Lecture on moleular diagnosis

7/29/2019 Lecture on moleular diagnosis

1/75


2/75

MOLECULAR DIAGNOSISOF HEREDITARY DISEASE

Arthur S. Schneider, M.D.Department of Pathology

Chicago Medical School at

Rosalind Franklin Universityof Medicine and Science


3/75

MOLECULAR PATHOLOGY

rapidly growing subspecialty area of

pathology practice and investigations

major applications in:

neoplasia (leukemias, lymphomas, and solidtumors)

hereditary disorders and disease predispositions

responsiveness to pharmacologic agents

identification of infectious agents

forensic applications (crime lab, paternity)

others (list is growing rapidly)


4/75


5/75

HEREDITARY DISORDERSCOMMONLY DIAGNOSED BY

MOLECULAR METHODS adult polycystic kidney disease

achondroplasia

1-antitrypsin deficiency

canavan disease

Charcot-Marie-Tooth disease

congenital adrenal hyperplasia

cystic fibrosis


6/75


MOLECULAR METHODS Duchenne/Becker muscular dystrophy

factor V Leiden mutation

familial adenomatous polyposis

familial hypercholesterolemia

fragile X syndrome galactosemia


7/75


MOLECULAR METHODS Gaucher disease

hemophilia a and b

Huntington disease

Marfan syndrome

mitochondrial disorders

myotonic dystrophy

neurofibromatosis types 1 and 2


8/75


MOLECULAR METHODS ornithine transcarbamoylase deficiency

phenylketonuria

spinal muscular atrophy

spinocerebellar ataxia

sickle-cell disease


9/75


MOLECULAR METHODS Tay-Sachs disease

- and -thalassemia tuberous sclerosis

von Hippel-Lindau disease

many others


10/75

Online Mendelian Inheritance inMan

OMIM is major source for information on

molecular changes in Mendelian disorders

http://www.ncbi.nlm.nih.gov/omim/
http://www.ncbi.nlm.nih.gov/omim/http://www.ncbi.nlm.nih.gov/omim/


11/75

TYPES OF MUTATIONS INHUMAN GENETIC DISEASE

deletions and insertions

entire gene, entire exon, multiple bases,

repeat sequences, etc.

codon deletions and insertions (number of

bases is a multiple of 3)

frameshift mutations with premature

termination of translation (number of bases

deleted or inserted is not a multiple of 3)


12/75

SUBSTITUTIONS (POINTMUTATIONS)

nonsense mutations (e.g., TCA (serine)

to TAA (stop))

missense mutations (amino acid

substitutions)

conservative e.g. TCA (serine) to ACA

(threonine)

non-conservative e.g. TCA (serine) to CCA

(proline)


13/75

SUBSTITUTIONS (POINTMUTATIONS)

silent mutation e.g. TCA (serine) to TCC

(serine)

mutations affecting promoter

RNA splicing mutations (intron/exon

splice sites or cryptic sites)


14/75

ALTERATIONS IN NON-CODINGRNAS

inhibit translation of mRNAs into specific

proteins (see text, pages176 and 218)

microRNAs, (miRNAs)

long non-coding RNAS (lncRNAs) (very large

number: greatly exceeds number of coding

mRNAs

Small interfering RNAs (siRNAs)


15/75

CYSTIC FIBROSISDELETION OF AN ENTIRE

CODON one of the most common autosomal

recessive disorders

one in 22 Caucasians carries a mutation in

CFTR gene (more than 500 mutations)

defect in gene that encodes CFTR (cystic

fibrosis transmembrane conductance

regulator)


16/75

CYSTIC FIBROSISDELETION OF AN ENTIRE

CODON delta-F508 mutation in 70 percent of cases

deletion of three base pairs that code for

phenylalanine CFTR protein made by the cell but not

transferred to cell membrane


17/75

Normal DNA CF DNA

Ile . Ile . Phe . Gly . Val..ATC ATC TTT GGT GTT...

TT

G

T

G

G

T

T

T

CT

A

C

T

A

T

T

G

T

G

G

TT

A

C

T

A

Ile . Ile . Gly . Val.

.. ATC ATT GGT GTT...

DELTAF 508

MUTATION


18/75

Factor IX promoter

CTAATCGACCTTACCACTTTCACAATCTGCA

G

mutation

start of transcription

start oftranscription

POINT MUTATION IN PROMOTER


19/75

Normal HEXA allele

Tay-Sachs allele

CCAGGCTCTG gtaagggt.

