2000MSC Molecular Genetics Lecture Notes 2015
Underg Undergrad raduat uate e Cours Course, e, School School of Medica Medicall Scienc Science, e, Grith Grith University, University, Gold Coast campus, Queensland, Australia.
1
TOPIC 1 MENDELIAN GENETICS 1.0 INTRODUCTION Genetics is the study of heedity heedity. Thee !"in #"nches$
1. Transmission genetics: the the study of transmission transmission of traits traits from generation to generation. Pioneered by Gregor Mendel in 1865 2. Molecular Molecular Genetic Genetics: s: the study study of the composit composition ion and role role of gene ge neti tic c mate materi rial al ba base sed d on th the e cent centra rall do dogm gma a th that at !" !" is th the e temp templa late te for for #!" #!" $hic $hich h en enco code dess prot protei eins ns.. This This e%pl e%plai ains ns th the e transmission of traits &rst described by Mendel. '.
Popu opulat lation ion Genetic Genetics: s: the study study of (ariat (ariation ion and e(oluti e(olution on in
populations. )e *no$ that !" accumulates changes +mutations, $hic $hich h resu result lt in ph phen enot otyp ypic ic (ari (ariat atio ion n be bet$ t$ee een n indi indi(i (idu dual alss in a populatio population. n. !atural !atural selection selection can then then act on that (ariat (ariation ion and the population $ill change o(er time- ie the population $ill e(ol(e.
% E
S&OULD AL% AL%A'S (EEP IN MIND T&E RELATIONS&IP
)ET%EEN T&E T&REE )RANC&ES
1.1 MENDELS E*PERIMENTS (Refer text chapter 3, pages 43-55)
•
Mendel 1856
•
rossed pea plants / easy to gro$- crossbred arti&cially- gro$s to maturity in one season
•
0sed (isible features +traits,- each $ith 2 (isible alternate forms +phenotypes,- used purebreeding strains
2
3Ple 3Pleas ase e re(i re(ise se ho ho$ $ chro chromo moso some mess mo(e mo(e in meio meiosi siss to prod produc uce e gametes: try youtube.com4$atch(79f youtube.com4$atch(79f;%au"< ;%au"<
1.1.1 MENDEL+S O)SER,ATIONS O- PLANTS %IT& ONE TRAIT The Monohy#id Coss
•
rossing t$o purebreeding plants resulted in one phenotype in the =1 progeny
>g.. plan >g plantt he heig ight ht tall tall
•
%
shor shortt
tall
nbr nbree eedi ding ng =1 prog progen eny y +sel +selff fert fertil ilis isat atio ion, n, resu result lted ed in t$o t$o phenotypes in the =2 generation
•
The The ph phen enot otyp ype e seen seen in th the e =1 prog progen eny y $as $as ' time timess more more common in the =2 progeny than the other phenotype
•
$1 /henoty/ic "tio 1$$1 2enoty/ic "tio
3
•
This led to Mendel?s &rst ' postulates
1.1. MENDELS -IRST POSTULATES
1. 0nit factors e%ist in pairs +unit factors control genetic characters4traits, •
Prior to this- thought that one per trait $ith parental traits blending in o@spring
•
=actors separate into germ cells +obser(ed traits of seedsA round4$rin*led- green4yello$ etc,
•
=ertilisation results in indi(iduals $ith one factor from each parent
2. ominance4#ecessi(eness •
n the pair of unit factors for a single trait in an indi(idual- if each is unli*e the other- one unit factor is dominant and the
4
other is recessi(e . L"3 of Se2e2"tion$ •
In the fo!"tion of 2"!etes4 the t3o f"ctos fo "ny t"it se/""te 5se2e2"te6 "ndo!7y4 so th"t e"ch ends u/ in " di8eent 2"!ete.
•
Se/""tion of f"ctos 2i9es 2"!etes 3ith e:u"7 ch"nce of e"ch f"cto due to "ndo! union of 2"!etes
1.1. MENDEL+S O)SER,ATIONS O- PLANTS %IT& T %O TRAITS The Dihy#id Coss
•
To see if the la$ of segregation applied to more than one traitMendel crossed plants that $ere purebreeding +dominant and recessi(e, for t$o traits eg. ;eed colour and shape
•
•
Be found: o
=1 / one phenotype of each separate trait
o
- ; <$$$1 /henoty/ic "tio
=2 ratio due to combination of t$o independent ':1 ratios
This may be done $ith a Punnett sCuare to allo$ the genotypes and phenotypes from a cross to be (isualised easily +#eg. Punnett ambridge- founded ournal of Genetics 1D1E- de(eloped Punnet s$Cuare 1DE5- disco(ered lin*age 1DE6,.
5
< di8eent 2enoty/es4 = /henoty/es in " <$$$1 "tio •
This led to Mendel?s second la$: The Fa$ of ndependent "ssortment
MEIO1.1.= L A% O- INDEPENDENT A SSORTMENT
•
;egregation of t$o factors for a trait are independent of the 6
segregation of other factor pairs in the formation of gametes •
Indi9idu"7
!e!#es
of
/"is
of
"77e7es
se2e2"te
inde/endent7y of one "nothe duin2 2"!ete fo!"tion
>g.
"ab )here " and a alleles separate independently of and b alleles
"
•
"b
a
ab
This gi(es H possible types of gametes- therefore if you cross H gametes $ith another H gametes
you get 16 possible
combinations CONCLUSIONS
•
Mendel determined la$s that demonstrate inheritance of traits
•
L"3 of se2e2"tion$ t3o f"ctos /e ce77 fo " /"ticu7" t"it se/""te inde/endent7y into 2"!etes
•
L"3 of inde/endent "ssot!ent$ se/""tion of /"is of "77e7es "e inde/endent of e"ch othe
•
Mendel had no *no$ledge of chromosomes or genes
•
0sed maths and probabilities to determine la$s
•
"lso persistence- repetition to (erify results and logic to reach conclusions
•
)e can correlate MendelIs Postulates $ith the beha(iour of chromosomes during meiosis
7
)ere his results too good •
Fuc* / did not use lin*ed genes or incomplete dominance
•
1D'6 / =isher e(aluated Mendel?s data to determine if Mendel $as neutral or obJecti(e in his studies or had his conclusions in mind
•
=ound that results $ere closer to e%pected freCuencies than should ha(e been
•
Foo*ing at $hole series of e%periments- there $as a 141HEEE chance of getting results as Mendel had claimed
•
Bo$e(er- others repeated- gi(ing ratios similar if not better than Mendel?s
8
•
0nconscious bias >%perience modifying e%pectations ;ome subJecti(e interpretation =isher $as incorrect Medel?s paper abbre(iated
1. TERMINOLOG' TERMINOLOG' I
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Genes: Mendel?s hereditary factorsA segment of !" specifying #!" or polypeptide
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"lleles: alternate forms of a gene- Knormal? $ildtype o
!: "lleles are not restricted to t$o- there may be many alleles for a gene
•
Genotype: genetic constitution
•
Phenotype: characteristics that result from genotype and en(ironment o
dentical t$ins: same genes- therefore di@erent phenotype from en(ironment
•
Focus: position of gene on chromosome
•
iploid: t$o copies of a gene +t$o sets of chromosomes,
•
Baploid: one copy of a gene +one set of chromosomes,
•
Bomologous chromosomes: same genes- may be di@erent alleles
•
Bomo9ygote: genotype $ith same allele +eg. "",
•
Betero9ygote: genotype $ith di@erent alleles +eg. "a,
TERMINOLOG' II
•
Purebreeding: parent that produces only one progeny li*e themsel(es
•
=1: &rst &lial generation
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•
=2: formed by crossing $ith = 1
•
Testcross: cross of hybrid = 1 $ith homo9ygous recessi(e to determine un*no$n genotype
•
ac*cross: cross of hybrid = 1 $ith parent o
•
!: testcross is a type of bac*cross
Monohybrid: an organism $ho is hetero9ygous at one gene locus "a
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ihybrid: an organism $ho is hetero9ygous at t$o gene loci "ab
CON,ENTIONS •
Genes on di@erent chromosomes: "abcd
•
Fin*ed genes:
"
abcd
•
ndicating haplotypes or arrangement of lin*ed alleles
•
apital letter for dominant genes
•
;mall letter for recessi(e genes
1. TESTCROSSES " testcross may be used to detect the genotype of an indi(idual $ho e%presses a dominant trait. >g. Bo$ $ould you determine if a tall plant is or d =or a testcross- cross $ith homo9ygous recessi(e to disco(er genotype of un*no$n 1..1 TESTCROSS % IT& ONE TRAIT 10
•
#esults gi(e un*no$n genotype
1.. TESTCROSS % IT& T %O TRAITS
n this case- it may be used to detect the genotype of an indi(idual $ho e%presses t$o dominant traits ""
%
aabb
+",
+ab, gametes
"ab
%
aabb
+",+"b,+a,+ab,
+ab, gametes
"ab 1
:
"abb 1
aab :
1
:
aabb 1
11
H phenotypes- H genotypes- in eCual ratios $hen the un*no$n is a dyhybrid ie "ab f un*no$n is ""- then 1 phenotype and 1 geneotype ie "ab 3o the Punnet sCuare<
1.= T&E -OR(ED>LINE •
MET&OD -OR -1 * -1 CROSSES
onsider that each gene pair mo(es separately during gamete formation
•
n "a % "a cross += 1 generation, $e *no$ that L $ill ha(e yello$ and $ill ha(e green seeds and in b % b cross $e *no$ that L $ill ha(e smooth and $ill ha(e $rin*led seeds
•
The product la$ is applied ie. $hen 2 independent e(ents occur simultaneously- the probability of the t$o outcomes occurring in combination is eCual to the product of their indi(idual probabilities of occurrence
12
•
The for*ed line method can also be used for a trihybrid crosses. Study text gure 3.9
1.? E*TENSIONS
O- MENDELISM
(Refer text chapter 4, pages 74-86)
•
>%tensions of Mendelian analysis include: o
that a gene can e%ist in many di@erent allelic states
o
that a particular gene can a@ect se(eral di@erent traits
o
that a particular trait can be a@ected by se(eral di@erent genes
o
genetic and en(ironmental factors inNuence phenotypic (ariation
COMPLETE DOMINANCE
•
Phenotype of homo9ygote same as that of a hetero9ygote o
>g. PO0 +Phenyl*etonuria,
o
o(erproduction of phenylpyru(ic acid +in PO0 patients,
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due to inacti(e phenylalanine hydro%ylase o
uild up of phenylalanine leads to mental retardation
o
ue to alleles of en9yme phenylalanine hydro%ylase
o
Bomo9ygote dominant: Phy Phy +PP, / una@ected
o
Betero9ygote: Phy phy +Pp, / una@ected
o
Bomo9ygote recessi(e: phy phy +pp, a@ected
P ARTIAL 5INCOMPLETE 6 DOMINANCE
•
Betero9ygote sho$s intermediate phenotype
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>g. ;napdragons
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!e$ phenotype seen in hetero9ygote
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!ot seen in Mendels? results
CO>DOMINANCE
•
oth phenotypes in hetero9ygote
•
>g. " human blood groups
•
ue to en9yme that adds sugar to blood group antigens on #
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" / !acetyl sugar
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/ 0nacetylated sugar
•
/ !o sugar
•
"lleles: " - - i
14
•
lood groups: "- - "-
•
Genotypes: " " - " iA - i A " - ii
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" / " indi(iduals produce " and antigens
Epistasis •
Gene interactions
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Phenotype of one gene inNuenced by genotype of one or more other genes
•
#esults in (ariations from simple Mendelian ratios
•
May be dominant or recessi(e epistasis- di@erent ratios for each
•
nteraction of genes / changes simple Mendelian ratios
•
>g.
Pigment in (iolets- eg of recessi(e epistasis $here dd is
epistatic to G and g Pin* D
g
/ acti(e en9yme d / inacti(e en9yme
#ed
/ en9yme g Q pin* G
/ en9yme G Q
$hite )hite GG
%
dGg
ddgg
%
dGg
7G7 : 7gg : dd7 7 D
:
'
: H
15
Penetrance •
ncomplete penetrance: some indi(iduals $ith a particular genotype do not al$ays e%press the associated phenotype
•
omplete penetrance: genotype al$ays e%pressed in associated phenotype 1EER penetrance- incomplete penetrance is $hen the genotype is e%pressed in the associated phenotype S1EER of the time
Variable Expressivity •
Genes e%pressed to di@erent degrees in di@erent indi(iduals
•
Buman disease- se(ere to mildly a@ected $ith same disease gene
•
>g. Polydactyly
Pleiotropy •
Multiple phenotypic e@ects from single defecti(e gene
•
>g 1. Porphyria (ariegate: buildup of porphyrins in the brain due to inadeCuate metabolism of haemoglobin porphyrins
•
=eatures include abdominal pain- fe(er- muscular $ea*nessracing pulse- insomnia- headaches- (ision problems- deliriumcon(ulsions- photosensiti(ity
•
>g. 2. Marfan syndrome =ibrillin1 mutation that a@ects many organs
Polygenic raits •
More than one gene contributes to one phenotypic trait eg. eye
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colour •
ften en(ironmental inNuence eg. human height- $eight- s*in colour
•
uantitati(e traits are polygenic
Let!al "lleles •
May be recessi(e or dominant
•
>g. Buntington?s disease
•
May change simple Mendelian ratios
1.@ UANTITATI,E IN&ERITANCE •
ontinuously (arying traits such as height in humans are inNuenced by a large number of genes- each segregating di@erent allelesA termed polygenic
•
These traits are also often inNuenced by a host of en(ironmental factors- each ha(ing a small e@ect
•
This combination of genetic and en(ironmental factors often creates a $ide range of phenotypes- described in Cuantitati(e terms ie. uantitati(e inheritance
•
e.g. height in humans- genetic U en(ironment such as nutrition
1.B PEDIGREE A NAL'SIS
•
an deduce modes of inheritance and genotypes of indi(iduals by loo*ing at pedigrees
•
;tandard con(entions to represent pedigrees
17
1.B.1 MODES O- IN&ERITANCE
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"utosomal / not on se% chromosomes
•
Vlin*ed / (ery rare
• •
;e%lin*ed / generally refers to Wchromosome "lso note (ariations $ithin the normal categories due to epistasis-
(ariable
e%pressi(ity-
incomplete
penetrance-
pleiotrophy
"utoso#al $o#inant
•
"t least one parent a@ected
18
•
Matings usually gi(e half a@ected o@spring
n general: •
Bas (ertical pedigree pattern
•
Fess se(ere disorders than recessi(e
•
ften sho$ (ariable e%pressi(ity
•
May sho$ XanticipationY- ie. earlier onset- more se(erity $ith each successi(e generation
•
>%amples: o
rachydacttly +short &ngers,
o
Polydactyly
o o
"chondroplastic d$ar&sm Buntington?s disease
•
"utoso#al %ecessive
•
Parents not usually a@ected
•
ousin marriages more common in parents of a@ected
•
nherited from both parents
n general: •
Fess (ariable e%pressi(ity since t$o copies of allele needed to see phenotype
•
Bas hori9ontal pedigree pattern
•
>%amples: o o
PO0 "lbinism
o
ystic &brosis
o
;ic*lecell anaemia
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TOPIC LIN(ED AND SE* LIN(ED GENES .1
CELL C 'CLES
(Reve! text chapter ")
.1.1 PRO(AR'OTES
•
ell cycle in(ol(es !" replication $ith cell di(ision by binary &ssion immediately after
•
!o special structures for di(ision- no !" condensation
•
;ingle circular !" molecule replicated
• "ttached to membrane cell $all di(ides
.1. EU(AR'OTES
•
!ucleated cells
•
Mitosis Z meiosis
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Most cells4organisms are diploid +2n,
•
Gametes are haploid +n,
•
iploid [ Baploid [ iploid
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meiosis
fertilisation
•
Ploidy number of chromosome sets
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nterphase phase bet$een cycles +cells most acti(e time,
•
Mitosis can occur in cells of any ploidy
•
Meiosis can only occur in e(en ploidy cells
•
Meiosis needs homologous chromosomes
•
Meiosis results in \"#"T! of o@spring o
Meiosis homologous chromosomes separate
o
Meiosis chromatids separate
.1. CONSEUENCES SE*UAL R EPRODUCTION
Meiosis •
Bal(es ploidy
•
ndependently assorts chromosomes
• "llo$s recombination bet$een lin*ed genes
&ertilisation •
#andom union of gametes from parents
• ,"i"tion in chromosome combinations Z gene combinations
on chromosomes in the o@spring
Men'els( La)s 1. ;egregation one allele in any gamete 2. ndependent "ssortment segregation of alleles- independent of allele segregation for other genes
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2*2
Genes +n $i,,erent C!ro#oso#es
•
;ho$ independent assortment- segregation
•
Pro(iding the follo$ing
1. !ot lin*ed 2. omplete dominance '. !o lethal genes H. !o epistasis
TESTCROSS -1
"ab
%
aabb
+",+"b,+a,+ab,
+ab,
"ab : "abb : aab :aabb 1
:
1
:
1
: 1
22
DI&')RID CROSS O- -1
=1]=1
"ab % "ab
"77: "7bb : aa7 : aabb D
.
:
'
:
'
:
1
LIN(ED GENES
(Refer text page #$6-9)
Lin-age •
Fin*age:
(ariation
in
gametes
due
to
di@erent
combinations of lin*ed genes •
rossing o(er occurs $hen homologous chromosomes align during Prophase - pieces of chromosomes may be e%changed
•
Proposed by Thomas Morgan: fruit Nies- 1DE8 olumbia 0ni(ersity- !e$ Vor*- found genetic crossing o(er
•
hiasmata disco(ered in 1DED by ansessns- esuit priest and Professor- a physical e%planation for crossing o(er hiasmata are points
of
chromosomes during
Prophase
•
Fin*ed
genes on the same chromosome
•
o !T sho$ independent assortment of alleles
•
Tend to be inherited together
•
Genes (ery close little crossingo(er
•
genes
are
o(erlap
on
homologous
23
•
Genes further apart more crossingo(er
•
1R crossingo(er 1cM +centimorgan,
•
1cM chromosome map unit
•
rossingo(er recombination
•
Ma%imum etectable 5ER
..1 DETECTION O- LIN(AGE
etected by ie.
de(iation
\ariation
from e%pected ratios
from
1:1:1:1
testcross
Z
D:':':1
dihybrid =2 >g.
