genetic disorders and topics molecular diagnosis disorders and molecular diagnosis...

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Genetic disorders and Molecular diagnosis

นายแพทย ดร. ณตพล ศุภณัฐเศรษฐกุล

ภาควิชาพยาธิวิทยา คณะแพทยศาสตร

มหาวิทยาลัยนเรศวร

Email : [email protected]

http://www.uga.edu/srel/kidsdoscience/images/genetics-puppets.gif http://www.uwo.ca/biology/images/undergraduate/chromosome.jpg 27 พฤศจิกายน พ.ศ. 2556

Topics

Introduction Genetic disorders

Single gene disorders Chromosomal disorders

Multifactorial diseases Somatic cell genetic disorders

Molecular diagnosis

http://history.nih.gov/exhibits/nirenberg/images/photos/01_mendel_pu.jpg

http://www.scq.ubc.ca/wp-content/mendel.jpg

Gregor Mendel ค.ศ.1822-1884

http://tesla.desy.de/~rasmus/media/Coherence/PDFs%20and%20high%20resolution/watson%20and%20crick.jpg

James D. Watson 1928 -

Francis H.C. Crick 1916 - 2004

Structure of DNA 1953

Nobel prize in 1962

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http://i61.photobucket.com/albums/h53/mocost/watsoncrick.jpg http://www.sanger.ac.uk/Info/Press/gfx/030414_hgp_300.jpg

1990 –

2003

Structure of DNA

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

Nucleotide = Nucleoside + Phosphate Nucleoside = Ribose sugar + Nitrogenous Base

http://th.wikipedia.org/wiki/%E0%B9%84%E0%B8%9F%E0%B8%A5%E0%B9%8C:Nucleotides_1.svg

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“Central Dogma”

DNA RNA Protein

Translation Transcription

Replication

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%

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

http://tainano.com/Molecular%20Biology%20Glossary.files/image040.gif http://homepage.mac.com/enognog/cell%20cycle.jpg

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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

http://www.robbinspathology.com/content/figure.cfm?ImageSeqNo=112100&screenWidth=1280&screenHeight=800

Genetic disorders

Single gene disorders Mendelian inheritance

Autosomal dominant (AD) Autosomal recessive (AR) X-linked recessive (X-linked)

Non-Mendelian inheritance Chromosomal disorders

Numerical chromosomal abnormality Structural chromosomal abnormality

Polygenic or multifactorial diseases or complex genetic disorders Somatic cell genetic disorders (neoplasia)

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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://bio1151.nicerweb.com/Locked/media/ch14/14_06PhenotypeVsGenotype_L.jpg

http://naturalsciences.sdsu.edu/ta/classes/lab2.4/trait.jpg


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Terminology Locus = position of DNA sequences, or gene on a chromosome Allele = possible alternative form of a given gene DNA polymorphism = the occurrence in a population of two or more alternative genotypes Gene pool = the study of frequency of total alleles on a given locus Population genetics = the study of the genetic composition of populations

http://ghr.nlm.nih.gov/handbook/illustrations/chromosomallocation.jpg Cystic Fibrosis Transmembrane Regulator, CTFR

http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/C/Chromo7.gif

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

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


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

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http://www.geneticsandhealth.com/wp-content/pedigree%20image_2.gif http://www.migeneticsconnection.org/genomics/Family%20History/pedigreeSymbols.jpg

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 (AD) Autosomal recessive inheritance (AR) X-linked recessive inheritance (X-linked) X-linked dominant inheritance Y-linked (Holandic) inheritance

A “dominant phenotype” is one that is expressed in heterozygotes, whereas a “recessive trait” is expressed only in homozygotes If the expression of each allele can be detected in the presence of the other, the two alleles are termed “co-dominant”

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http://www.uic.edu/classes/bms/bms655/gfx/pedigree1.gif

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

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 Achondroplasia

Sporadic type and Inherited type (AD) Fibroblast growth factor receptor 3 (FGFR3) gene mutation Abnormality in cartilage formation Affected people have one normal copy of the FGFR3 gene and one mutant copy Two copies of the mutant gene (homozygous) are fatal

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

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

Marfan syndrome

Marfan syndrome Antoine Marfan, a french pediatrician, 1896 Fibrillin-1 (FBN1) gene mutation on chromosome 15 Fibrillin-1 is an extracellular matrix glycoprotein, secreted by fibroblasts Structural component of microfibrils in the formation of elastic fibers in connective tissue Skeletal abnormality, subluxation of the lens (ectopia lentis), CVS abnormalities (mitral valve prolapse, aortic aneurysm, aortic dissection)

Examples

Skeletal: Marfan syndrome, Achondroplasia, Osteogenesis imperfecta Nervous system: Huntington disease, Neurofibromatosis Urinary: Polycystic kidney disease (Adult) Gastrointestinal: Familial polyposis coli Hematopoietic: Von Willebrand disease Metabolic: Familial hypercholesterolemia, Acute intermittent porphyria

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

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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

If a carrier has an unaffected partner, there is a 50% chance of the children being carriers Only mating between heterozygotes (carrier) 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

http://healthnet.md.chula.ac.th/text/forum1/thalassemia2/picture/thalassemia.jpg

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Thalassemia Hemoglobinopathy Hemoglobin (Hb): iron-containing oxygen transport protein in the red blood cells Four globular protein subunits – Heme group containing Fe Two alpha(α) and two beta(β) subunits: [Heterotetramer; α2β2 = Hemoglobin A] Alpha thalassemia: HBA1 and HBA2 (16p13.3) Beta thalassemia: HBB (11p15.5)