CCAGGCTCTG ctaagggt.

nosplicing

normalsplicesite

POINT MUTATION AFFECTING

SPLICE-SITE


20/75

Asp - Asp - Ala - Lys -Arg -GlnGAT GAT GCC AAA CGA CAA

GAT GAT GCC AAA TGA CAAAsp - Asp - Ala - Lys -Stop

Normal allele

NF1 allele

NONSENSE MUTATION


21/75


22/75

FRAME-SHIFT WITH

PREMATURE TERMINATION OFTRANSLATION

ABO A allele Leu - Val - Val - Thr - Pro ..

CTC GTG GTG ACC CCT T

ABO O allele CTC GTG GTA CCC TT

Leu - Val - Val - Pro -

altered readingframe


23/75


24/75

SICKLE CELL ANEMIA

most common hereditary anemia inpersons of African lineage

change in codon 6 of -globin gene

GAG (glu) --> GTG (val)

abolishes a restriction site for MstII

readily diagnosed by PCR or Southern blot

PCR reaction can be used


25/75

SICKLE CELL ANEMIA


26/75

1 2 3 4 5 6 7Val - His - Leu - Thr - Pro -Glu -Glu

GTG CAC CTG ACT CCT GAG GAG

GTG CAC CTG ACT CCT GTG GAGVal - His - Leu - Thr - Pro -Val -Glu

Abeta

globin

Sbetaglobin

MISSENSE MUTATION

SICKLE CELL ANEMIA

H l bi S


27/75

Pro . Glu . GluCCT GAG GAG

Pro . Val . GluCCT GTG GAG

1.15 kb 0.2kb

1.35 kb

A beta-globin

S beta-globin

Hemoglobin SLoss of restriction site


28/75

HUNTINGTON DISEASE

autosomal dominant inheritance severe neurological disorder

motor, cognitive, and psychiatric

manifestations characterized by

involuntary movements, mental

deterioration, and death after 5-20 years


29/75

HUNTINGTON DISEASE

progressive neurodegeneration withneuronal depletion mainly in striatum

(caudate nucleus and putamen) and

frontal cortex

delay of clinical abnormalities until age

30-40


30/75

HUNTINGTON DISEASE

increased numbers (more than 11-34)of CAG trinucleotide repeats in HD

(huntingtin) gene on tip of short arm of

chromosome 4 (4p16.3)


31/75

HUNTINGTON DISEASE

paternal transmission results inincreased number of CAG repeats and

earlier onset of disease in successive

generations (anticipation)


32/75

HUNTINGTON DISEASE

CAG trinucleotide repeat expansioncodes for expanded polyglutamine tract


33/75

HUNTINGTON DISEASE

gain of toxic function as a result of anexpanded polyglutamine tract can cause the

protein huntingtin to interact abnormally with

a variety of proteins, resulting in the complexof neuropathological changes seen in

Huntington's disease


34/75

HUNTINGTON DISEASE

several huntingtin-interacting proteinsmight be associated with normal

function of huntingtin and/or involved in

the pathology of Huntington's disease


35/75

HUNTINGTON DISEASE

CAG repeats common to at least nineinherited neurodegenerative diseases.


36/75

HUNTINGTON DISEASE

Since these diseases show distinctneuropathological changes, it has been

suggested that protein environment and

protein-protein interactions may play an

important role in the specific

neuropathology of these diseases


37/75

PRENATAL DIAGNOSIS

techniques

cytogenetics

FISH for trisomies, etc.

molecular analysis

specimen sources

amniotic fluid cells

chorionic villus sampling

umbilical cord blood


38/75

MOLECULAR TECHNIQUES

List of new techniques is rapidly increasing

PROCEDURES UTILIZING


39/75

PROCEDURES UTILIZINGGENOMIC DNA

electrophoretic and blotting techniques

restriction analysis and southern blotting

amplification techniques

polymerase chain reaction, ligase chain

reaction, several others

DNA sequencing methods

manual and automated sequence analysis

SOURCES OF HUMAN


40/75

SOURCES OF HUMANGENOMIC DNA

blood and other body fluids oral swabs and washings

amniotic fluid cells, chorionic villus sampling,and umbilical cord blood

urine sediment freshly isolated tissue samples in frozen surgical

specimens

preserved tissue samples (including formalin-

fixed/paraffin-embedded tissues) cultured cells

forensic samples (clotted blood, hair, semen)


41/75

SOUTHERN BLOT

identifies gene fragments and

demonstrates molecular size of

fragment

multistep procedure

digestion

electrophoresis

southern transfer

hybridization

detection


42/75

http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/R/RestrictionEnzymes.html