T>;T#;; "
%
ab
"
ab
=1
"
%
ab
ab
ab
"
ab
"b
a
ab
ab
ab
ab
24
3< crossing o(er +non parentals, f genes are 2EcM apart- then: recombinants 2ER parentals 8ER
Reco!#in"tion due to crossingo(er •
Phenotypes di@erent to original parents- therefore sometimes called nonparentals
•
Non>Reco!#in"nts no crossingo(er
•
Phenotypes same as original parents- are called parentals
•
>g. f genes 1EcM apart o
1ER recombinants +5R for each type,
o
Z DER nonrecombinants +H5R for each type,
Proportions estcross R recom.
Parental
!on
Parental 1EcM
E.H5
E.H5
E.E5
E.E5
2EcM
E.HE
E.HE
E.1E
E.1E
5EcM
E.25
E.25
E.25
E.25
!ot Fin*ed
1.EE
1.EE
1.EE
1.EE
2.3.2 Determining Distance
0se testcross to determine distance bet$een lin*ed genes
25
=1
]
bd
bd
+,
+bd, gametes
]
bd
bd
bd
+,+d,+b,+bd,
+bd, gametes
bd !os
bd
16
R recombination
bd
d
bd
bd
1DE 16
b 18
16U18 total 8.5cM
.. LIN(AGE M APS • "mount
of crossingo(er used to determine distances
bet$een genes •
Termed Fin*age Maps
•
istance is additi(e
•
HcM
5cM
" add distance " DcM
26
•
Genome Maps in (arious organisms use lin*age data
•
Z somatic cell hybridi9ation Z insitu hybridi9ation
•
ata to combine results to gi(e maps of (arious chromosomes
.=
GENE M APPING
Three Methods i,
;omatic ell Bybridi9ation =ollo$s segregation of gene product $ith a particular
chromosome. Te%t P 12' ii,
n;itu Bybridi9ation ind cloned gene probe to chromosome directly
iii,
Fin*age ;tudies •
Probability that particular pedigree4s $ith 2 traits reNects lin*age bet$een them.
•
an be detected on autosomes and se% chromosomes
•
alculate probability that 2 traits independently assort according to nonlin*age.
•
Then calculate probability that same data results from lin*ed genes.
•
alculate ratio of these probabilities and con(ert to logarithm
•
This generates F scores +F Fog of the odds for lin*age,
LOD SCORES
•
Fin*age bet$een mar*ers detected by computer analysis of gene ratios
•
=: 27
o
Fod score of ' 1EEE:1 odds for lin*age
o
Fod score of 2 1EE:1 odds against lin*age
o
Fod scores can be added o(er families
.?
SE*>LIN(ED IN&ERITANCE
(Refer text chapter 4 pages 88-9#)
•
;e% can be used as a mar*er for a disorder
•
Genes located on the se% chromosomes sho$ se%lin*ed inheritance o
#efers to genes in the di@erential segment of W or V
•
Genes on W are called Wlin*ed
•
Genes on V are called Vlin*ed +holandric,
•
Mostly WFin*ed
•
Mode of inheritance $ill indicate Wchromosomal localisation of the gene in Cuestion
•
ie. !o male to male transmission in Wlin*ed Z obligate male to female transmission in Wlin*ed
•
;ince Wchromosome is much larger Z therefore has much larger di@erential segment- there are many more Wlin*ed disorders +Wlin*ed disorders almost al$ays recessi(e,
•
=irst documented by Thomas Morgan 1D1E
•
Males hemi9ygous for for Wlin*ed genes
.?.1 *>LIN(ED R ECESSI,E
•
Tends to s*ip generations
•
More a@ected males than females +a@ected female reCuires
28
mother Z father a@ected $hich is rare, •
Balf sons of carrier females are a@ected
•
Fethal only in males
•
>%amples: o
Baemophilia " Z
o
uchenne Muscular ystrophy
o
#edGreen olour lindness
.?. *>LIN(ED DOMINANT • "t least one parent a@ected • "@ects males and females eCually • "@ected female transmits to half sons • "@ected male transmits to none of his sons but "FF
daughters •
n general- males ha(e more se(ere Z less (ariable forms of the disease
•
>%amples: o
\itamin resistant ric*ets
o
Golt9Is syndrome- usually lethal in males- multiple abnormalities
.?. SE*>LIMITED AND SE*>IN-LUENCED IN&ERITANCE
•
Genes not on se% chromosome
•
;e% plays a role in e%pression of a phenotype
SE*>LIMITED C&ARACTERISTICS
•
n ;e%limited inheritance- e%pression limited to one se% eg. Plumage in domestic fo$l- mil* production in female dairy cattle 29
SE* IN-LUENCED C&ARACTERISTICS
•
n se%inNuenced inheritance- se% inNuences e%pression o
•
May be hormonal inNuence
>g. Pattern baldness in humans +also called premature S'5yrs baldness, o
#are in females
o
#elated to testosterone le(els
o
ald allele B- normal allele B!
o
B dominant males- recessi(e females
!ormal
ald
Males:
B!B!
B!B-BB
=emales
B!B
BB
B!B!
.@
SE* C&ROMOSOMES
(Refer text chapter 7 pages #78-#88)
[email protected] C&ROMOSOMAL ) ASIS O- SE* DETERMINATION &UMANS 5*'6
•
2n H6
•
HH autosomes U WW or WV
•
Gametes +male, 22" U W or 22" U V
+heterogametic,
•
Gametes +female, 22" U W
+homogametic,
30
MOST INSECTS • %r&te'&r method of se% determination +WWW, o
Male- 2n 1'
12" U W
o
=emale- 2n 1H 12" U WW
• ygaeus method of se% determination +WWWV, o
Male- 2n 1H
12" U WV
o
=emale- 2n 1H 12" U WW
N). MALES NOT AL%A'S T&E &ETEROGAMETIC SE*
•
>g. ;ome birds- butterNies- &sh- reptiles- chic*ens +^^4^),
•
=emales e%hibit either %r&te'&r or ygaeus method of se% determination
•
0se notation ^^ +male,4^) +=emale, $here ^) is female heterogametic
.@. DISUNCTION
•
uring "naphase of meiosis the se% chromosomes separate called dysJunction
•
W Z V chromosomes ha(e short homologous pairing segment
•
This allo$s se% chromosomes to pair at metaphase. They then separate at "naphase
•
Resu7ts in male gametes : 22" U W or 22" U V
•
n females- WW pair homologously Z separate at "naphase gi(ing : 22" U W gametes
.@. NON>DISUNCTION O- SE*>C&ROMOSOMES
31
•
n females : occasionally the Wchromosome do not separate at "naphase of meiosis
•
#esults in t$o abnormal gametes:
•
22" U WW
•
22" U
•
=ertilisation can then result in:
•
i, HH" U V
lethal
•
ii, HH" U W
TurnersI ;yndrome
•
iii, HH" U WWV
OlinefeltersI syndrome
•
i(, HH" U WWW
Triple W female
Male
=emale 22" U WW HH U WWV
22" U
22" HH U V
V 22" U
+lethal, HH U W
+Olinefelters, HH U WWW
W
+Turner,
+Triple W,
.@.= &UMAN SE* C&ROMOSOMAL A )NORMALITIES TURNER S 'NDROME 5*6 RE-ERRED TO AS 5*O6
•
;terile- female phenotype
•
0nde(eloped breasts- rare menstruation
•
)ebbing of the nec*
•
;hort stature
•
!ormal ntelligence
( LINE-ELTER S 'NDROME 5**'6
32
•
Male phenotype
• \aries
normal phenotype [ sterile male $ith breast
de(elopment •
road pel(is- breast enlargement
•
Generally reduced intelligence but not al$ays
TRIPLE * -EMALE 5***6
•
!ormal female phenotype- including fertility- maybe tallermaJority ne(er detected- 141EEE births.
Non.$ys/unction can also +ccur in Males •
•
!ondysJunction at "naphase can lead to o
HH U WWV
or
o
+Olinefelters,
HH U W +Turners,
!ondysJunction at "naphase can lead to o
22WW or 22VV
o
U normal 22" U W
o
HH U WWW or
o
+Triple W,
HH U WVV
+WVV male,
*'' M ALE •
Male phenotype- normal
•
Tall due to increase ;BW genes- cm on a(erage- may be fertile
• " fascinating e%ample of bad data and instant social
stigmati9ation based on genetic testing. ;o called WVV super male criminal / no$ considered false.
33
.@.? SE* DETERMINATION •
IN M AMMALS
se% is determined by the dominant e@ect of the V chromosome
•
presence of V maleA absence of V female
•
M"7eness is " esu7t of$
• V chromosome loci that cause di@erentiation of embryonic
gonads to form testis •
T= +Testis etermining =actor, is a gene on the V chromosome in a regions called ;#V +;e%determining #egionV, $hich is Just outside the pseudoautosomal region
•
;#V $as found by studying indi(iduals $hose se% $as inconsistent $ith their chromosome constitution eg WW males and WV females.
•
WW males carry a small piece of the V chromosome in one to their W chromosomes
•
WV females ha(e a small region of their V chromosome deleted
• V chromosome has a small Xpseudoautosomal regionY that
has genes found on W
.@.@ A NDROGEN INSENSITI,IT' S 'NDROME • "*a. testicular feminisation synfrome •
H6- WV normal *aryotype but sterile female phenotype
•
!ormal male hormone +testosterone, le(els- but testes 34
de(elop internally •
Male se% characteristics do not de(elop
•
ue to defecti(e W chromosome gene for androgen receptor protein $hich is acti(ated by testosterone to de(elop male phenotype- therefore testosterone fails to act Z female se% characteristics de(elop
• "ndrogen receptor protein o
oded for by "# gene on W- defecti(e gene Tfm +testicular feminisation mutant,
•
Therefore WV females can respond to testosterone if the hormone is administered- resulting in male characteristics
[email protected]
T&E SE* R ATIO Male
=emal
W_
W WW_
V W_
WW_
V W__
_
V
e W__
•
>%pect
• "ctually
1:1 male:female Male
=emale
•
conception S1.2 :
1
•
birth
1.E5 :
1
•
2E yrs
1
:
1
•
85 yrs
E.62 :
1
•
#easons: greater mobility of smaller Vbearing sperm
•
More male deaths in utero Z later- maJor reason- mas*ing of unfa(ourable Wgenes in females- not males
35
[email protected] *>INACTI,ATION DOSAGE COMPENSATION IN -EMALES •
;ince females ha(e t$o chromosomes- $hy donIt they produce t$ice as many W chromosome gene products compared to males o
"ns$er: Wnacti(ation +Fyon Bypothesis, results in dosage compensation
•
n females- interphase somatic cells- one W condenses into small dar* obJect )" )ody +named after disco(erer Murray arr,
• " arr body is composed of Iinacti(eI condensed chromatin-
therefore female Z male- W gene products are eCual o
!umber f arr odies !o. f W hromosomes 1
o
>g. WWW female has 2 arr bodies
•
0sed as se% test eg athletics
•
n
mammals-
Winacti(ation
occurs
during
early
embryogenesis $hen the embryo consists of Just a fe$ thousand cells •
>ach cell ma*es an independent decision to randomly silence one of its W hr?s
•
This W hr remains inacti(ated in all the descendants of that cell leading to clones of cells $ith di@erent acti(ated W chromosomes
•
n cells that become oocytes- W inacti(ation is re(ersedtherefore cells entering meiosis ha(e 2 acti(e W chromosomes
•
Thus female mammals contain 2 types of cell clones- some $ith the maternally inherited W inacti(ated- others $ith the paternal
• " female that is hetero9ygous for an Wlin*ed gene is
therefore able to sho$ 2 di@erent phenotypes •
>g.
=ur
colouration
in
cats
and
mice
are
Wlin*ed 36
+hetero9ygote tortoiseshell,
TORTOISES&ELL C ATS
•
Gene for colouring in cats on W chromosome
)hy do Turner ;yndrome +W, patients sho$ phenotypical abnormalities $hen WW female cells inacti(ate one W chromosome normally?
Ans3e$ " small number of genes on the arr body are still acti(e
and are thought to be essential in a double dose for proper gro$th and de(elopment. "dditionally both W?s are reCuired +arr body becomes unmethylated4acti(e again, for normal o(arian function.
37
TOPIC MEDICAL GENETICS =.1 C&ROMOSOME STRUCTURE
4.1.1 Chromosomes •
omposed of !" core $ith (arious proteins
•
!" Z proteins is termed cho!"tin
•
;eCuence of bases in !" determine types of genes
•
Genes lie in linear fashion along chromosomes
4.1.2 STRUCTURE
(Refer text gure #".9)
•
hromatin is !" coiled around histone proteins +$hich are U(e charged,
•
8 histones comple% forms nucleosomes
•
1H bp of !" coiled t$ice around histone core containing:
2%B2"- 2%B2- 2%B'- Z 2%BH
•
B1 associated $ith !" that lin*s nucleosomes
•
!ucleosomes are then coiled $ith 6 nucleosomes per turn
•
=urther pac*ing Z coiling prior to cell di(ision associated $ith nonhistone proteins
•
!" must be pac*ed to &t into cells- also to allo$ mo(ement
•
1 human diploid cell 6.H picograms !" ` 2 metres length
•
This &ts onto a nucleus $ith a si9e of 51E m
38
4.1.3 Chromatin Remodeling •
hromatin remodeling must occur to allo$ the !" to be accessed by !" binding proteins for gene e%pression and replication.
•
Bistones play a role in chromatin remodeling
•
Bistone tails may be acetylated or methylatedA related to increased gene acti(ity
•
0T methylation of bases T0#!; == genes
•
n(ol(ed in normal de(elopment eg. W inacti(ation
•
)e call this >PG>!>T modi&cation ie. changes in gene e%pression not related to !" seCuence
4.1.4 Chromosome Morphology •
"t metaphase most tightly coiled to allo$ mo(ement
•
In"cti9e in terms of gene e%pression
•
>ach chromosome has 1 molecule of !" di@erent length Z
type of !" seCuences
di@erent genes on each
chromosome •
0T ho!o7o2ous chromosomes ha(e same genes +may ha(e
di@erent alleles, •
Prior to cell di(ision- metaphase chromosomes ha(e 2
cho!"tids Joined at the •
Cento!ees mediate chromosomal migration during mitosis
and meiosis- along $ith •
cento!ee
*inetochore
Cento!ees contain much repetiti(e seCuence in higher
eu*aryotes •
"ll chromosomes ha(e a short arm and a long arm separated by
the centromere o
;hort arm is designated p (pett) 39
o
Fong arm is designated (ueue)
•
Tips are referred to as te7o!ees
•
Te7o!ee !" seCuences consist of short tandem repeats
in(ol(ed in stability and o
integrity of the chromosome.
ther$ise chromosome ends $ould resemble ; brea*s and become degraded by nucleases or fuse to other chromosomes
•
Te7o!e"se pre(ents telomere shortening
•
!umbering from centromere to telomere: cen ter- based on
bands of di@erent staining •
hromosome forms: Metacentric- submetacentric- acrocentric-
telocentric •
"bnormal chromosome types: o o
"centric- no centromere +generally lost, icentric- 2 centromeres
4.1.5 Chromosome Banding •
entromere position and arm ratios can be identical in di@erent chromosomes
•
Therefore limited ability to identify all chromosome pairs
•
yes can be used to produce reproducible patterns of bands on chromosomes
•
hromosome banding is a standard and indispensable tool for cytogenetic analysis
•
G- - # and banding- chromosome painting
•
>ach chromosome has a uniCue Kbar code?
