/bms655/gfx/figure18.gif (Tendency)

9/12/2011

37

Example

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

9/12/2011

38

Clinical genetics and Molecular Diagnosis

Genetic testing for carrier detection Prenatal diagnosis Newborn screening Molecular diagnosis Genetic counseling

Genetic Testing for Carrier Detection

To identify asymptomatic heterozygotes for AR traits, or AD disorders that have limited penetrance or late onset Confined to small ethnic populations in which there is an anomalously high incidence of a particular disease Examples : Thalassemia, Cystic fibrosis, ADPKD, Cascade screening

9/12/2011

39

Prenatal diagnosis

To assure of having unaffected children when the risk of having an affected child is unacceptably high NOT equivalent to the assurance of having normal children, It does not address all possible birth defects Noninvasive techniques : ultrasonography, maternal serum screening Invasive technique : amniocentesis, chorionic villus sampling, cordocentesis DNA analysis (PCR, Southern blot), Cytogenetic analysis

http://www.nlm.nih.gov/medlineplus/ency/images/ency/fullsize/1062.jpg

9/12/2011

40

http://www.pennhealth.com/health_info/pregnancy/graphics/images/en/19177.jpg http://www.pennhealth.com/health_info/pregnancy/graphics/images/en/19175.jpg

Cordocentesis

http://www.nlm.nih.gov/medlineplus/ency/images/ency/fullsize/19163.jpg http://www.pennhealth.com/health_info/pregnancy/graphics/images/en/19166.jpg

Chorionic villi sampling

9/12/2011

41

Newborn screening

To identify treatable genetic disorders such as phenylketonuria, galactosemia, maple syrup urine disease, and congenital adrenal hyperplasia in newborn infants Dietary management is usually an early intervention to eliminate clinical symptoms that would otherwise lead to severe disability, mental impairment, or dealth Prevalence, severity of a condition, availability, effectiveness of a treatment, and cost will determine which disorder will be screened

9/12/2011

42

http://www.health.state.mn.us/divs/fh/mcshn/ncfu/images/pkupath.gif

Molecular techniques DNA , RNA Extraction Gel Electrophoresis Polymerase Chain Reaction (PCR) Direct Hybridization Method

Southern blot analysis Northern blot analysis

Fluorescence In situ Hybridization (FISH) DNA Microarrays Cytogenetics

Karyotyping Spectral karyotyping (SKY analysis)

9/12/2011

43

http://www.favorgen.us/sitebuilder/images/Blood_Genomic_DNA_Mini_chart-437x463.jpg

DNA Extraction

http://oceanexplorer.noaa.gov/explorations/03bio/background/molecular/media/gel_plate_600.jpg http://universe-review.ca/I11-50-gelectrophoresis.jpg

DNA Gel Electrophoresis

9/12/2011

44

Polymerase Chain Reaction (PCR)

http://www.obstbau-kompetenzzentrum.de/arbeitsbereiche/Folder.2006-11-07.1358/Thermocycler_.jpg http://www.bio.miami.edu/~cmallery/150/gene/c7.20.7.pcr.jpg

Taq DNA Polymerase

http://users.ugent.be/~avierstr/principles/pcrcopies.gif

9/12/2011

45

http://images.google.co.th/imgres?imgurl=http://users.ugent.be/~avierstr/principles/pcrcopies.gif&imgrefurl=http://users.ugent.be/~avierstr/principles/pcr.html&usg=__kCFieOdf4YXbyVmdNjY2uR9bquI=&h=306&w=598&sz=14&hl=th&start=9&um=1&tbnid=3Ku-pvmv7CJ2gM:&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

9/12/2011

46

Blot Detection Method 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

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

9/12/2011

47

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

9/12/2011

48

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

9/12/2011

49

http://www.virtualsciencefair.org/2004/mcgo4s0/public_html/t2/dwn_chromo.gif

Cytogenetics : Karyotyping

Spectral Karyotyping (SKY) Analysis

http://www.cityofhope.org/PublishingImages/Research/Shared%20Resources%20-%20Equipment/Cytogenetics%20Core%20Laboratory/HumanSKY_Large.jpg

9/12/2011

50

Genetic Counseling

Indications for genetic counseling Information conveyed in genetic counseling

http://www.gen.vcu.edu/images/counseling.jpg

Indications for Genetic Counseling

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

9/12/2011

51

Information Conveyed in Genetic Counseling

The magnitude of the risk of occurrence or recurrence The impact of the disease on the patient and the family The possibility of modification of either the impact or the risk Anticipated future development

References คัมภิรานนท, อมรา., “พันธุศาสตรมนุษย (Human Genetics), พิมพครั้งที่ 2.” เท็กซ แอนด เจอรนัล พับลิเคชั่น, กรุงเทพมหานคร, 2546.

บุญแสง, วิชัย., และคณะ., “ลายพิมพดีเอ็นเอ..จากสารพันธุกรรมสูเทคโนโลยีพิสูจนบุคคล, พิมพครั้งที่ 2.” สํานักงานพัฒนาวิทยาศาสตร และเทคโนโลยีแหงชาติ, 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.

Documents

Genetic and Molecular อ.สู้