RESTRICTION ENZYMES

Southern Blot


43/75

Southern Blot

Restriction enzyme

DNA ofvarious sizes

Electrophorese on agarose gel

gel

Denature - transfer tofilter paper.

blot

http://www.asip.org/edu/hs/Power%20of%20MBT.ppt


44/75

Hybridize to probe

Visualize

Denature- transfer tofilter paper.

blot



45/75

Southern Blot



46/75


47/75


48/75

southern blot -- DNA

major deletions, insertions

mutations affecting restriction sites

northern blot -- RNA

measure of gene expression

western blot -- proteins

specific antibody used as probe

ALLELE SPECIFIC


49/75

ALLELE SPECIFICOLIGONUCLEOTIDE

HYBRIDIZATION detects single nucleotide alterations

useful for mutations that do not causealterations of restriction sites

ALLELE SPECIFIC


50/75

ALLELE SPECIFICOLIGONUCLEOTIDE

HYBRIDIZATION oligonucleotide probes

minor mismatch detected bydestabilization of complex of probe and

target DNA

vary temperature or salt concentration to

detect destablization


51/75

ALLELE-SPECIFIC

OLIGONUCLEOTIDEHYBRIDIZATION

ASOH IN CYSTIC FIBROSIS


52/75

Normal

allele

CF

allele

ASOH IN CYSTIC FIBROSIS


53/75

IN-SITU HYBRIDIZATION

visual identification of sequences in tissue

sections

localization of genes on chromosome

important widely used variation is

fluorescent in situ hybridization (FISH)


54/75


55/75

RESTRICTION FRAGMENT


56/75

RESTRICTION FRAGMENTLENGTH POLYMORPHISMS

(RFLP's) genetic linkage to DNA "marker"

sequences

based on DNA polymorphisms

approximately one in every 200 to 500 bp is a

heritable polymorphism

most in non-coding areas

create or abolish restriction sites



57/75


(RFLP's)

fragments of differing lengths reflectpolymorphisms (RFLP's)

proper combination of restriction endonuclease

and probe for a sequence physically "near" genepermits gene "tracking"



58/75


(RFLP's) useful for diagnosis

facilitates localization of unknown genes



59/75


(RFLP's) requires samples from individuals

displaying the genetic phenotype andkey family members

reliability increased the nearer the

marker sequences to the gene ofinterest


60/75

RFLP ANALYSIS

VARIABLE NUMBER OF TANDEM


61/75

Restrictionsite

Restrictionsite

Restrictionsite

Restrictionsite

Restrictionsite

VARIABLE NUMBER OF TANDEMREPEATS

SINGLE NUCLEOTIDE


62/75

SINGLENUCLEOTIDEPOLYMORPHISMS (SNPs)

much more common than RFLPs

occur throughout genome

used in similar fashion to RFLPs

now considered to be marker of choice


63/75

DNA SEQUENCE ANALYSIS

chemical degradation or chain termination multiple fragments of DNA terminating

alternatively at either G, A, T, or C

gel resolution of fragments differing byonly one nucleotide


64/75

DNA SEQUENCE ANALYSIS

four separate lanes each representing oneof the G, A, T, or C termination sites

read nucleotide sequence directly

definitive tool for identifying sequencevariations

DIDEOXY CHAIN TERMINATION


65/75

DIDEOXY CHAIN TERMINATION

SEQUENCING

Enzymatic

Elongation

Reactions

C

G

G

A

T

C

GA

T

A

T

ddGTP

dNTPs

ddATP

dNTPs

ddCTP

dNTPs

ddTTP

dNTPs


66/75


67/75


68/75


69/75

POLYMERASE CHAIN


70/75

POLYMERASE CHAINREACTION (PCR)

oliogonucleotide primers bracket desiredarea of amplification

POLYMERASE CHAIN


71/75


synthesis of new complementary strandsmediated by heat resistant DNApolymerase

multiple cycles of annealing, extension,and denaturation

controlled by sequentially raising and loweringtemperature

exponential amplification, twenty-five cyclestheoretically amplifies DNA segment 106 fold

POLYMERASE CHAIN


72/75


permits use of extremely small samples

prior DNA purification not required

facilitates use of molecular techniques in

clinical setting


73/75

RT-PCR

variation of PCR using RNA as starting

material

RNA is first converted to c-DNA

(complementary DNA) by treatment withreverse transcriptase

Then PCR is performed

PCR


74/75

3

3

primers

DNA synthesis

5 35

3

5

5

3 5

5 3

PCR


75/75

Documents

Lecture on moleular diagnosis