•
btain chromosomes from blood etc
•
ulture and stimulate cell di(ision
• "rrest in metaphase- colchicine may be used
40
4.1.5 Karyotyping •
#epresentation of chromosomes $ithin a cell
•
hromosomes paired and in order of si9e
•
an be used to study chromosome number and morphology
•
!ormal *aryotype: 22 pairs autosomes +numbered 122, and a pair of se% chromosomes +WW or WV,
•
hromosomes identi&ed based on si9e- morphology and banding patterns
•
Oaryotypes are presented in standard form
•
The total number of chromosomes follo$ed by a comma and then the se% chromosome constitution
•
an be follo$ed by a short hand (ersion of any abnormalities
•
>g. female dog $ith increased length of the short +p, arm of chromosome 2: 8- WW- 2pU
=. PLOID' •
Baploid one set of chromosomes +n,
•
iploid 2 sets +2n,
=..1 EUPLOID' (Refer text chapter 8)
•
Multiples of complete haploid sets- includes o
iploid
o
Polyploid more than 2 haploid sets
ommon in crop plants: bananas- cotton- peasco@ee
ften gi(es cells larger si9e- therefore larger- more
41
(igorous plants
\ertebrates rare- occasionally in in(ertebrates
!eed e(en no. polyploidy in nature : Hn [ 2n gametes
o
Triploid / ' haploid sets
Mitosis : no problems
Meiosis : une(en [ infertile gametes
ut useful sometimes eg triploid bananas $ith small infertile- edible seeds
Mechanism •
=ailure of chromosome separation during mitosis
• "utopolyploidy o o
Polyploidy from single ancestor set of chromosomes "utotriploids can be produced e%perimentally by crossing diploids $ith tetraploids +gametes n U 2n 'n,A used commercially
in
some
bananas-
potatoes-
seedless
$atermelons o
Tetraploidy produced e%perimentally using colchicine $hich inhibits microtubule formation or cold4heat shoc* +=ig 8.D,
• "llopolyploidy o
hromosome sets from 2 or more ancestral species +hybrids from distant diploid species,
o
Bybridi9ation Z allopolyploidy important in plant e(olution Z breeding
•
>ndopolyploidy o
)hen certain cells in an other$ise diploid organism are polyploidy eg. human li(er cells
=.. A NEUPLOID'
42
•
Polysomy e%tra chromosome copies- not sets Q abnormal phenotypes o
•
Trisomy 2n U1
Monosomy missing chromosome copy Q harmful- often lethal 2n1
=. NORMAL ( AR'OT'PES
•
hromosome type Z composition *aryotype
•
Buman Male HH autosomes U WV
•
Buman =emale HH autosomes U WW
•
0sually stained by Giemsa G anding
• •
"rranged in pairs 1 [ WV The
analysis
of
stained
chromosomes
$ith
respect
to
chromosome number and structure is a discipline referred to as cytogenetics
=.= A )NORMAL C&ROMOSOMES AR'OT'PES =.=.1 A )NORMAL (
Trisomy 21 - Down yndrome o
!ondisJunction 21- usually oogenesis- associated $ith age of mother
o o
•
148EE li(e births "lso #obertsonian translocation in familial cases
=eatures / heart malformations- malformations of the GT-
mental retardation- characteristic facial features- increased incidence of leu*emia +1E / 2E% higher,- hearing loss- many de(elop neuropathology of "l9heimer disease
43
Mother?s age and ris* of o$n ;yndrome in child 2E2D ` 142EEE 'E'D ` 146EE HEHH ` 141EE H5HD ` 14HE fetal loss rate `'ER of o$ns syndrome
Trisomy 1! - "dwards yndrome o
148EEE li(e births
o
;ur(i(al a(erage ' months- multiple defects- 1R reach age 1E.
o
H8R fetal loss rate
Trisomy 13 - #ata$ yndrome o
1 in 1DEEE li(e births
o
;ur(i(al a(erage ' months- cyclopia- proboscismultiple defects
o
•
6DR fetal loss rate
uplication
larger
chromosomes
usually
not
(iable
Q
miscarriage •
!ondisJunction se% chromosomes Q W +Turner,
•
WWV +Olinefelters,-
•
WVV +WVV male,-
•
WWW +Triple W female,
=.=. C&ROMOSOME A )ERRATIONS
•
eletions o
Barmful
o
Farger deletions most harmful
44
uplications
•
o
Fess harmful than deletions
o
ut larger duplications [ less sur(i(al
n(ersions
•
o
#e(ersed order of genes can Q uneCual crossingo(er
Translocations
•
o
>%change of chromosome parts in nonhomologous chromosomes chromosome rearrangement
=ragile sites eg. =ragile W
•
o
Mental retardation- (ariable
o
ue to abnormal trinucleotide repeat number $hich inacti(ates nearby gene
o
;ho$s genetic anticipation
Cri-d$-Chat yndrome
"n e%ample of a disease caused by chromosomal abberationsA Xat / ryY
•
o
(oice abnormalities- multiple oddities
o
retardation and muscular defects
o
ue to deletion of small part chr 5 ie. H6- 5p
=.=. C&ROMOSOME A )NORMALITIES A SSOCIATED %IT& C ANCER
• •
"bnormalities associated $ith cancer types >g. Philadelphia chromosome o
22 U D U +DA22,
o
Part of 22C translocated to D
o
•
"ssociated $ith particular type of leu*emia +MF,
ften brea*points at oncogenes 45
•
>g. abl oncogene U +DA22,: leu*emia
•
>g. ras oncogene 11p: )ilmIs tumour
•
>g. myb oncogene U +6A1H,: lymphoma
•
>g. mos oncogene U +8A21,: leu*emia
•
>g. myc oncogene U +8A1H,: leu*emia
=.=.= &UMAN GENETICS
%re&$en'y () Birth De)e'ts
ominant Z WFin*ed #ecessi(e
1.E E.1
hromosomal
E.H
ongenital Malformation
D.E 1E.5R
•
Therefore 1E.5R human ne$borns carry genes [ disorders during lifetime
•
DR malformations such as cleft lip- palate Z polygenic disorders eg diabetes- ulcer etc de(elopmental disorders
•
1.5R Mendelian disorders Z chromosomal abnormalities
Inf"nts "nd Inf"nt De"ths 5P"ents 3ou7d #e c"ndid"tes fo " 2enetic efe"76$ •
'5R of all births result in congenital malformations
•
E.5R of all ne$borns ha(e a chromosomal abnormality
•
R of all stillborns ha(e a chromosomal abnormality
•
2E'ER of all infant deaths are due to genetic disorders
•
'E5ER of postneonatal deaths are due to congenital
46
malformations
ingle *ene Disorders •
1D86 McOusic*- \: Mendelian nheritance n ManA contained HEEE Mendelian traits- most associated $ith disease
• "utosomal ominant
: 5ER
• "utosomal #ecessi(e
: H'R
•
WFin*ed
: R
" +,M#"
ystic =ibrosis PO0
"# "#
Glu6PBase =amilial
141EEEE WF
"
1425EE 141EEE
145EE
Bypercholesteremia Thalassaemia
"#
1416EEE
Baemophilia
WF
148EEE
•
ften freCuency hard to determine due to: o o o
Penetrance \ariable e%pressi(ity Beterogeneity Q many syndromes $ith similar symptomsA di@erent mutations Q same disorder
o
"lso- treatment Z early inter(ention can a@ect freCuency- eg. PO0: early inter(ention +diet, allo$s indi(iduals $ho are a@ected to li(e Z reproduce
o
thers ha(e freCuency changes due to pressure +selection, against reproduction or reduced fertility
47
M$ltigene Disorders •
;ome genetic disorders due to se(eral genes interacting polygenic
•
;ome comple% polygenic plus en(ironmental factors o
•
>g. Bypertension- diabetes
;ome disorders sho$ familial aggregation- indicating a genetic in(ol(ement o
>g. ;pina bi&da ris* increases $ith pre(ious child a@ected child or parent +to 142E, also for anencephaly- $ith a pre(ious a@ected child increases ris* to 142E
pontaneo$s Mis'arriages •
1st Trimester
•
5ER chromosomal abnormalities
•
W +Turner,- 2ER of miscarriages total +D5R Turners miscarried,
• "utosomal trisomies 5ER abnormal chromosome conceptions o
Many ne(er seen li(eborn
#realen'e () *eneti' Conditions •
2E'ER of all infant deaths are due to genetic disorders
•
'E5ER of postneonatal deaths are due to congenital malformations
•
E.5R of all ne$borns ha(e a chromosomal abnormality
•
R of all stillborns ha(e a chromosomal abnormality
•
11.1R of paediatric hospital admissions are for children $ith
48
genetic disorders •
5ER of indi(iduals found to ha(e mental retardation ha(e a genetic basis for their disability
•
12R of adult hospital admissions are for genetic causes
•
1ER of the chronic diseases +heart- diabetes arthritis, $hich occur in the adult populations ha(e a signi&cant genetic component
R&*'s&' +. a'd 'de' . #993. /'ca/ e'etc 0a'd*&&1, 2&st&', 2/ac1!e// Sce'tc %u*/cat&'s. eathera// . #985. he e! e'etcs a'd /'ca/ %ractce, Sec&'d dt&'. xf&rd: xf&rd ;'versty %ress.
=.? GENETIC DIAGNOSIS •
;ymptoms- family history
•
etermination of mode of inheritance
•
etermination of ris* to preclinical- prenatal situations
•
Therefore #is* "ssessment
•
Genetic counselling in(ol(ed
=.?.1 PREIMPLANTATION TESTING
•
=or \=
•
n recurrent miscarriages- maternal age- prior chromosomally abnormal child or fetus- heritable medical condition- gender selection
=.?. PRENATAL TESTING
49
ULTRASOUND
an detect de(elopmental defects eg. o
Bead
anencephaly
o
;pine
spina bi&da
o
"bdomen polycystic *idneys
o
Beart
congenital heart disease
o
Fimb
deformities- shortening
Pre(ention of disorders by o
ntrauterine treatment in some cases
o
>g. ;urgical treatment of urinary tract obstruction or chemical treatment such as (itamin therapy
o
r termination
SCREENING -OR C&ROMOSOMAL A )NORMALITIES
16 NUC&AL TRANSLUCEN' TEST
•
` 12 $ee*s
•
Measures thic*ness of Nuid layer at le(el of foetal nec* by ultrasound
•
ncrease ris* of chromosomal abnormalities or heart defects $ith increased thic*ness
•
U blood test for maternal free βBG and P"PP" +pregnancy associated plasma protein", or other mar*ers in second trimester
•
βBG higher and P"PP" lo$er in trisomies
•
#is* calculated for chromosomal abnormality in foetus- not
50
diagnostic •
f greater than 14'EE and4or patient older than '5V- diagnostic tests recommended chorionic (illus sampling or amniocentesis
6 C&ORIONIC , ILLUS )IOPS'
•
`12 $*s
•
;ample ta*en transabdomen under 0; guidance
•
ut 141EE ris* of miscarriage
•
"lso chance of sampling maternal cells and not detecting
abnormality •
ells separated Z used for testing- =;B for trisomies and W V*aryotyping- biochemical testing
6 A MNIOCENTESIS
•
1H1 $*s
•
;ample of amniotic Nuid containing foetal cells through
abdomen •
=oetal cells must be cultured for cytochemical tests or used for
biochemical or !" testing •
#esults ta*e longer
•
142EE ris* of miscarriage
•
More accurate as only foetal cells sampled
6 CELL -REE -OETAL DNA
•
!onin(asi(e
•
=rom plasma of pregnant $omen
•
!ot as accurate
51
TESTS PER-ORMED ON -OETAL CELLS A 6
-IS&
•
ytochemical testing
•
=luorescently labelled probe hybridises to fetal chromosomes
•
an pic* up trisomies- Turner?s monosomy
)6 ( AR'OT'PE A NAL'SIS
•
ytochemical testing
•
ulture fetal cells- arrest in metaphase on slides- stain- obser(e microscopically for abnormalities
C6 )IOC&EMICAL TESTING
•
etter to use amniocentesis Z analyse Nuid for:
•
nborn errors of metabolism eg PO0
•
;tructural protein disorders eg osteogenesis imperfecta +fragile bone disorder,
•
!eural tube defects eg spina bi&da by chemical monitoring
D6 DNA TESTING
•
y direct mutation analysis of genes- eg. Thalassaemia
•
y analysis of lin*age mar*ers eg BuntingtonIs
•
Therefore defect *no$n or lin*ed +defect close to mar*er,
•
etect !" seCuence changes by ;outhern lot or P#
R-LPS
52
•
#estriction =ragment Fength Polymorphisms
•
!" seCuence changes in restriction en9yme sites
•
;table- Mendelian inheritance
•
an occur $ithin genes or $ithin noncoding regions
•
0seful !" mar*ers
• "lleles can be tested for lin*age or association •
etect by ;outhern lotting
•
r P# +useful for #=FPs in(ol(ing insertions or deletions,
•
#=FP may cause defect such that indi(iduals inheriting one allele $ill get disease- eg sic*lecell anaemia
•
r #=FP lin*ed to disease ie !" containing #=FP on same chromosome eg Wlin*ed muscular dystrophy
.?. NEONATAL TESTING
*$thrie Test •
Beel pric* blood test
•
Test for PO0 +acterial inhibition assay,- = +P# and mutation analysis, and other disorders
•
>arly detection useful for pre(ention of eg mental retardation and genetic counselling
.@ TREATMENT 1. ietary >%clusion >g. Phenylalanine in PO0 2. ietary "ddition >g.
0ridine
supplement
in
oroticaciduria
+abnormal
pyrimidine metabolism,
53
'. rug "(oidance >g. Glu6PBase antimalarial drugs Z fa(a bean cause # haemolysis H. ody >limination >g. Baemochromatosis- iron accumulation [ heart- li(erpancreas disorders #emo(e iron by (enesection 5. ofactor ;upplementation >g. >n9yme error- increase cofactor eg 12- 6 cause en9yme to act normally 6. >n9yme #epression or nhibition >g. ;upply substrate stopping o(erproduction of metabolite by feedbac* inhibition . #eplacement Therapy >g. lood in Thalassaemia =actor \ in haemophilia- missing en9ymes- hormones 8. rgan Transplantation >g. ystic disease- *idney transplantation D. Pre(entati(e ;urgery >g. #emo(al of colon in hereditary colon cancer +during early stages, 1E. Pre(entati(e Therapy >g. "nti#h antibody inJection into #h mother after birth of 1st #hU child Oills #hU blood cells before immune system is stimulated +inJection decays before ne%t pregnancy
54
55
opic $N" ec!nology .1 R ECOM)INANT DNA TEC&NOLOG'
#ecombinant !" refers to the Joining of !" moleculesusually from di@erent biological sources- that are not found together in nature
#ecombinant !" technology is used to isolate- replicate- and analy9e genes
The introduction of recombinant !" technology has allo$ed genetic engineering
asis is technology of ma*ing recombinant molecules Ν
nserting !" into (ector Z ma*ing multiple copies- ie. loning
.1.1 CLONING STEPS 1. ut donor !" 2. ut (ector !" '. oin fragments H. ntroduce into host 5. ;creen for desired gene
. TOOLS
*2*1 %estriction Eny#es
Most useful en9yme for !" $or* are #estriction >n9ymes +#>, Ν
also called restriction endonucleases
These are en9ymes that cut !" at speci&c nucleotide 56
seCuences
"ct as means of remo(ing foreign !" in bacterial cells Ν
acteria ha(e a modi&cation system that methylates o$n chromosomal !" to stop #> digestion
*2*2 %ecognition sites
Most recognition seCuences e%hibit a form of symmetry described as a palindrome- and restriction en9ymes cut the !" in a characteristic clea(age pattern. palindromes- eg G""TT TT""G
Mostly four to si% nucleotides long
;ome contain eight or more nucleotides
ut to gi(e blunt or stic*y ends
.. OT&ER ENH'MES 1. !" Figase Ν
oins !" molecules
2. "l*aline Phosphatase Ν
#emo(es 5P- stops religation
Ν
0seful after #> digestion to stop selfligation
'. !" Polymerase Ν
opies ;; !" to gi(e ; !"
H. #e(erse Transcriptase Ν
Ma*es !" from #!" template
57
Ν
0seful for c!" library construction
5. Terminal Transferase Ν
0seful for ItailingI !" fragments ie attaching bases at 'end to create stic*y ends
. , ECTORS
\ectors are carrier !" molecules that can replicate cloned !" fragments in a host cell
)ide (ariety generally deri(ed from plasmids- phage
Many ne$ arti&cial constructs
..1 DESIRED C&ARACTERISTICS
apable of autonomous replication in host cell
;mall si9e +usually '*b, generally
!ormally capable of amplifying cloned seCuence
"t least one uniCue #> site for insertion Mar*ers
for
easy
identi&cation
Z
selection
+"ntibiotic
resistance- colour- Nuorescence,
"ppropriate signals of e%pression desired +olour- Nuorescencelin*ed gene metabolism,
\ectors: Many Types nclude: 1. Plasmids 2. Fambda Phage '. >%pression \ectors H. osmids 5. ;huttle 6. V"
hoice of (ector depends on:
58
Ν
Type of e%periment
Ν
;i9e of insert
Ν
"(ailable #> sites
**2 Plas#i's
;mall- circular- selfreplicating
>%tra chromosomal
1many copies4cell +ampli&cation,
ther genes for drug resistance genetic mar*ers
Most $idely used are p#'22 Z deri(ati(es
Many single #> sitesA tet and amp mar*ers
"n arti&cial construct Ν
nserts: up to 15*b +51E*b stable inserts,
Ν
;elect recombinants by insertion into antibiotic resistance gene
Ν
>g. utting $ith P(u or Pst- then $ill be tetracycline resistant but ampicillin sensiti(e
.. ) ACTERIOP&AGE L AM)DA 5 6
>.coli (irus
HD *b circular structure
59
;ingle #> insertion sites or double sites for replacement !"
Most commonly used types are Ν
gt1E
all purpose (ector
if form plaCues- recombinant Ν
gt11
e%pression system
if insert [ hybrid gene- select by antibiotics
nserts : 1E2E *b +S2'*b,
Fambda contains essential genes U a replacement region. Finear molecule circularised by annealed cohesi(e ends cos site
;elect by plaCue formation if no replacement region or !" insert here $ith plaCue formation
** 3"Cs
acterial "rti&cial hromosomes
Fo$ copy number plasmids
Good for large inserts
an accept 1EE'EE*b of !"
**5 4"Cs
Veast "rti&cial hromosomes
#eplicate Z segregate ;T"FV during mitosis Z meiosis
;maller than 5E*b unstable- therefore insert [5E*b
2 origins of replication
Ν
ori bacterial origin
Ν
ars autonomous replicating seCuence +yeast,
ther structural features Ν
>! centromere seCuences- attachment sites for spindle
Ν
T>F telomere seCuences for integrity of chromosome ends
;electable mar*ers 60
Ν
ampr +plasmid,
Ν
T#P1 0#"' 3gro$ in T#P +tryptophan, 0#" +uracil, yeast host cell<
** Expression Express ion Vectors
These contain promoters that allo$ e%pression of gene products These contain origins of replication- for replication in >. coli. 0suall 0sually y conta contain in select selectab able le mar*e mar*ers rs to select select transf transform ormant antss in bacteria +>.coli, +eg "mp# , and eu*aryotic cells +GH184!eomycin,. ontain a multiple cloning site +M;, do$nstream of the promoter to insert !" to be e%pressed.
.= &OST CELLS
-OR C CLONING
acterial cells Veast Veast cellsA gro$ li*e bacterial cells but eu*aryoteA can modify proteinA genetics $ell *no$nA safe for production of (accines etc.
Plants
nsects
Buman cell lines
.? R ECOM)INANT ECOM)INANT DNA LI)RARIES
ollection of cloned !" seCuences from a single source eg. ell type- tissue type- indi(idual
*5*1 Library Construction
1. ut source !" 2. ;elect Z cut (ector 61
'. Figate desired !" into (ector H. ;elect recombinants recombinants 5. ;creen for desired clone eg. probes
.?.