ควรมีรูป Hb

http://gassama.myweb.uga.edu/hemoglobinmolecule.gif

Hemoglobinopathy Alpha thalassemia (decreased α chain) α chain mutation (4 loci) αα/αα, -α/αα, -α/-α, --/αα, --/-α, --/--

Beta thalassemia (decreased β chain) β chain mutation (2 loci) β/β, β/β0 , β/β+ , β+β+ , β0/β+ , β0/β0

Sickle-cell anemia β globin chain point mutation [missense mutation, Glu -> Val] Hemoglobin S (HbS) α2βS

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http://home.kku.ac.th/acamed/kanchana/p7.jpg

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http://www.microscopyu.com/galleries/pathology/images/sicklecell/sicklecellanemia40x02small.jpg

http://www.irondisorders.org/News/SickleCell.jpg

Example

Metabolic: Cystic fibrosis, Phenylketonuria, Hemochromatosis, Glycogen storage diseases, Galactosemia, Homocystinuria, Lysosomal storage diseases Hematopoietic: Thalassemia, Sickle cell disease Endocrine: Congenital adrenal hyperplasia Nervous: Friedreich ataxia

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)

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

Example

Musculoskeletal: Duchenne muscular dystrophy, Becker muscular dystrophy Blood: Glucose-6-phosphate dehydrogenase (G6PD) deficiency, Hemophilia A and B Metabolic: Diabetes insipidus, Lesch-Nyhan syndrome Nervous: Fragile-X syndrome (Atypical) Immune: Agammaglobulinemia

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

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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.transtutors.com/Uploadfile/CMS_Images/4026_Karyotype%20of%20human%20(Male).JPG


http://www.asklenore.info/miscarriage/bick/images/fig3.jpg

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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


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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)

Numerical Chromosome Abnormalities

Concern : Extra single chromosome, Missing single chromosome, Extra haploid sets Mechanisms

Polyploidy Trisomies Monosomies

Polyploidy

Fertilization by two sperm A diploid sperm (2n) due to failure in meiosis A diploid ovum (2n) due to failure in meiosis

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Trisomies (aneuploidy) Failure of separation (nondisjunction) of homologous chromosomes at meiosis I Failure of separation of chromatids in meiosis II Advancing maternal age is associated with increased incidence of trisomy

http://www.anselm.edu/homepage/jpitocch/genbio/nondisjunction.JPG

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


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Down’s syndrome

CVS : Endocardial cushion defect (Atrioventricular septal defect) most common GI : increase risk of Hirschprung’s diseases

(intestinal stenosis) Down’s syndrome facies Simian crease Gap between 1st and 2nd toes

http://www.suriyothai.ac.th/en/node/1005

http://upload.wikimedia.org/wikipedia/commons/c/c3/Trisomy21_graph.jpg http://cas.bellarmine.edu/tietjen/HumanBioogy/bills_developmental_abnormalities.htm

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http://www.robbinspathology.com/content/figure.cfm?ImageSeqNo=109400&screenWidth=1280&screenHeight=800 http://cas.bellarmine.edu/tietjen/HumanBioogy/bills_developmental_abnormalities.htm

Structural Chromosome Abnormalities

Result from breakage and limitations of DNA repair systems Mechanisms

Translocation Inversion Duplication Deletion and ring chromosome Isochromosome


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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 (cat-like cry) : Deletion of the region on 5p15.2 or the whole short arm of chromosome 5

III. Multifactorial Disorders

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

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

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

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Threshold model for Multifactorial disorders

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

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 Genetic disorders

Single gene disorders Mendelian inheritance

Autosomal dominant (AD) Autosomal recessive (AR) X-linked recessive (X-linked)

Non-Mendelian inheritance Chromosomal disorders

Numerical chromosomal abnormality Structural chromosomal abnormality

Polygenic or multifactorial diseases or complex genetic disorders Somatic cell genetic disorders (neoplasia)

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http://images.google.co.th/imgres?imgurl=http://www.web-books.com/eLibrary/Books/B0/B10/OPS/images/image023.jpg&imgrefurl=http://www.web-books.com/eLibrary/ON/B0/B10/23MB10.html&usg=__BPWTVs-v3b_lv3aPmVYlIh6BMlQ=&h=427&w=570&sz=26&hl=th&start=7&um=1&tbnid=XfWmaKv8PLBt0M:&tbnh=100&tbnw=134&prev=/images%3Fq%3Dtranslocation%2Bin%2Bleukemia%26ndsp%3D18%26hl%3Dth%26um%3D1

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

Southern blot analysis Northern blot analysis

Fluorescence In situ Hybridization (FISH) DNA Microarrays Cytogenetics

Karyotyping Spectral karyotyping (SKY analysis)

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

DNA Extraction

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

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http://users.ugent.be/~avierstr/principles/pcrcopies.gif http://oceanexplorer.noaa.gov/explorations/03bio/background/molecular/media/gel_plate_600.jpg http://universe-review.ca/I11-50-gelectrophoresis.jpg

DNA Gel Electrophoresis

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

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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

http://en.wikipedia.org/wiki/File:FISH_(Fluorescent_In_Situ_Hybridization).jpg http://www.nature.com/modpathol/journal/v18/n12/images/3800465f1.jpg

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DNA Microarrays

http://img.medscape.com/fullsize/migrated/430/330/cc430330.fig2.gif http://en.wikipedia.org/wiki/File:NA_hybrid.svg http://en.wikipedia.org/wiki/File:Affymetrix-microarray.jpg

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

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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

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.

genetic disorders and topics molecular diagnosis disorders and molecular diagnosis...

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