GENOMIC ,S ENOMIC ,S CDNA LI)RARIES
Genomic all !" seCuences from source- coding Z noncoding
c!" c!" on only ly seCu seCuen ence cess used used in ma*i ma*ing ng m#!" m#!" tran transc scri ript ptsstherefore only coding !" from source
=or ge geno nomi mic c libr librar arie iess- tend tend to use use pa part rtia iall dige digest stss to ge gett o(er o(erla lapp ppin ing g frag fragme ment ntss to ge gett inta intact ct ge gene ne in at leas leastt some some fragments
"lso allo$s I$al*ingI to get get to desired gene gene To represent library fully better to insert into (ector that can ta*e bigger !" fragments- that $ay less clones to screen for desired gene >g. phage or cosmid rather than plasmid
.?.
CDNA
LI)RAR' C CONSTRUCTION
solate m#!"
62
.?.=
LI)RAR' S SCREENING
dentify recombinant containing desired insert by hybridi9ation to labelled ;; probe 63
Probe may be partial seCuence oligonucleotide or related seCuence from similar species or related gene +eg pseudogene,
.@ MOLECULAR GENETIC TEC&NIUES
[email protected]
PCR
Polymerase hain #eaction
Process for producing large amounts of a speci&c !" fragments in (itro- enabling use of this !" seCuence for (ariety of e%periments
e(eloped in 1D8' by O.Mullins Z =.=aloona started use 1D85 86 Reuires
Template ; !" or #!"
2 oligoprimers +not complementary,
d!TPs- bu@er- Mg2U TaC Polymerase
!>>: Ν
;eCuence *no$n at ends of region to be ampli&ed
Ν
;eCuence for ampli&cation !CR Method
; !" denature- anneal oligoprimers JKKK KKK
TaC Polymerase ``````` JKKK 64
ycle 1
2 ; !"
KKK```````
denature- anneal- TaC... ``````S```` ````[`````` H ; !" ``````S```` ```[``````` ... and so on +up to 'E cycles ` 2'E,
65
!CR !oints "AQ !#$%M&RAS&
!" Polymerase isolated from her
!ot denatured by heating to DED5
ptimum temperature 5 AM!$'('CA"'#) !$A"&AU
'E'5 cycles
2'E2'5 copies !" seCuence
!o further ampli&cation
Cyc7e > ste/s
+i, enaturing Ν
; !" [ ;; !"
Ν
D5- 'E secs
+ii, "nnealing Ν Ν
"nneal primers to ;; !" 1 minute '65
+iii, >%tension
Ν
;; [ ; !"
Ν
1 minA 6E2
TaC Polymerase allo$ed automation Ν
>n9yme can be cooled repeatedly heated Z cooled
Ν
Bigh temperature optimum [ high stringency
Primers not complementary Ν
f $ere could hybridise to each other
P# can be used to amplify genes or noncoding fragments Ν
>g. etect insertion4deletion4mutation by P#
>%tract !"- P#
#un !" on gel
bser(e bands
Farger or smaller bands depending on si9e of !" 66
bet$een primers
[email protected]
R EAL>TIME PCR
"llo$s Cuanti&cation of gene e%pression
;e(eral di@erent types eg. #TP# or Cuantitati(e realtime P#
n Cuantitati(e realtime P# m#!" is con(erted to c!"
=luorescent dyes and probes are used $hich bind to the !" bet$een the primersA e%tension causes emission of light $hich is detected by a laser
Greater Nuorescence higher gene e%pression
.@.
ELECTROP&ORESIS
O- NUCLEIC A CIDS
Gel electrophoresis is transfer through a porous gel or membrane under charge. •
;ubmarine gel electrophoresis is conducted in a submerged gel +under bu@er,.
•
apillary gel electrophoresis is conducted in a capillary
;eparation of molecules based on si9e Z charge +eg protein gel electrophoresis can also be performed,
;mallest fragments mo(e farthest in the gel
=ragments can be (isuali9ed $hen stained $ith ethidium bromide and illuminated by 0\ light
;tandard mar*ers are used to determine si9e of un*no$n product.
.@.
SOUT&ERN
)LOTTING
!amed after >d$ard M. ;outhern
0sed to identify $hich clones in a library contain a gi(en !" seCuence and to characteri9e the si9e of the fragments from
67
restriction digest
Transfer of !" from gel to membrane +usually nylon and usually positi(ely charged,
Transfer occurs (ia i, electroblottingA ii, (acuum andA iii, capillary
#!" and protein can also be transferred from gel to membrane: referred to as !orthern and )estern lotting respecti(ely.
.@.=
MICROARRA'S
=unction using the same systems as ;outhern lot +Probe annealing,
hips or slides co(ered in &%ed !"4#!" probes "llo$s detection of concentration as $ell as presence of speci&c seCuences.
#ecent ad(ances in technology allo$ multiple millions of probes on a single chip.
.@.?
-IS&
=luorescent in situ hybridi9ation +=;B, in(ol(es hybridi9ing a probe directly to a chromosome or #!" $ithout blotting
=;B can be carried out $ith isolated chromosomes on a slide or in situ in tissue sections or entire organisms
.@.@
DNA
SEUENCING
!" obtain by cloning or P#
!: P# more prone to error +HW more,- therefore do not use 68
P# deri(ed clone. 0se direct P# product to gi(e mi%ture of errors SA)G&R*+'&#-% S&QU&)C')G
;; !" Pol template
!" Pol to e%tend P'2 primer
d!TPs plus dd!TPs Ν
ideo%y!TPs do not form phosphodiester bonds- therefore stop chain
[ fragments of di@erent lengths H reactions- H di@erent dd!TPs [ gel +polyacrylamide, U autoradiography
M;T MM! "nimation
at
http:44$$$.dnalc.org4resources4animations4sangerseC.html )&-" G&)&RA"'#) S&QU&)C')G
ften referred to as H5H ;eCuencing- or onic ;eCuencing
#elies on binding of P# product4!" fragment to a bead $hich can be isolated in a speci&c chamber.
ound !" to be seCuenced is rendered single stranded prior to seCuencing
;ample is then seCuenced by seCuentially adding one base and a !" polymerase and detecting addition +or not, of speci&c bases.
etection can be indirect- by detection of liberated "TP as in pyroseCuencing- or direct- as in detection of BU released by base addition- as in ionic seCuencing.
@ers ad(antages in terms of throughput and ability to determine comple% genotypes.
!ot currently common- but has potential to o(erta*e ;anger ;eCuencing.
69
TOPIC ? DNA MUTATION ?.1 DNA MUTATION (Refer text chapter #5) • " mutation is an alteration in a !" seCuence. •
nduced mutations result from the inNuence of an unrelated factor or agent- either natural or arti&cial.
•
;pontaneous mutations happen naturally and randomly- arising
70
from replication errors and base modi&cations •
Mutations can occur in any cell: (arious changes in phenotype ranging from minor to maJor changes
?.1. SPONTANEOUS MUTATIONS
•
Mutations
occur
randomly
but
$ith
characteristic
rate
depending on: o
Gene
o
rganism
•
Mutations are not adapti(e
•
>g. Gro$ bacteria that are sensiti(e to antibiotic in antibiotic Q resistant bacteria o
0T replica plating sho$ed that mutations already in these
cells-
ie.
mutations
random:
Lede#e2
e/ei!ents •
Lui">De7#c Nuctuation test also sho$ed that mutations are
spontaneous •
ther e%amples: nsecticide resistance- antibiotic resistant bacteria- fungus resistant plants
?.1. CLASSI-'ING MUTATIONS • \arious $ays to classify •
y location i, Germinal Q gametes- inherited ii, ;omatic Q other cells- not generally inherited iii, "utosomal / $ithin genes on autosomes i(, Wlin*ed / $ithin genes on W chromosome
•
y molecular change o
=rameshift mutation
o
Point mutation: nucleotide substitution
71
•
y phenotypic e@ects o
Fethal mutations
o
onditional mutations phenotype changes- dependent on en(ironment
eg temperature sensiti(e ;iamese cats fur colouring
?.1..1 POINT MUTATIONS • "*a. ase substitutions >g. "n " substituted by a •
M"V be noncoding or coding- $hich may result in change in codon $hich may change an amino acid +missense mutation, or may not +silent mutation,- # lead to chain termination +nonsense mutation,- if altered to 0""- 0"G or 0G"
•
0sually no physiological e@ect- but occasionally there may be
•
>g 1. Baemoglobin \ariants !" m#!" Protein
GG" T T 0 G"G G"G Pro Glu Glu
!" Bb m#!" cell anaemia Protein
GG" A T 0 G0G G"G
a
Bb
s
•
Pro
\al
normal
has
sic*le
Glu
>g 2. =P =ibrodysplasia ssi&cans Progressi(a
T$o types of point mutations
6i7 ransition •
Pyrimidine Q pyrimidine TQ QT
72
•
Purine Q purine GQ"
"QG
5II6 TRANS,ERSION
•
Pyrimidine Q purine or (ice (ersa o
•
8 (ariations
;pontaneous mutations of this sort biased to transition because of I$obbleI ie 'rd nucleotide of codon can (ary gi(ing same amino acid Z often transition mutation gi(es same amino acid Si7ent Su#stitution
5*1**2 &ra#e.S!i,t Mutation •
nsertion or deletion of a nucleotide can Q maJor aa change
•
Through alteration of the reading frame o
>g. eletion
•
she c"5n6 sc e"! 7ou d7y
•
she c"s ce "!7 oud 7y. o
•
"lso possible- ne$ aa is ;TP codon Q short protein
>g. uchenne muscular dystrophy- " blood system
?.1.. MUTATION T 'PES
1. ;ilent no change to aa seCuence 2.
Missense
substitution
Q
changed
aa
seCuence
eg.
"chondroplasia '. !onsense substitution Q ;top codon eg. Marfan syndrome
73
H. "ddition4eletion add or remo(e one or more bases ften occurs $ith short tandem repeats or runs of particular nucleotides eg. ystic &brosis 5. =rameshift addition4deletion can change reading frame 6. Transposon Mutations inserts into gene . >%pansion of repeat seCuences eg. =ragile W- Buntington isease •
;ome diseases can arise from a large number of mutations
•
>g. eta thalassemia
?.1..= )OM)A' P&ENOT'PE • "@ects inheritance of the " blood type • " rare recessi(e mutation in the =;# +fucosyl transferase,
gene- in(ol(ed in synthesi9ing the B substance- pre(ents the addition of the terminal sugars of the " and alleles •
ndi(iduals are functionally and phenotypically Type although genotype may be "4
?. PROCESSES •
!" replication errors
•
ase mispairing Ν
Tautomeric shifts in nucleotides
Ν
epurination- deamination
•
%idati(e damage
•
ntegration of transposons
?..1 TRANSPOSA)LE ELEMENTS
•
=ound in pro*aryotes and eu*aryotes
• umping genes
74
•
an create mutations or silence genes by insertion into functional genes or alter e%pression
•
an trigger duplications- deletions or translocations
•
May be germline
•
n(ol(ed in human disease
?. INDUCED MUTATIONS 16 )"se An"7o2s •
ompounds similar to bases
•
ause mutations because prone to mispairing o
>g. 5bromouracil T analog but can change to enol form Z pair $ith G +instead of ", during replication
•
!e%t #eplication Q G formation
2, A7y7"tin2 A2ents • "dd chemical groups to bases Q di@erent pairing or base
distortion o
>g.
!itrosamines-
nitrogen
mustard-
ethylmethane
sulfonate +>M;, 6 Intec"7"tin2 A2ents •
>thidium bromide- hemotherapeutic agents
•
nsert bet$een adJacent bps o
Q Misaligned template4daughter stands replicated !"
o
Q "ddition4deletion in daughter strands
6 Adduct ;fo!in2 "2ents •
mutationcausing chemicals- bind to !" and alter its conformation- thereby interfering $ith replication and repair 75
• "cetaldehyde o
•
omponent of cigarette smo*e
Beterocyclic amines +B"s, o
ancercausing chemicals created during coo*ing of meats +beef- chic*en- and &sh,
o
1 di@erent B"s lin*ed to cancers of stomach- colon- and breast
=6 U, 5U7t" ,io7et6 •
0\ Q dimer formation
Transcription U replication bloc*s ADIATION ?..1 R
•
W#ays dental- medical
• "lpha- beta- gamma rays radioisotopes- fallout•
0\ lo$er energy- ;un
8oniing %a'iation •
!" amage
•
Q ;; brea*- sugarPbac*bone
•
Q ; brea*
•
Q ase alteration o
• "lso
Fast t$o most damaging chromosome
brea*s
in
eu*aryotesQ
translocations-
in(ersions- duplications- deletions "mount of damage depends on: i6 Tot"7 Dose
Mutation rate directly proportional to dose recei(ed in rosophila
76
# #oentgen units for e%posure dose ii6 Dose>R"te •
;ame rate dose gi(en Cuic*ly is more damaging than gi(en slo$ly
•
>g. Mice DE#4min HW more mutations as DE#4$ee* for same total dose o
;uggests some repair mechanisms
o
ut no safe le(el- ie. threshold dose belo$ $hich all damage repaired
$+93L8NG $+SE •
Bumans- H5# WraysA doubles the mutation rate o
1D6E chest Wray : E.1#
o
1D8E chest Wray : E.E1#,
TUMOUR T&ERAP' R ATIONALE
•
nterphase cells less susceptible to chromosome brea*
•
Therefore cancer cells more mitosis
•
Therefore more lethal chromosome brea*age induced by radiation
?.. DNA POL'MORP&ISMS
•
!ormal (ariations in the genome
•
e&ned as occurring in [1R of population
• "rise point mutation •
#=FPs : #> site
77
?.= DNA R EPAIR !" repair systems maintain the integrity of genetic material susceptible to spontaneous and induced damage- protecting against disease and cancer ?.=.1 T 'PES O- DNA R EPAIR S 'STEMS
i@erent en9yme systems a(ailable for repair of speci&c errors. !ot all a(ailable in humans. MISMATC& R EPAIR
•
;ystem for detecting Z correcting after proofreading
•
0ses e%cision Z resynthesis
•
Mismatched base detected
•
ne strand cut
•
#egion around mismatch e%cised o
!" Polymerase &lls gap +uncut strand template,
o
!" Figase seals gap
n pro*aryotes- $hen detect mismatch- en9ymes preferentially cut undermethylated +daughter, strand to remo(e error- therefore sometimes called methyldirected mismatch repair +methylation slo$er than replication,
Error %ates : "verage •
•
#eplicationmismatch: 1 in 1E>5 o
ie. Mismatched base for each round of replication
o
hance 1 in 1E>5 $rong
Proofreading chance of not correcting: 1 in 1E>2 78
o
•
DDR $rong corrected
Mismatch #epair chance of not correcting: 1 in 1E>' o
DD.DR those remaining corrected
1E>5 ] 1E>2 ] 1E>' 1E>1E +chance that base not correct, POST>R EPLICATION R EPAIR
•
#esponds after damaged !" has escaped repair and failed to be completely replicated
•
#ecombinational #epair
•
rea* in ; !"
•
#ec " protein binds- initiates strand e%change $ith homologous !" Q double recombination Joint
•
!" Pol &lls gaps
•
#ecombination Joints resol(ed
•
#ecombination is so that each ha(e one good strand
SOS R EPAIR
•
Fast resort
•
>%treme !" damage
gaps in ; !" in both daughter
strands •
ie. Bomologous chromosome damaged
•
Therefore no recombination repair Q ;; #epair
•
` 2E ;; genes $hich insert bases into gaps $ithout template therefore ' out of H bases $rong
•
Q >%tensi(e mutagenesis
P&OTOREACTI,ATION R EPAIR
79
•
Fight dependent
•
#emo(es thymine dimers caused by 0\ light.
•
The process depends on the acti(ity of the photoreacti(ation en9yme called Photolyase
•
Photolyase captures energy from light and brea*s co(alent bonds bet$een thymine dimers
•
Bumans and other organisms lac* photoreacti(ation repair
E*CISION R EPAIR
•
Fight independent
•
>%ists in all pro*aryotes and eu*aryotes
•
amaged region cut out by nuclease- gap &lled by !" Pol Z sealed by ligase
•
ase e%cision repair remo(es bases
•
!ucleotide e%cision repair +!>#, remo(ed bul*y lesions
•
Buman autorecessi(e diseases that occur due to failure of !># path$ay 1. Weroderma pigmentosa o o
Fost ability to undergo !># "@ected indi(iduals e%hibit se(ere s*in abnormalities- s*in cancers- and a $ide range of other symptoms- including de(elopmental and neurological defects
2. oc*ayne syndrome +;, o
e(elopmental
and
neurological
defects-
sunlight
sensiti(ity- but no increase in cancers o
Premature aging and death by age 2E
'. Trichothiodystrophy +TT, o
$ar&sm- retardation- brittle hair and s*in- facial deformities
o
;ensiti(ity to sunlight but no increase in cancers
o
Median life span of si% years 80
DOU)LE STRANDED )REA( R EPAIR
•
Bomologous recombinational repair
•
ccurs during the late ; or early G2 phase of the cell cycle
•
>%change of chromosome parts due to brea*age Z reunion of intact double helices
•
Therefore H strands in(ol(ed in crosso(er $ith chiasma +cross o(er points, (isible before Metaphase
•
!onhomologous end Joining is acti(ated in G1
?. MITOC&ONDRIAL DNA MUTATION
•
=or a human disorder to be attributed to mitochondrial !"o
the inheritance must e%hibit a maternal inheritance pattern
o
the disorder must reNect a de&ciency in the bioenergetic function of the organelle
o
•
there must be a speci&c mutation in a mitochondrial gene
Buman mt!" contains 16-56D base pairs coding for 1' of o(er E proteins reCuired for aerobic cellular respiration
•
mt!" is (ery susceptible to mutations
•
oesnIt ha(e histones to protect from mutations
•
Mitochondria ha(e high concentrations of reacti(e o%ygen species +#;, generated by cell respiration
•
#; damages organelle contents +proteins- lipids- mt!",
• " 9ygote recei(es a large number of organelles through the eggA
a mutation in one or a fe$ $ill be diluted out by many mitochondria that lac* the mutation and function normally •
Beteroplasmy is the condition in $hich adult cells ha(e a (ariable mi%ture of normal and abnormal organelles
•
T$o disorders arising from mt!" are
81
o
Myoclonic epilepsy and ragged red &ber disease +M>##=, in $hich indi(iduals e%press ata%ia- deafness- dementia and sei9ures
o
FeberIs hereditary optic neuropathy +FB!, $hich is characteri9ed by blindness
•
efecti(e mt!" is implicated in the aging process
?.? USE
O- MUTATION
?.?.1 MUTATION R E,ERSAL
)ild Q mutant for$ard mutation Mutant Q $ild re(ersion •
asis of "mes Test to test mutagenicity of compounds
•
uses
any
of
a
do9en
+mutated,
strains
of
Sa/<&'e//a
typh
Therefore more lethal chromosome brea*age induced by radiation ?.?. -OR%ARD GENETICS
•
Mutants used to determine gene function
•
Mutagenise organism by mutagen such as chemical or transposon
•
solate mutant
•
Map loci o
>g. 0se complementation analysis to determine if t$o mutations causing similar phenotype are in same gene
82
83
TOPIC @ POPULATION GENETICS @.1 %h"t is Po/u7"tion Genetics
Population genetics / the study of genetic (ariation in groups of organisms uilds on Mendel?s &ndings of inheritance of traits in groups of pea plants due to genetic elements n(ol(es study of genetic (ariation $ithin and bet$een groups to e%plain e(olutionary forces eg. mutation- selection- migrationmi%ing Population genetics can help understand human disease patterns / genetic epidemiology 6.2 Buman Genetic \ariation +the spice of life, Genetic (ariation is important to sur(i(al of species because it allo$s a population to respond to en(ironmental change. >g. "ntibiotic and (accine resistance s the basis of ar$in?s theory of natural selection. !b. hange in genetic (ariation o(er time is slo$ relati(e to social change and fast relati(e to geographic change @..1 Geno!ic ,"i"tion A!on2 Indi9idu"7s
Genomics is the study of an organisms entire hereditary information +!" ma*eup, !o 2 humans ha(e e%actly the same genomic ma*eup +e%cept identical t$ins,. n a(erage the seCuences of all human genomes are DD.DR the same This lea(es only E.1R of the genome for seCuence (ariation among humans. E.1R of the genome ' million sites +loci,. The (ariation at these ' million loci contributes to (ariation in human traits +eg. appearance- disease susceptibility,. @.. Re7e9"nce of Geno!ic ,"i"tion 84
0nderstanding genomic (ariation in humans is important for researchers becauseA !" (ariants form the direct basis of heritable traits. !" (ariants can be used as informati(e mar*ers in: Bealth and medicine Mapping genes for comple% disease Predicting a patient?s response to medication =orensics science ndi(idual identi&cation Genetic "nthropology Buman migration and e(olution @.. Ty/es of Geno!ic ,"i"nts
There are t$o main types: 1. ;ingle base change e(ents
Mutations ;ingle nucleotide polymorphisms +;!Ps, . nsertion4deletion of segment of !"
\ariable number tandem repeats +\!T#, Minisatellites +repeat units of 1E / 5EE base pairs, Microsatellites +repeat units of 2 / 6 base pairs, opy !umber \ariants +!\, copies or deletions of large !" fragments +[1EEEbp, @...1 DNA Se:uence ,"i"nts 5"" Po7y!o/his!s6
There are di@erent categories of !" seCuence (ariations: ;ingle nucleotide polymorphisms +;!Ps, agcttctatct agcttctctct
;hort Tandom #epeat polymorphisms +;T#s, agtctctctctctctctctctctctatacg (CT)11 agtctctctctctctctctatacg (CT)8
opy !umber \ariants +!\s,
85
cattcaaaggagaaaggagaggtctc cattcaaaggagaggtctc
[;ingle !ucleotide Polymorphisms +;!iPs, 0sually de&ned as ha(ing a minor allele freCuency [ E.E1 +1R, in a population Most common !" (ariants by far (er 2 million ha(e been identi&ed to date. n the human genome there is on a(erage 1 ;!P e(ery 1EE 'EE bp 0seful for disease gene mapping in human populations.
Ty/es of SNPs
"nonymous ;!Ps / ;!Ps that ha(e no *no$n e@ect on gene function. These are (aluable as mar*ers only. oding ;!Ps +c;!Ps, / ;!Ps present in the gene coding region of the chromosome. These may represent disease causing (ariants. !onsynonymous ;!Ps / ;!P results in a di@erent amino acid +most li*ely to be functional, ;ynonymous ;!Ps / ;!P does not result in an amino acid change. ;!Ps are preferred for population genetic analysis o(er other types of mar*ers because: ;!Ps are most abundant genetic mar*er +polymorphism, in the human genome and pro(ide the best genome co(erage Modern ;!P chip technology allo$s rapid and ine%pensi(e genotyping Most ;!Ps are XbiallelicY $hich ma*es statistical analysis more tractable [;hort Tandem #epeats +Microsatellites,
86
Byper(ariable highly polymorphic due to high mutation rate. 0p to HE repeat (ariations for one locus ;paced appro%. 1 e(ery Mb +1-EEE-EEE bases, in the genome [opy !umber \ariants +!\s, Gains or losses +insertions or deletions, of large segments of !" 1EEEbp / 5million bps an include $hole genes and therefore can cause pathology and human disease "bout 2EEE !\s currently *no$n and may be 1EEs more @.= Genetic M"e M"/s
0seful for mapping position of genes. 0seful for disease gene mapping. Three main types i, ytogenetic Maps +hromosomal position indicated by cell staining bands, ii, Genetic Map +istance in entimorgans, ii, Physical Map +istance in base pairs, #ule of thumb 1cM ` 1Mb @.=.1 Geno!e M"/ of CN,s
Genomic \ariation atabases Mutation +isease, atabases MM $$$.ncbi.nlm.nih.go(4mim4 BGM $$$.hgmd.org B0G $$$.genomic.unimelb.edu.au4mdi ;!P atabases db;!P $$$.ncbi.nlm.nih.go(4;!P T; $$$.snp.cshl.org !\ atabase T"G http:44proJects.tcag.ca4(ariation4 Microsatellite atabases
87
G $$$.gdb.org Marsh&eld maps $$$.reseach.marsh&eldclinic.org
@.? Genoty/e "nd A77e7e -e:uency An"7ysis
0nderstanding human genomic (ariation relies on genotype and allele freCuency analysis of polymorphisms. "llele di@erent (ersions of a genetic locus or !" seCuence (ariant +" or a, iploid indi(iduals +humans, ha(e 2 possible alleles homo9ygotes same 2 alleles +"" or aa, hetero9ygotes 2 di@erent alleles +"a, Genotype freCuencies proportion of indi(iduals in the population ha(ing a particular genotype "llele freCuencies proportion of alleles in the population
@.@ Po#"#i7ity "nd Genetics
"nalysis of allele and genotype freCuencies relies on probability la$s Probability is the chance of an e(ent occurring and (alues range from E / 1 Probability of &rst child being a boy is E.5 Prob of 2 boys is E.5 ] E.5 E.25 +e(ents are independent / product la$, Prob of 2 boys or 2 girls is E.25 U E.25 E.5 +e(ents mutually e%clusi(e / sum la$,
@
[email protected] Po#"#i7ity "nd A77e7es
n terms of allele freCuencies probability that siblings $ill inherit identical alleles from parents is independent- therefore multiply probabilities.
88
Probability of one child inheriting a disease allele is E.5 or 5ER chance. Probability of ' children inheriting same disease allele isA +E.5,' E.125 +or 12.5R chance, Probability of H children inheriting same disease allele isA +E.5,H E.E625 +or 6.25R chance,
6.6.2 Calculating Genotype and Allele Frequencies
/.0 Allele (reuencies 1ary Among !opulations -In population genetics a population is a group of individuals dened ! so"e c#aracteristic$ -%roup t#oug#t to s#are a co""on set of genes &alleles' -(llele fre)uencies var! in space and ti"e ecause of evolutionar! forces* -a ne+ "utation arises spontaneousl! -selection occurs +#en one allele is advantaged &or disadvantaged' in particular iron"ent -"igration a"ong individuals occurs t#roug# separation and "i,ing of individuals -ne+ populations gro+ out of a s"all nu"er of founders &genetic drift' -(nal!sis of allele fre)uencies #elps understand t#ese evolutionar! forces and population #istories$ /.2 Relationship 3et4een allele and genotype freuencies
89
-#eoretical relations#ip et+een t#e proportion of alleles and fre)uenc! of genot!pes +as descried in 1900s ! .ard! and /einerg$ -.ard!-/einerg a+ is a "at#e"atical e)uation for predicting genot!pe fre)uencies fro" allele fre)uencies in an ideal population$ -innitel! large -not suect to evolutionar! forces -rando" "ating /.2.5 6hat is 7ardy*6ein3erg $a48 -ased on proailit! and ino"ial e,pansion rules &see c#apter 3' -or a pol!"orp#is" +it# 2 alleles &( and a' -re)uenc! of allele ( or f&(' p -re)uenc! of allele a or f&a' ) ∴ p + q = 1 (p+q)2=1
-.ard!-/einerg a+ p2 2p) )2 /.2.9 7ardy*6ein3erg &uili3rium +76& -.ard!-/einerg )uiliriu" p2 2p) )2 1 -./ e,ists +#en allele fre)uencies re"ain constant fro" generation to generation ut onl! under ideal circu"stances -no "utation selection "igration and large rando" "ating population -In realit! no population "eets t#e ./ assu"ptions perfectl! ut "ost #u"an populations do not deviate signicantl! fro" t#e "odel /.2.: Use of 7ardy*6ein3erg $a4 g$ (linis" is a recessive disorder a:ecting 1;20000 individuals$ /#at is t#e fre)uenc! of
-.ard!-/einerg )uiliriu" p2 2p) )2 1 +#ere p2 nor"al genot!pe &((' 2p) carrier genot!pe &(a' )2 alino genot!pe &aa' 1;20000 0$00005 -If )2 0$00005 t#en ) 0$007 -If ) 0$007 t#en p 1 0$007 0$993 -#erefore carrier fre)uenc! 2&0$993?0$007' 0$014 -#erefore 1$5@ of population are carriers &A1;70 people' /.; 7aplotypes analysis is also important in population genetics -( #aplot!pe is a se)uence of BC alleles stretc#ing along an e,tended seg"ent of DC( essentially a s$per alleleE
90
-.aplot!pes are usuall! in#erited as a single unit fro" parents ie$ sa"e as allelesE Ff course reco"ination can #appen$ .aplot!pe distriution represents t#e nal product of natural selection$ /.;.5 Association Among Ad less c#ance t#at reco"ination +ill separate t#e alleles of BCs t#at are close toget#er ie$ reaG up #aplot!pesE -(ssociation of BCs t#at are p#!sicall! close toget#er &ie$ alleles on sa"e #aplot!pe' is called linGage dise)uiliriu" &D' Some !ractice Questions -/#! stud! t#e genetics of populations> give so"e reasons -/#! is geno"ic variation i"portant in nature> -/#at is t#e "ost co""on t!pe of DC( se)uence variant> -/#! do geneticists favour BCs over BHs> 2 "ain reasons -Ca"e and descrie a t!pe of genetic "arGer "ap -/#at is t#e general purpose of a genetic "arGer "ap> -/#at is t#e proailit! t#at a couple +ill #ave 2 o!s and a girl> -/#at is a CJ> -Ca"e 3 evolutionar! forces t#at can cause allele fre)uencies in a population to c#ange -/#at is t#e .ard!-/einerg a+ &understand concept e)uation and application' -/#at part of t#e la+ corresponds to t#e fre)uenc! of #eteroK!gotes in a population> -./ is ased on ino"ial e,pansion rules &;' -r! doing role"s in t#e te,t ooG -Dene #aplot!pe and linGage dise)uiliriu"
91
TOPIC B GENETIC EPIDEMIOLOG' AND MULTI-ACTORIAL TRAITS Genetic e/ide!io7o2y B.1 %h"t is Genetic E/ide!io7o2y
>pidemiology the study of the distribution and determinants of disease in populations. Genetic >pidemiology The epidemiology of diseases $ith an inherited +genetic, component. n(ol(es sur(eying the genome B. DNA Se:uence ,"i"tions
There are di@erent categories of !" seCuence (ariationsA ;ingle nucleotide polymorphisms +;!iPs, agcttctatct agcttctctct
;imple Tandom #epeat Polymorphisms +;T#s, agtctctctctctctctctctctctatacg (CT)11 agtctctctctctctctctatacg (CT)8
nsertions4eletions +indels, cattcaaaggagaggtctc cattca…………ggtctc
B. Dise"se Co!/7eity
;imple isorders e%hibit predictable inheritance patterns / Mendelian 0sually due to single gene mutations rare eg. ystic &brosis- sic*le cell anemia omple% disease 92
disease does
not
e%hibit classical
Mendelian
patterns
of
inheritance. ie. genotype4phenotype correspondence brea*s do$n ue to multiple factors +genetic and en(ironmental, ie. multifactorial traits B.= -"ctos Co!/7ic"tin2 Dise"se Inheit"nce in &u!"ns
ncomplete penetrance / indi(idual inherits disease gene but does not de(elop disease Phenocopy / indi(idual has disease but does not ha(e disease gene +en(ironmental factors, Genetic heterogeneity / di@erent genes +or alleles, inNuence same disease trait Genetic nteraction / multiple genes Join forces to inNuence the disease +epistasis, ther transmission mechanisms / mitochondrial inheritance and genetic imprinting B.? Ty/es of Co!/7e t"its
ontinuous Traits eg. measurements such as height- M- bp follo$ a distribution $ith statistical mean and (ariance parameters iscrete Traits categorical +meristic, eg. mole count threshold eg. besity .6 Bo$ do $e *no$ a disease has a genetic component bser(ation the disease tends to run in families ie. familial clustering Bo$e(er- this alone is not enough to conclude that genetic factors are in(ol(ed [email protected] ScientiQc 3"ys of Dete!inin2 Genetic In9o79e!ent in Dise"se
alculating ris* to relati(es
93
T$in4adoption studies alculating Beritability [email protected] Re7"ti9e Ris 56
The magnitude of degree of familial clustering of disease indicating genetic component dentifying genetic components of disease is much easier for traits $ith high (alues of . [email protected]. &eit"#i7ity
Bo$ can $e Cuantify the relati(e contributions of genetics and en(ironment for a comple% disease 0se a measurement called heritability +B, B estimates the R of the phenotypic (ariation for a trait that is due to genes alculating heritability relies on genetic model [email protected]. Phenoty/ic ,"i"nce "nd the Genetic Mode7
Mathematical model describing the relationship among factors that e%plain (ariation in the phenotype ie. (ariance components \Phenotype \Genotype U \>n(ironment U \G]> Beritability relates to Cuantifying \ of genetic factors 2 types of heritability road ;ense +B2, !arro$ ;ense +B, road ;ense Beritability +B2, measures contribution of genetic (ariance +factors, to phenotypic +trait, (ariance B2 \G4\P !arro$ ;ense Beritability +B, more speci&cally measures contribution of
additi(e action of
di@erent alleles +additi(e (ariance \",
94
\" thought to be largest main source of genetic (arianceA others are dominance (ariance +\, and interacti(e (ariance +\, \G \" U \ U \ ;o !arro$ ;ense heritability- B \"4\P [email protected].= T3inAdo/tion Studies
Measurements of heritability rely on studies of families- especially t$ins an discriminate bet$een genetic or en(ironmental inNuences on disease traits "doption studies ha(e sho$n that the ris* of an adoptee ha(ing a disease depends more on biological parents T$in studies comparing M^ and ^ t$ins may sho$ increased similarity rates of disease +concordance, in M^ o(er ^ t$ins indicating genetic in(ol(ement. T$in ;tudies +see page 68, Mono9ygotic +M^, t$ins deri(e from single egg and ha(e identical genotypic ma*e up. ie share 1EER of genome i9ygotic +^, t$ins ha(e separate eggs and share about 5ER of genome The di@erences in disease concordance (alues bet$een M^ and ^ t$ins can be used to estimate heritability and $hether or not a disease has a strong genetic component. B.B Genetic E/ide!io7o2y
The epidemiology of diseases $ith an inherited +genetic, component. n(ol(es sur(eying the genome B.B.1 Rese"ch A//o"ches in Genetic E/ide!io7o2y
1. andidate gene targeting
95
n(ol(es targeting speci&c !" (ariants $ithin genes of suspected physiological importance #eCuires *no$ledge of disease pathophysiology 2. Genome ;canning or Mapping 0tilises adJacent !" mar*ers in the genome to indicate a region harboring a disease gene oes not reCuire *no$ledge of disease pathophysiology " na(e4unsuper(ised approach
B.B. Study Desi2ns in Genetic E/ide!io7o2y
1 "ssociation ;tudies / Tests for associations bet$een !" mar*ers and disease traits in unrelated population groups.. 2. Fin*age ;tudies / tests for coinheritance of !" mar*ers and disease traits in a@ected pedigrees B.B..1 Associ"tion Studies
iseasemar*er association e%ists $hen alleles at the mar*er locus occur $ith di@erent relati(e freCuencies in a@ected and una@ected +control, patient groups: nb see lecture notes for more detail about study design as $ell as e%amples %h"t "#out 2ene>2ene inte"ctions
Typically genetic association studies ha(e focused on only one gene +(ariant, at a time. =or comple% diseases any single gene +(ariant, is only li*ely to confer a relati(ely small e@ect on the trait ie. has minor clinical importance The combined e@ect of multiple genes is more li*ely to be more important medically. Therefore- strategies are reCuired that consider genegene interactions
96
Geno!e>%ide Associ"tion Sc"ns
0seful $henA family +inheritance, information is not a(ailable eg. !" ban*sadult onset disorders +usually more common, =ocuses on unrelated population groups not pedigrees 0seful for &nding multiple genes each ha(ing a small impact on disease +lo$ penetrance, >g.
diseases
include
type
2
diabetes-
rohn?s
isease-
schi9ophrenia- breast cancer- migraine Most popular approach these days because of ;!P genotyping technology G)"; are based on !" mar*ers called ;ingle !ucleotide Polymorphisms +;!Ps, B.B.. Genetic Associ"tion Study Desi2n
To perform G)"; it is estimated that `5EE-EEE !" mar*ers +;!Ps, spanning the genome reCuire genotyping. 1EEE cases and 1EEE controls may be needed to &nd lo$ penetrance genes in G)"; 1 billion genotypes ost of Genome$ide "ssociation studies B.B.. %h"t do 3e do 3hen 3e Qnd the 2enes
1. 0nderstand the genetic mechanisms that cause the disease pathology. 2. etermine the strength of disease gene association ie. Bo$ $ell does genotype predict disease phenotype '. etermine ho$ the disease gene association is modi&ed by non genetic
+en(ironmental,
factors
+ie.
genebyen(ironment
interaction,. So!e P"ctice uestions
97
e&nitions of epidemiology ompare and contrast simple (s comple% disease Ono$ the factors that complicate disease inheritance 0nderstand section on heritability )hat are the 2 main approaches to mapping genes and $hat is the di@erence )hat is the de&nition of association in genetic epidemiology 0nderstand casecontrol analysis strategy
including testing for
genotype4phenotype association using chisCuared test. )hat has happened to facilitate the $hole genome association scanning approach )hat do $e do $hen $e &nd disease genes
98
TOPIC F EPIGENETICS F.1 DE-INITIONS •
>pigenetics is the study of gene e%pression changes that do not in(ol(e changes to the !" seCuence
•
>pig >p igen enet etic ic trai traits ts are are stab stably ly inhe inheri rite ted d ph phen enot otyp ypes es th that at are are transmitted (ia mitosis and meiosis
•
The epigenome is the cell speci&c epigenetic state of a cell $hich can be modi&ed throughout an organism?s lifetime
F.1.1 EPIGENETIC P AT&%A AT&%A'S 'S
•
Thre Three e cate catego gori ries es of pa path th$a $ays ys th that at esta establ blis ish h an and d main mainta tain in epigenetic state 1. E/i2en"tos:
en(ir n(iron onme ment ntal al sig signa nals ls th tha at stim stimul ula ate
an
intracellular path$ay 2. E/i2en E/i2eneti etic c
initi" initi"to tos s:
produce
the
response
to
the
epig ep igin inat ator or sign signal alss an and d de de&n &ne e th the e loca locati tion on incl includ ude e !" !" binding proteins- noncoding #!"s and proteinprotein signal transduction path$ays '. M"int"ines: maintain the response once the modi&cations ha(e occurred / include !" methylases- histone acetylases and deacetylases
99
F. EPIGENETIC MEC&ANISMS F..1 MET&'LATION
•
The re(ersible modi&cation of !" by the addition or remo(al of methyl groups
•
n mammals- methylation of !" ta*es place after replication and during di@erentiation of adult cells
•
Methylation in(ol(es the addition of a methyl group catalysed by methyltransferase methyltransferase en9ymes
•
This occurs on cytosine bases adJacent to guanine called pG dinucleotides- $hich are clustered in regions called pG islands
•
pG pG isla island ndss are are loca locate ted d in an and d ne near ar prom promot oter er seCu seCuen ence cess adJacent to genes
•
0nm 0nmeth ethylat lated
pG
islan sland ds
adJace Jacen nt
to
essen ssenti tia al
gen enes es
+house*eeping genes, and cellspeci&c genes are a(ailable for transcription •
th ther er gen enes es $ith $ith adJace Jacent nt meth ethyla ylated ted pG pG isla islan nds are are transcriptionally transcriptionally silenced
•
The bul* of methylated pG dinucleotides are found in repetiti(e
100
!" !" seCu seCuen ence cess loca locate ted d in he hete tero roch chro roma mati tic c regi region onss of th the e genome including the centromere •
Bete Be tero roch chro roma mati tic c
meth methyl ylat atio ion n
also also
main mainta tain inss
chro chromo moso some me
stabil stability ity by pre(en pre(entin ting g transl transloca ocatio tion n and other other chromo chromosom somal al abnormalities F.. . . * INACTI,ATION
•
ne of the W chromosomes in each somatic cell of mammalian females is inacti(ated by con(erting them into heterochromatin $ith altered patterns of methylation
•
This pro(ides dosage compensation for W chromosome genes
•
r Mary Mary Fyon Fyon propo proposed sed Winact inacti(a i(atio tion n ie. Fyoni Fyoni9at 9ation ion in the 1D6Es
• "n essential element of FyonIs hypothesis $as the random
nature of the inacti(ation process •
;igna ignall from rom W ina inacti( cti(at atio ion n cent centrre +W +W,, at WC1' WC1' cau ause sess inacti(ation
•
Gene called >?S $ithin $ithin W coats inacti(e W
F.. &ISTONE MODI-ICAT ION
•
hromatin is composed of !" $ound around an octamer of histone proteins to form nucleosomes
• "mino acids in the !terminal region of the histones can be
co(alently
modi&ed
by
acetylation-
methylation-
and
phosphorylation •
Modi&cations occurs at conser(ed amino acid seCuences in the !terminal histone tails- $hich protrude from the nucleosome
•
hem hemic ical al modi modi&c &cat atio ion n of hist histon ones es alte alters rs th the e stru struct ctur ure e of
101
chromatin-
ma*ing genes accessible or
inaccessible for
transcription • "cetylation by histone acetyltransferase +B"T, opens up the
chromatin structure- ma*ing genes a(ailable for transcription •
#emo(al of the acetyl groups by histone deacetylase +B", closes the con&guration- silencing genes by ma*ing them una(ailable
•
The sum of the comple% patterns and interactions of histone modi&cations that change chromatin organi9ation and gene e%pression is called the histone code
F..= SMALL NON>CODING RNA S 5SIRNA S6
•
si#!"s also participate in epigenetic regulation of gene e%pression
• "fter transcription- si#!"s associate $ith protein comple%es to
form #!"induced silencing comple%es +#;s, •
si#!"s can silence genes by interfering $ith transcription initiation
•
si#!"s complementary to promoter regions bind to a promoter that bloc*s the assembly of preinitiation comple% by pre(enting the binding of transcription factor T= and #!" polymerase
•
;hort #!" molecules can also associate $ith protein comple%es to form #!"induced transcriptional silencing +#T;, comple%es
•
#T;
comple%es
initiate
formation
of
facultati(e
heterochromatin that silences genes located $ithin these ne$ly created heterochromatic regions •
This is re(ersible and can be con(erted to euchromatin- $hich is accessible for transcription.
F. EPIGENETICS AND IMPRINTING
102
•
Genomic imprinting in mammals results in the e%pression of the alleles of a gi(en gene being dependent on their parental origin ie. some genes are turned on +acti(e, only on the copy that is inherited from a personIs father $hile others are acti(e only on the copy from the person?s mother. This parentspeci&c gene acti(ation
is
caused
by
a
phenomenon
called
genomic
imprinting. •
!" methylation is a *ey mechanism of imprinting
•
mprinted genes play maJor roles in controlling gro$th during embryonic and prenatal de(elopment
• ?=" gene encoding the insulinli*e gro$th factor2 is an
e%ample of an imprinted gene •
n humans +and other mammals li*e mice and pigs, the ?=" allele inherited from the father +paternal, is e%pressedA the allele inherited from the mother is not.
•
f both alleles should begin to be e%pressed in a cell- that cell may de(elop into a cancerous cell.
•
Most human disorders associated $ith imprinting ha(e their origins during foetal gro$th and de(elopment eg.
•
Prader)illi syndrome occurs due to loss of function of genes in a particular region of chromosome 15- $hich are e%pressed only in the paternal allele. ommon characteristics include abnormal gro$th and body composition +small stature- (ery lo$ lean body mass- and earlyonset childhood obesity,- hypotonia +$ea* muscles, at birth- insatiable hunger- e%treme obesity- and intellectual disability.
• "ngelman?s syndrome occurs due to loss of function of genes in
a particular region of chromosome 15- $hich are e%pressed only in the maternal allele. t is characteri9ed by de(elopmental disabilities- sei9ures- speech de&cits- and motor oddities. •
ec*$ith)eidmann syndrome is caused by altered methylation
103
of the regulatory regions of genes in(ol(ed in abnormal gro$th such as ?=". ommon characteristics include enlargement of some organs and tissues- large prominent eyes- large tonguesei9ures. •
>%ternal or internal factors that disturb the epigenetic pattern of imprinting or the e%pression of imprinted genes can ha(e serious phenotypic conseCuences
•
n (itro fertili9ation +\=, in humans can cause problems $ith imprinted genes.
•
hildren born after \= and other "#T procedures are at ris* of ha(e (ery lo$ birth $eight and ha(e an increased ris* of "ngelman?s syndrome and ec*$ith)eidmann syndrome
F..1 UNIPARENTAL DISOM'
•
0niparental disomy +0P, occurs $hen an indi(idual recei(es both copies of a chromosome from one parent only
•
Problems occur $hen the chromosome in(ol(ed in the 0P is imprinted
F.= EPIGENETICS AND C ANCER •
Bypomethylation is a property of all cancers e%amined to date
•
=or some comple% diseases- there are strong lin*s to some en(ironmental factors- such as smo*ing and lung cancer
•
n the 1D8Es =einberg and \ogelstein obser(ed that colon cancer cells had much lo$er le(els of methylation than normal cells deri(ed from the same tissue
•
The epigenetic states of normal cells are greatly altered in cancer cells- and other epigenetic changes- including selecti(e hypermethylation and gene silencing- are also present in cancer
104
cells •
ancer is no$ (ie$ed as a disease that in(ol(es both epigenetic and genetic changes that lead to alterations in gene e%pression
•
!" hypomethylation re(erses the inacti(ation of genes- leading to unrestricted transcription of many gene sets including oncogenes
•
)hile
hypomethylation
is
a
hallmar*
of
cancer
cells-
hypermethylation at pG islands and inacti(ation of certain genes- including tumoursuppressor genes are also found in many cancers • 2R+ # is hypermethylated and inacti(ated in breast cancer and
o(arian cancer •
The combination of mutation and hypermethylation occurs in familial forms of cancer eg. @"+ mutation in bladder cancer
•
ancer cells also sho$ disrupted histone modi&cation pro&les
•
Mutations in genes encoding members of the histonemodifying proteins
histone
acetyltransferase
+B"T,
and
histone
deacetylase +B", are lin*ed to the de(elopment of cancer eg. #ubensteinTaybi syndrome F.=.1 STEM CELLS IN CANCER
•
;ince
methylation
transformation
patterns
process-
it
occur $as
(ery
proposed
early that
in
the
initiating
epigenetic changes leading to cancer may occur in stem cells residing in normal tissue •
Three lines of e(idence support stem cell in(ol(ement in epigenetic changes
•
1st: >pigenetic changes can replace mutation in silencing indi(idual tumour suppressor genes or acti(ating oncogenes.
•
2nd: Global hypomethylation may cause genome instability and the largescale changes characteristic of cancer.
105
•
'rd: >pigenetic modi&cations are more e@ecti(e than mutations in transforming normal cells into malignant cells
•
The focus of epigenetic therapy is the reacti(ation of genes that ha(e been silenced by methylation or histone modi&cation
•
The =" in the 0;" has appro(ed a drug +decitabine- mar*eted as \ida9a, for treatment of acute myeloid leu*emia and myelodysplastic syndrome- a precursor to leu*emia. t is a hypomethylating drug.
F.? EPIGENETICS AND )E&A,IOUR •
>pigenetic patterns-
changesand
including
histone
alterations
modi&cation
of
may
be
methylation important
components of beha(ioural phenotypes •
n mice- t$o regions of the brain sho$ preferential e%pression of parental genes
•
Parentoforigin e@ects ha(e been sho$n in more than 8EE genes- supporting the idea that imprinting in di@erent regions of the brain may represent a maJor form of epigenetic regulation
•
n humans- epigenetic changes ha(e been documented in the progression
of
neurodegenerati(e
disorders
and
neuropsychiatric diseases $ith altered beha(ioural phenotypes eg. "l9heimer disease- Par*insonIs disease- Buntington iseaseschi9ophrenia- and bipolar disorder • " contro(ersial theory related to epigenomics concerns the idea
that epigenetic alterations lin*ed to en(ironmental signals during de(elopment or early in life inNuence beha(iour +and physical health, later in adult life •
;e(eral e%periments sho$ed that beha(ioural changes $ere mediated by epigenetic methylation of !" and modi&cation of
106
histones that alter chromatin con&guration leading to altered le(els of gene e%pression •
>n(ironments e%perienced by pregnant animals or ne$borns a@ected the beha(iour and health of the o@spring as adults.
F.@ EPIGENETICS AND
•
T&E EN,IRONMENT
>n(ironmental agents including nutrition- chemicals- and physical factors such as temperature can alter gene e%pression by a@ecting the epigenetic state of the genome
•
The clearest e(idence for the role of en(ironmental factors comes from studies in e%perimental animals
[email protected] DIET • " reduced protein diet fed to rats during pregnancy results in
permanent changes in the e%pression of se(eral genes in the = 1 and =2 o@spring eg "gouti •
n rodents- unmethylated agouti gene yello$ coat colourobese and prone to diabetes and cancer- methylated agouti bro$n coat and lo$ disease ris*
•
bese yello$ mice and Knormal? bro$n mice are genetically identical but the yello$ mice ha(e an epigenetic mutation.X ie. epimutation
•
#odents studies sho$ that e%posure to chemicals such as P" can also hypomethylate the agouti gene and cause the yello$ obese phenotype
•
These &nding ha(e applications to epigenetic diseases in humans
•
#is* of colorectal cancer is lin*ed directly to folate dietary de&ciency and acti(ity di@erences in en9ymes leading to the 107
synthesis of methyl donors •
)omen pregnant during the 1DHH/1DH5 Xutch Bunger )interY famine in the !etherlands had children $ith increased ris* of obesity- diabetes- and coronary heart disease
•
Bypo Bypome meth thyl ylat atio ion n of th the e impr imprin inte ted d ?=" gen ene e $as $as seen seen in chil childr dren en e%po e%pose sed d in ut uter ero o du duri ring ng th the e utc utch h Bu Bung nger er )inte interr compared $ith une%posed samese% siblings. The researchers also found that ?=" $as hypomethylated in indi(iduals $hose mother motherss $ere $ere pe peric riconc oncept eptual ually ly e%pose e%posed d to famine famine-- $here $hereas as other genes including leptin $ere hypermethylated.
•
The =2 generation also had abnormal patterns of $eight gain and gro$th
•
Pater aterna nall diet diet can can also also caus cause e an e@ec e@ect. t. ;tud ;tudy y on an isol isolat ated ed ;$ed ;$edis ish h comm commun unit ity y from from 1DD 1DD on on$a $ard rdss ba base sed d on an annu nual al har(ests sho$ed that food abundance during ;GP +slo$ gro$th peri pe riod od prio priorr to pu pube bert rty, y, $as $as asso associ ciat ated ed $ith $ith a shor shorte tene ned d lifespan $hile scarcity of food $as associated $ith an e%tended sur(i(al of grandchildren grandchildren
•
n animal studies epigenetic regulation has been sho$n to be induced by both maternal under and o(ernutrition o(ernutrition $ithin genes that control lipid and carbohydrate carbohydrate metabolism and $ithin genes in(ol(ed in the central appetite/energy balance neural net$or*. n many cases- the epigenetic status of the same genes is altered both bo th by mate matern rnal al un unde der r an and d o(er o(ern nut utri riti tion on-- alth althou ough gh th the e direction of the epigenetic change is di@erent.
•
;e(eral
proJects
are
no$
focussing
on
ho$
epigenetic
mechanisms can impact health and control disease processes. >g. The !B #oadmap >pigenomics ProJect
108
TOPIC < )IOTEC&NOLOG' <.1 )IOTEC&NOLOG' •
0sing li(ing organisms to create products to enhance Cuality of life
•
#elies on genetic engineering ie. recombinant !" technologycloning- genomics techniCues
•
Bas potential to address global problems ie. health issues- food shortage 0T
•
#aises ethical- social- economic Cuestions
ECOM)INANT DNA T EC&NOLOG' <.1.1 R ECOM)INANT
•
reates arti&cial !" molecules from di@erent sources
•
May be from di@erent species
•
;e(era ;e(erall techni techniCu Cues es used used to introd introduce uce foreig foreign n !" into into host host genomes
1. DROSOP&ILA
•
Mobile transposon termed the P element-
•
P element transposons
109
o
entral region coding for transposase $ith Nan*ing '1bp #
o
Tran Transp spos osas ase e sect sectio ion n de dele lete tedd- !" !" frag fragme ment nt of inte intere rest st inse insert rted ed Z inJe inJect cted ed into into ge germ rm cell cellss- alon along g $ith $ith inta intact ct P element +ma*ing transposase, as helper
. M AMMALS
•
0sually use retro(iral (ectors
•
nJect (ector !" into fertili9ed egg
•
Transfer egg to mother for de(elopment
•
#etro(iral (ector
•
#!" code for re(erse transcriptase that con(erts to ; !" Z inserts into host genome Transgenic animals o
0T disad(antages.
o
#andom insertion
o
an inacti(ation of genes
o
!eed inducible promoter to s$itch on
o
Prone to rearrangement Z deletions
o
May infect other cells
o
ther indi(iduals not in need of therapy
• "lternati(ely "lternati(ely- alter embryonic stem cells +cells in blastocyst, in
cult cultur ure e by muta mutati ting ng or intr introd oduc uce e (ect (ector or-- micr microi oinJ nJec ectt- into into blastocyst in(ol(ed in germ line . PLANT TRANS-ORMATION
•
Plasmid large Ti +2EE*b, o
ontains a transposable element
T !" element +'E*b,
=lan*ed by 25bp #s
•
Plasmid found in soil bacteria: +gr&*acteru< +gr&*acteru< tu
•
nfe nfect ctss susc suscep epti tibl ble e plan plants ts +mos +mostt comm common on No$e No$eri ring ng plan plants ts 110
16E-EEE species, •
Tumours
at entry site +usually a $ound,
•
T !" codes for protein- stimulating di(ision of infected cells
tumours •
Transposase function in plasmid !" +not T !" directly,. Plasmid transposase inserts T !" into plant genome
•
an modify to remo(e tumour genes and add other genes eg. herbicide resistance
=. SITE>DIRECTED EU(AR'OTIC TRANS-ORMATION
•
>arly sitedirected systems used mutating chemicals fa(ouring rough areas of !"
•
)ere random- but useful $hen speci&c results not reCuired
•
More modern system uses 9inc &nger nucleases +^=!s, o
^inc &nger nucleases are themsel(es recombinant proteins made up of:
^inc&nger
domains
+speci&c
seCuence
recognition,
#estriction domain +e.g. =o*,
•
^=!s are used in pairs to impro(e speci&city
•
May be directly inserted into target cell or transiently e%pressed (ia plasmid
•
)hen bound to target seCuence- restriction domain causes a double strand brea*
•
ell acti(ates recombination based repair to replace damaged region $ith alternati(e copy
• "ddition of desired alternati(e seCuence in high concentration
facilitates production of speci&c alteration. •
an include signi&cant insertion of seCuence to ma*e fusion genes- as $ell as to delete genes or e@ect single nucleotide 111
changes in a permanent manner. •
Therefore has utility in a broad range of genetic engineering applications
•
(ercomes many of the problems associated $ith (iral (ectors. 0T o
May also result in nonhomologous endJoin repair
Gene deletion
ncogenic +cancer causing, fusion genes
May cause apoptosis
o
!eeds selection to identify properly modi&ed cells
o
eli(ery to host cells ' vv& di@icult
o
urrently still Cuite e%pensi(e
<.1. GENETIC ENGINEERING$ A PPLICATIONS
•
#esearch o
solate gene
o
;tudy gene regulation- de(elopment- function
>g. BP#T de&cient mice model for Fesch!yhan "lso mouse models for cystic &brosis- uchene Muscular ystrophy
Gene *noc*outs: yeast- drosophila- mice
Onoc*out gene in mouse cells- insert into blastocyst $ith di@erent coat colour- mate to establish homo9ygous *noc*out
•
Production o
f particular proteins
o
iochemicals- en9ymes- drugs- organic chemicals eg insulingro$th hormone
•
ommercial
112
o
!e$ phenotypes
o
>g. acteria designed to clean up oil spills- industrial $aste
o
>g. Plants disease resistance- yield
o
Berbicide resistance +*ills other plants,
•
Gene Therapy correcting de&ciencies- trials under$ay
•
;tem ells / tissue engineering- simple grafts and implants in use
•
isease iagnosis / eg. using !" probes deri(ed from recombinant !" methods
<. SPECI-IC E*AMPLES <..1 INSULIN PRODUCTION IN ) ACTERIA
•
Bumulin
•
Precursor polypeptide is preproinsulin- clea(ed to acti(e form " Z chains
• " Z subunits inserted into separate (ectors beside lac^ gene •
Fac^ and subunit transcribed and translated as unit fusion protein
•
=usion proteins clea(ed- subunits Join acti(e insulin
<.. PRODUCTION O- P&ARMACEUTICALS IN EU(AR'OTES
•
Fimitations of pro*aryote hosts: inability to modify eu*aryotic proteins- also misfolding
•
>u*aryotes hosts including cultured cells or farm animals
•
>g. α1antitripsin- de&ciency in heritable emphysema o
Gene
U
sheep
gene
promoter
into
cloning
(ector-
microinJect into sheep 9ygote 113
o
•
"dult sheep produces functional α1antitripsin
Bepatitis subunit (accine o
Gene encoding Bep surface protein cloned into yeast e%pression (ector
o
•
>%tracted- puri&ed- used as (accine
>dible (accines- not yet appro(ed- technical issues
<.. GENETIC ENGINEERING IN PLANTS
•
Prior to genetic engineering- selecti(e breeding used to enhance
•
an no$ add genes to confer resistance to herbicides and insects
•
>g. glyphosphate inhibits chloroplast en9yme >P;P and *ills plants o
nsert bacterial >P;P gene that is resistant to glyphosphate (ia Ti plasmid glyphosphate resistance
•
>g. t crops: insect resistance o
acillius thuringiensis +t, protein *ills certain insects
o
nsert gene into plants insect resistance
o
ontro(ersial because may *ill other insect species eg. Monarch butterNies
•
!utritional enhancement of plants eg. golden rice $ith enhanced betacarotene le(els
•
oncerns: safe to consume- gene transfer by crossbreeding $ith other plants- loss of natural species- builtin sterility +pos (s cons,
<..=
•
DISEASE DIAGNOSIS
#=FPs restriction fragment length polymorphisms 114
o
Polymorphism or point mutation alters clea(ing site of restriction en9yme
• ";s
allele speci&c oligonucleotides- probes detect base
changes caused by point mutations o
•
an use for \= preimplantation genetic diagnosis
ther methods eg. seCuencing
• "mniocentesis and \; allo$ prenatal sampling for genetic and
other tests •
Microarrays and genome scans can test for many mutations in genome.
•
oncerns: genetic discrimination- protection of informationcommunication of results and ris*s- PG and desirable characteristics designer babies
<..? P&AMACOGENOMICS
•
#eactions to drugs ha(e a genetic component eg. !" (ariations result in li(er en9ymes $ith di@erent capacity to metaboli9e and use certain drugs
•
#eactions to certain cancer treatment due to TPMT en9yme gene (ariants
•
linicians can tailor dosage after testing for gene (ariants
•
#ational drug design synthesises drugs that a@ect speci&c gene products +not trial and error,
<..@ GENE T&ERAP'
•
Transfer functional genes to patient (ia (ectors- usually modi&ed retro(iral
•
=irst trials ; 1DDE- "" gene into T cells
•
;ome
successful
but
problems
leu*aemia-
massi(e
inNammatory response 115
•
Many clinical trials but none appro(ed yet
•
ssues to o(ercome: inacti(ation of genes- need inducible promoter to s$itch on- prone to rearrangement Z deletions- may infect other cells or other indi(iduals not in need of therapy- ho$ to deli(er- multiple gene diseases- ho$ to control e%pression
<..B R EGENERATI,E MEDICINE
•
ased on genetic modi&cation- or more recently protein manipulation of cells to form pluripotent or multipotent stem cells. ;ome stem cells can be har(ested directly.
•
;tem cells can be induced to form speci&c cell and tissue types using signalling factors
•
;imple cultures may be su@icient for reimplantation +bone marro$ for blood system regeneration in cancer,
•
;imple structures ha(e been de(eloped for reimplantation +bone- s*in- cartilage- li(er,
•
Problems ha(e been encountered in de(eloping larger and more comple% tissues. ;ome of these ha(e been o(ercome in human and animal trials
• "rti&cial and recycled tissue sca@olds are being in(estigated to
help $ith the process
<..F NO,EL P&ARMACOLOGICAL USES
•
Production of humanised antibodies allo$s targeting of human proteins by human immune system. o
Bumanised anti\>G= antibody is used for treatment of cancer and retinal diseases in(ol(ing blood (essel gro$th.
116
o
"llo$s
suppression
of
cells
displaying
aberrant
phenotypes or absorb e%cess signalling molecules o
Made by adding human elements to speci&c antibody gene deri(ed from animal immune response to human protein and then made in cell culture.
•
!" origami methods being in(estigated for containing chemicals for deli(ery- as $ell as potential nanotechnological uses.
• "daptamers +!" fragments binding to speci&c substrates,
being in(estigated for drug targeting- cell binding and sensing proteins. <..< INDUSTRIAL USES
•
n addition to biomedical and food production- biotechnology is beginning to sho$ direct industrial uses.
•
ioremediation of to%ic substances +oil spills,
•
etection of chemicals or other organisms
•
=ermentation for alcohol production from nonfood biomass
• "rti&cial photosynthesis to produce fuel •
iological batteries and po$er generation
117
TOPIC 10 ET&ICS 10.1 ET&ICS
•
The discipline dealing $ith the consideration of $hat constitutes acceptable moral conduct
•
MaJor ethical theories: o
onseCuentialism:
moral
action
determined
by
conseCuences o o
eontology: dutybased system of analysis \irtuebased: "ristotleA role of inner character traits
o
=eminist: caring- empathy and sensiti(ity
o
#eligious: (aried- can be simple or comple%- typically ha(e many elements
o
Principlism: the principles of autonomy- bene&cence- non male&cence- Justice
10.1.1
•
)IOET&ICS
The study of the ethical- social- legal- philosophical and other related issues arising in healthcare- the biological sciences and from biotechnology
10.1.
•
% &' IS )IOET&ICS IMPORTANT
Xiotechnology operates in an en(ironment on $hich past e%periences and current norms may be insu@icient to guide our moral reasoning. The e(ere%panding possible applications of modern biotechnology and the often uncertain conseCuences ma*e it imperati(e to discuss the implications and issues arising 118
from the science.Y •
BTTP:44))).>TB; .G\ . "04
10.1.
•
)RIE- &ISTOR' O- )IOET&ICS AND MEDICAL ET&ICS
Prior to the ;econd )orld )ar- fe$ nations had la$s or e(en guidelines for the ethical conduct of human or animal research.
•
The Bippocratic ath $as often the closest thing to medical ethics in place.
•
=ollo$ing the ;econd )orld )ar- the identi&cation of atrocities ta*ing place in !a9i concentration camps $hich included medical4biological research led to the de(elopment of the !uremberg ode- the &rst maJor document to attempt to codify medical research ethics.
•
n 1D6H- the !uremberg ode $as replaced by the Belsin*i eclaration- $hich is the current international standard in describing medical ethics.
•
!either the ode nor the eclaration has force in international la$- but most countries $ith ethical la$s and systems in place use the eclaration to some degree.
•
0pta*e of the principles laid out in the ode4eclaration $as not uni(ersal and notable (iolations did occur after ))2 +e.g. the 1D'21D2 Tus*egee ;yphilis >%periment,
10. PRINCIPLES
O- MEDICAL )IOET&ICS
• Autono!y$ "ctions should not be subJected to controlling
constraints by others •
)eneQcence$ ne ought to pre(ent and remo(e e(il or harm.
ne ought to do and promote good. ne ought to $eigh and balance the possible goods against the possible harms of an
119
action •
Non!"7eQcence$ ne ought not to inNict e(il or harm.
• ustice$ eCual access to the goods in life that e(ery rational
person (alues •
+ased on the theory of principlism,
10..1
•
MEDICAL )IOET&ICS IN A USTRALIA
!ational Bealth and Medical #esearch ouncil "ct 1DD2 speci&es that !BM# $ill issue ad(ice and guidelines on ethics and related issues in the &elds of health and human and animal research.
• "ustralian Bealth >thics ommittee +"B>, o
ad(ises the !BM# on ethical issues relating to health Z de(elops guidelines for the conduct of research in(ol(ing humans
o
members include e%perts in philosophy- the ethics of medical research- public health and social science researchclinical medical practice and nursing- disability- la$religion and health consumer issues.
•
!ational ;tatement on >thical onduct in Buman #esearch +2EE,: four principles 1. #esearch merit and integrity 2. ustice '. ene&cence H. #espect
10..
•
PROCESS O- ET&ICAL R E,IE%
>thical re(ie$ for all biomedical research is pro(ided by Buman #esearch >thics ommittees +B#>s, and "nimal #esearch >thics ommittees +"#>s,.
120
•
These committees are o(erseen by the !BM# and composed of indi(iduals $ith scienti&c e%pertise as $ell as members of the community.
•
Most Buman and "nimal #esearch >thics ommittees in "ustralia use the !ational >thics "pplication =orm +!>"=, for their applications- or a modi&ed (ersion.
• "pplications reCuire the disclosure of all aspects of the intended
research- including:
•
o
Method of recruitment4)hat animals used
o
Bo$ many test subJects
o
Bo$ 'f&r
o
)hat is the scienti&c basis for the research
o
)hat tests $ill be done
o
)hat pro(isions there are for feedbac*
o
Bo$ data $ill be handled
o
)ho is pro(iding funding
o
)here $ill the research be done
o
)ho is doing the research
nce application is made- the B#> or "#> $ill discuss the merits of the application and reply to the researchers- acceptingreJecting or reCuiring modi&cation of the protocols.
•
>thical re(ie$ is for a ma%imum of &(e years +three years initially- $ith t$o years e%tension a(ailable,. ProJects ta*ing longer than this $ill need to apply multiple times.
10..
•
CLINICAL TRIALS
;peci&cally research aimed at testing a treatment in human beings.
;ome
Jurisdictions
also
include
diagnostic
and
obser(ational studies as clinical studies and may ha(e additional
121
subcategories.
•
o
Phase / ;afety testing
o
Phase / ase e@icacy and protocol testing
o
Phase / )idescale e@icacy testing
o
Phase \ / Postappro(al long term studies
>thical appro(al for these is some$hat more in(ol(ed than standard human research.
•
More codi&ed protocols are reCuired- $ith speci&c reCuirements for patient safety and reporting of ad(erse e(ents.
•
n "ustralia- the Therapeutic Goods "dministration +TG", must also appro(e a clinical trial before it can begin.
•
Most B#>s $ill also reCuire that any clinical trial is registered in a clinical trial database- such as the "ustralia and !e$ ^ealand linical Trials #egister.
10. SPECI-IC ISSUES 10..1
•
O- ET&ICAL INTEREST
ET&ICAL IMPLICATIONS O- )IOTEC&NOLOG'
There are many public concerns o(er numerous issues in biomedical research and use of biotechnology.
•
Many of these concerns are based on public perceptions of $hat is being done $ith these technologies- a not insigni&cant number
of
$hich
are
based
on
faulty
or
incomplete
understandings of the science in(ol(ed. •
!onetheless- there are a number of signi&cant and legitimate ethical
issues
in
biomedical
research
and
the
use
of
biotechnology. • "mong these concerns are: o
0se of animals in medical research +old and ongoing issue,
o
Production of genetically modi&ed food crops
122
•
o
Genetic discrimination
o
0se of genetically modi&ed organisms in industry
o
Production of arti&cial life forms
o
;tem cell research
o
Buman cloning
Many fears and concerns ha(e been ad(anced concerning these issues. These include potential dangers to human health from GM foods- the potential to create XsuperorganismsY as $ell as moral arguments o(er the use of embryonic stem cell lines.
• "s scientists- it is our responsibility to ensure that the public is
su@iciently educated about the speci&cs so as to allo$ a reasonable debate on the issues in(ol(ed.
10..
•
STEM CELL R ESEARC&
>mbryonic stem cells: pluripotent- can di@erentiate Z gro$ in culture- immune reJection problems- contro(ersial
• "dult stem cells may di@erentiate but not into all cells- di@icult
to gro$- a(oids reJection problems- can turn cancerous- found in bone marro$- umbilical cords and other tissues •
#esearch in the use of these is progressing constantly. ;ome early therapies are beginning to appear no$.
•
#esearch on mechanisms in embryonic stem cells ha(e allo$ed the alteration of somatic adult cells to resemble embryonic stem cells. >arly (ersions reCuired (iral transduction- but the most recently de(eloped protocols can be induced $ith proteins only.
•
#esearch into
organ
regeneration
has made
signi&cant
progression- based in part on earlier grafting techniCues. 10..
&UMAN CLONING
123
•
;omatic cell nuclear transfer +;!T, $as the techniCue used to create the &rst cloned mammal- IollyI the sheep. The nucleus of an oocyte is replaced $ith the nucleus of a somatic cell.
•
The resulting embryo could- in theory- lead to cloned human beings #eproducti(e cloning
•
;tem cells could be har(ested from the cloned embryo to form a ne$ embryonic stem cell line. The embryonic stem cells could be encouraged to de(elop into human tissue or +possibly in the future, a complete organ for transplant. Therapeutic cloning
•
;uggested alternati(es o o
"!T: "ltered nuclear transfer "#: octye assisted reprogramming
10..=
•
STEM CELL R ESEARC& AND CLONING IN A USTRALIA
n 2EE2- the =ederal Parliament passed the Pohi#ition of &u!"n
C7onin2 fo
Re/oduction
Act
00
and the
Rese"ch In9o79in2 &u!"n E!#yos Act 00 . • "mended in The Prohibition of Buman loning for #eproduction
and the #egulation of Buman >mbryo #esearch "mendment "ct 2EE6. •
The "ct states that only embryos that are left o(er from "#T +"ssisted #eproducti(e Technology, treatments can be used to deri(e human embryonic stem cell lines for research. >mbryos cannot be created purely for the purposes of research.
•
Buman cloning is banned in "ustralia under the Prohibition of Buman loning for #eproduction "ct.
•
t is no$ possible to apply for a license to conduct human cloning for ma*ing embryonic stem cells. The total ban on human cloning for reproduction $ill remain.
124
TOPIC 11 Genetic An"7ysis of C"nce 11.1 DE-INITION AND C AUSES 11.1.1
•
O- C ANCER
DE-INITION O- C ANCER
ancer is de&ned as the abnormal- uncontrolled proliferation of cells
•
Tumours can be classi&ed as benign +localised gro$th, or malignant +cancerous,
•
ancer cells di@er from normal cells in ' $ays
They e%hibit unconto77ed /o7ife"tion that is not regulated as in normal cells
They gro$ e%tensi(ely and in9"de surrounding tissue They are capable of !et"st"sis +resulting in formation of secondary tumours,
•
!ormal cells are under gene control- cancer cells de(elop due to mutations in genes controlling basic cellular function
•
ancer cells sho$ higher than normal rates of mutationchromosomal abnormalities and genomic instability
•
ancer cells are clonal
125
•
They originate from a common ancestral cell that accumulated numerous mutations
•
ancer stem cell hypothesis
on(entional hypothesis (s ancer ;tem ell Bypothesis
;tem cells ha(e important characteristics that distinguish them from other cell types
11.1.
•
GENETIC C AUSES O- C ANCER
hanges in genes that normally control cell gro$th and di@erentiation can result in cancer
Genes that control gro$th and di@erentiation &t into 2 broad categories: oncogenes +act in a dominant fashion, and tumour suppressor genes +act in a recessi(e fashion,
•
hanges in as little as 1 or 2 genes may result in canceralthough usually mutations in 612 di@erent genes reCuired
• "lthough a particular gene may not be directly responsible for
allo$ing uncontrolled gro$th- it may be in(ol(ed in signalling path$ays
to
indirectly
a@ect
gro$th-
additionally
some
susceptibility genes may not be in(ol(ed in gro$th but may be responsible for deto%i&cation of particular carcinogens • "poptosis +programmed cell suicide, usually destroys cells $ith
mutations- but sometimes mutations result in the inacti(ation of this process allo$ing damaged cells to proliferate
+ncogenes •
C"nce c"usin2 2enes
Genes $hose products are in(ol(ed in transforming cells in culture or inducing cancer in animals
May be caused by (irus or altered cellular gene
126
• "ct in a dominant fashion- therefore a mutation in only 1 allele
reCuired to cause cancer •
Protooncogenes are normal cellular genes that if mutated +altered, become oncogenes
Protooncogenes code for proteins that respond to signals from other cells to stimulate gro$th
)hen mutated to form oncogenes- gro$th path$ays are continuously acti(e and may resulting in uncontrolled cell gro$th
ancer may result from:
Point
mutations
of
protooncogenes
to
become
oncogenes
hromosome rearrangements such as translocations may fuse areas of chromosomes together to form oncogenes
"mpli&cation of protooncogenes may lead to increased e%pression +therefore beha(ing as oncogenes,
ouble minute chromosomes are acentric chromosome fragments that may contribute to the de(elopment of tumours by pro(iding ampli&ed oncogenes
Bomogenously staining regions are chromosomally integrated
forms
of
ampli&ed
!"
containing
oncogenes
\iral insertion may alter promoter regions- increasing transcription and leading to altered e%pression
•
There are appro%imately 1EE *no$n oncogenes- some common e%amples are:
0a-R+S +cellular membrane protein, associated $ith lung-
bladder- breast- *idney and colon cancers
A +factor in(ol(ed in acti(ating transcription of gro$th 127
promoting genes, associated $ith lung- breast- o(arian and colon cancers
yc/'s +regulate cell cycle, associated $ith lung and
esophagus cancers- many others •
ncogenes can act almost any$here on or $ithin the cellincluding cell surface receptors- nucleus- cytoplasm
u#our Suppressor Genes 6SGs7 •
Genes that control abnormal cell proliferation
Mutations result in a loss of acti(ity of these genestherefore a loss of gro$th control path$ays resulting in uncontrolled gro$th
•
Mutations can be caused by:
eletions
Point mutations
Methylation at pG islands
Onudson?s t$o hit hypothesis: T;Gs act in a recessi(e fashiontherefore mutations in both alleles are reCuired to cause cancer
;poradic cancers: t$o somatic mutations in same cell
=amilial cancers: one inherited mutation and one somatic mutation
•
>%amples
%53 pre(ents the cell progressing to the ; phase of gro$th
and is li*ely to play a role in transcriptional control- it is associated $ith o(er half of all cancers including lung- s*inbladder- breast and brain cancers
R2# also pre(ents cell progressing to ; phase of gro$th by
inacti(ating cellular transcription factors and is associated 128
$ith retinoblastomas- sarcomas- breast and bladder cancer
2R+" in(ol(ed in !" repair- associated $ith breast-
o(arian- prostate cancers.
11.1.
•
CELL C 'CLE AND C ANCER
ancer ells ontain Genetic efects "@ecting ellycle #egulation
•
Gro$th and di@erentiation of cells are strictly regulated.
•
n cancer cells- many of the genes that control these functions are mutated or aberrantly e%pressed- leading to uncontrolled cell proliferation.
11.1.=
•
EN,IRONMENTAL C AUSES O- C ANCER
Many cancers sho$ no form of inheritance and are li*ely due to e%posure to carcinogens
•
>%posure to carcinogens usually causes mutations in the !" resulting in cancer formation
•
>%amples of this are common:
Fung cancer has been associated $ith asbestos- smo*ingradon
;*in cancer has been associated $ith 0\ radiation- coal tar and petroleum products and certain drugs and antibiotics
Fi(er cancer has been associated $ith P\ +only in $or*ers,- iron o(erload- alcohol induced li(er damage and arsenic
ladder cancer has been associated $ith aniline dyescigarette smo*ing and ingestion of analgesics or arti&cial
129
s$eeteners •
t is sometimes hard to distinguish bet$een genetic Z en(ironmental components of speci&c cancers and many cancers are due to a combination bet$een genetic predisposition and en(ironmental carcinogen e%posure +causing mutations in cell cycle related genes,
• "ge plays a role in many cancers due to an accumulation of
mutations o(er the years 11.1.?
CLASSI-ICATION O- C ANCER
•
Feu*emia: blood
•
arcinoma: from endoderm +gut- intestines, or ectoderm +epidermis- ner(ous system,
•
;arcoma: from mesoderm +muscle- blood- connecti(e tissue,
11. C ARCINOGENESIS AND METASTASIS 11..1
•
C ARCINOGENESIS 5C ANCER -ORMATION6
ancer is caused by mutations at the molecular le(el that allo$ for uncontrolled gro$th and di@erentiation and is characterised by three steps:
nitiation: a permanent and inheritable change in the cell?s genome
Promotion: proliferation of initiated cells
Progression: further genetic alterations that cause genomic instability and transformation into a malignant phenotype
130
8nitiation •
May be inherited or caused by en(ironmental factors
•
>arly initiation may be epigenetic- later &%ed by mutation
•
=or the initiation process to be &%ed +irre(ersible,- one or t$o rounds of cell di(ision $ith the mutation must occur
•
nitiated cells appear the same as normal cells
•
Genetic mutation is passed on inde&nitely to cell?s progeny
•
Most initiated cell?s remain dormant throughout a host?s lifetime
Pro#otion •
Promoting agents +carcinogens, stimulate the rate of cell di(ision and can inhibit apoptosis in initiated cells
•
Promotion is a re(ersible process
f the promoting agent is remo(ed- clonal e%pansion $ill cease and regression $ill occur
•
ncreases li*elihood that cell may be further altered in progression phase
Progression •
May be caused by random somatic mutations or by e%posure to additional
mutagenic
agents
or
epigenetic
modi&cations
fa(ouring gro$th •
ells appear di@erent to normal cells
•
ncreased gro$th rate- in(asi(eness and metastatic capability as $ell as biochemical and molecular changes occur $ithin the cells
131
11..
•
METASTASIS 5C ANCER SPREADING 6
Metastasis is de&ned as a neoplasm arising from another neoplasm that it is no longer in continuity $ith
•
n simple terms- it is the spreading of cancer to other areas of the body by the circulatory or lymph systems
•
To
undergo
metastasis-
cells
adJust
genetically
and
epigenetically- resulting in a (ariety of biological properties •
Metastasis is an acti(e process- resembling $ound healing
•
The process may commence earlier than pre(iously belie(ed
•
;uccessful metastasis in(ol(es 6 steps:
;eparation
n(asion and intra(asation
irculation
"rrest
>%tra(asation and in(asion
Gro$th
Separation •
ell detaches from neighbouring cells
•
ell must sur(i(e $ithout the usual cell to cell interactions
•
t is belie(ed that tumour cells de(elop a mechanism- $hich brea*s do$n cellular adhesion
•
Many separated tumour cells are not (iable- those that are ha(e usually made adaptions such as production of gro$th factors
132
8nvasion an' 8ntravasation •
n(asion is penetration
through
e%tracellular matri% +eg.
asement membranes- interstitial stroma- cartilage and bone, into (essels
ccurs through receptor attachment of tumour cell to tissue- secretion of hydrolytic en9ymes- chemota%is and motility factors
•
ntra(asation is repeated in(asion- many cells mo(e from tissue to (essels
ften accompanied by angiogenesis +recruitment of ne$ blood (essels,
•
ells may remain in (essels- proliferate and circulate at another time or may circulate immediately
•
#ole of the e%tracellular matri% and its constituents
Circulation •
ells enter circulation +blood or lymphatic (essels,
•
Bostile en(ironment +immunological and mechanical forces destroy many tumour cells,
•
Most rate limiting step +SE.E1R circulating tumour cells initiate colonies
Bomotypic or heterotypic aggregation may be used as a defence mechanism to protect a small number of cells
"rrest •
Fodging of tumour cells in the (essels of the target organ 133
•
Mechanical $edging- entrapment or attachment
May be nonspeci&c or sitespeci&c
8nvasion an' Extravasation •
egradation and mo(ement into the tissue +same as in(asion and intra(asation, *
;uccess depends on immune status of cancer cells and
induction of angiogenesis
Gro)t! •
Tumour gro$th occurs $ith the aid of tumour gro$th factors
•
;uccessful metastasis allo$s for subseCuent metastases $ith impro(ed e@iciency
•
This may be the limiting factor for successful metastasis- as other factors may occur early.
11..
C ANCER STEM CELL & 'POT&ESIS
•
Bypothesis that cancers may harbour a form of stem cell.
•
ancer stem cells beha(e li*e transformed normal stem cells$ith some cells remaining relati(ely Cuiescent $hile others rapidly reproduce. The XtissueY they regenerate is a tumourperhaps retaining (estigial function.
•
ue to Cuiescence- cancer stem cells are not a@ected by common treatments.
•
They may e%plain ability of tumours to self rene$ and be a component of tumour immortality.
•
;till some$hat contro(ersial as to the nature of the stem nature
134
of the cells. •
ebate as to $hether the cells are transformed stem cellstransformed progenitor cells- or mutated somatic cells that ha(e regained stem cellli*e functions.
11. C ANCER STUDIES 11..1
•
- AMIL' STUDIES
n maJority of cases- there is no general predisposition to cancerbut there is to s/eciQc c"nces
3reast Cancer •
f ha(e 1st degree relati(e $ith disease- 1ER chance female $ill de(elop by 85yrs +controls 5R chance by 85yrs,
•
>n(ironmental inNuence: childbearing reduces ris* of breast cancer
Sto#ac! Cancer •
1st degree relati(e a@ected results in 'R chance +control group 1.5R,
•
>n(ironmental inNuence: lo$er socioeconomic group increases stomach cancer ris*
11..
T %IN STUDIES
•
an be used to determine genetic (s en(ironmental components
•
reast Z 0terus ancer
oncordance higher in t$ins- ie more chances of both siblings a@ected if identical than nonidenticalA therefore larger genetic component
135
•
;tomach ancer
Most identical t$ins discordentA therefore en(ironmental factors more important than breast cancer but still must be genetic component since more concordant t$ins than general population
11..
•
A NIMAL STUDIES
0sually use mouse models
reed strains susceptible to de(eloping particular cancers
>g. 'B strain +DER or more, de(elop breast cancer
e(elopment reduced by dietary restrictions
11..=
•
, IRAL STUDIES
n mice sho$n that mil* transmits a (irus to ne$born that can greatly increase cancer in cancer prone strains
•
>(idence of oncogenic (iruses in(ol(ed $ith speci&c cancers
>g. abl oncogene chronic myeloid leu*aemia
11..?
•
)IOC&EMICAL STUDIES
#ecent biochemical lin* genotype Z en(ironment $ith lung cancer
• "BB: en9yme aryl hydrocarbon hydro%ylase
rea*s do$n speci&c polycyclic hydrocarbons +from tobacco smo*e, to carcinogenic metabolites
"bility to induce this en9yme is (ariable Z genetically controlled +multifactorial,
&i2h induces - greater ris* of lung cancer if smo*e
136
11..@
•
IN UTERO E*POSURE
ertain cancers sho$ strong lin* to inutero e%posure
>g. ;tilboestrol +gi(en to mothers $hen may abort, (aginal cancer in female o@spring
>g. Phenytoin neuroblastoma
11..B
MOLECULAR STUDIES O- C ANCER
•
Many studies no$ on genes Z !" studies in(ol(ed in cancer
•
an in(ol(e family based studies on speci&c mutations or studies on molecular characteristics and classi&cation
•
;tudies for molecular biology and classi&cation can lead to impro(ed treatment
•
=amily based studies gi(e insights into particular cancer syndromes or the acti(ity of speci&c genes
•
>g. #etinoblastoma
"uto dominant +HER of cases, =ound en9yme- esterase - from 1'C1H- sho$ed lin*age in autosomal
dominant
families
used
to
identify
retinoblastoma gene- $hich is a tu!ou su//esso 2ene
A#sence of gene leads to tumour
=ound single mutation inherited as auto dominant- $ith sporadic somatic e(ent in de(eloping cells of retina +due to uneCual crossingo(er as an e%ample,
f not inherited- get double sporadic de(elopment- therefore less li*ely Z later onset Z more chance unilateral
11.= E*AMPLES
O-
C ANCER
%IT&
PRIMAR' GENETIC
C AUSES
137
11.=.1
POL'POSIS COLI
• "utosomal dominant polys of large bone •
!ormally- polyps in the bo$el are not a problem- polyposis coli is a large number of polyps in the bo$el- ma*ing the person susceptible to cancer
•
Bigh ris* of polyps becoming cancerous [ DER a@ected die of bo$el cancer
•
#=FPs lin*ed to gene at 5C 2122
•
Mutations +single base and small insertions and deletions, $ithin the gene result in the disease- also di@erent forms of se(erity
• " similar pattern may be seen in s*in cancer +solar *eratosis
sCuamous cell carcinoma,
11.=.
*ERODERMA PIGMENTOSUM
• "uto recessi(e •
Bypersensiti(ity to 0\ light
•
nability to correct dimer formation resulting in s*in carcinoma +as early as H5 years old,
• "lso ocular and neurological problems •
WP repair genes ha(e been identi&ed +WP" to WPG,
efects in these di@erent repair genes result in di@erent se(erities of the disease
11.=.
NEURO-I)ROMATOSIS
• "uto dominant • "lso called \on #ec*ling*ausensI disease •
Tumours of peripheral Z central ner(ous system
138
•
;e(erity may di@er
•
T$o forms !=1 +located chromosome 1,- !=2 +located chromosome 22,
11.=.=
R ETINO)LASTOMA
• "uto dominant +in HER cases,- other cases sporadic
nherited cases: 1 germline mutation- 1 somatic mutation
;poradic cases: 2 somatic mutations
•
oth eyes- highly malignant cancer of retinal cells of the eye
•
0sual onset before 5 yrs
•
#1 gene located 1'C1H.12
11.? TREATMENT 11.?.1
O- C ANCER
CURRENT TREATMENT
Prevention •
•
Pre(enting e%posure to carcinogens
>g. !ot smo*ing to pre(ent lung cancer
>g. "(oiding sunlight to pre(ent s*in cancer
one before cancer originates
C!e#ot!erapy •
0se of drugs to treat cancer
•
ommonly used to treat cancers $hen metastasis is li*ely +treats XescapedY cells,
139
%a'iot!erapy •
0sing ionising radiation to treat cancer
•
0sed at some point during treatment of more than half cancers
•
amages !" of tumour but not of surrounding tissue
Surgery •
#emo(al of the cancerous tissue
•
Preferred method of treatment for some cancers- such as colorectal cancer
11.?.
POTENTIAL TREATMENTS AND T ARGETS
8##unot!erapy •
0se natural biological substances to acti(ate the immune system to treat cancer
•
Three main categories: immune response modi&ers- monoclonal antibodies and (accines
•
an attac* cells that ha(e XescapedY from the tumour +in process of metastasis, and is li*ely to be more selecti(e than chemotherapy
•
>g. reast cancer (accine
n mice: shrin*age of solid tumours
Trying to replicate in humans
"ngiogenesis 8n!ibitors
140
• "ngiogenesis is the formation of ne$ blood cells- these feed the
cancer cells $ith o%ygen and nutrients •
!atural and synthetic angiogenesis inhibitors may stop the gro$th of cancer
•
n animal studies angiogenesis inhibitors ha(e stopped tumour formation
•
urrently being trialled in breast- prostate- brain- lung- o(ary and other cancers
%esveratrol •
#es(eratrol +found in red $ine, has been sho$n to inhibit initiation- promotion and progression stages of cancer
;tudies performed on mice and cell lines
Preclinical trials
141
>%ample Cuestions: 1. )hat are the three *ey aspect of cancer 2. Bo$ may the cancer stem cell hypothesis a@ect treatment strategies '. f a cancer cell has )T p5' is this a good or a bad thing H. Bo$ is cell cycle chec*point control inNuenced 5. Bo$ can $e determine $hat phase of the cell cycle a cell is in 6. )hat role does the >M play in normal tissue . )hen the >M is dysregulated- ho$ can this a@ect cancer progression +Gi(e t$o e%amples,. 8. Bo$ is a person?s relati(e ris* of cancer determined D. )hat determines treatment 1E.
an +and ho$, cancer treatment be impro(ed
142
opic 12 3e!avioural Genetics
1.1 )E&A,IOURAL
GENETICS
eha(ioural genetics aims to identify genes and polymorphisms responsible for types of beha(iour- as $ell as determining ho$ they are inheritied and e%pressed
eha(ioural traits are incredibly comple% to unra(el due to multifactorial genetic and en(ironmental factors $hich a@ect beha(iour: •
Phenotypic (ariance +\ P, is the (ariation in a trait or beha(iour among indi(iduals in a sample or population.
•
Genetic (ariance +\ Gen, is the proportion of phenotypic (ariation due to genetic factorsA can be additi(e +\ " , $here alleles ha(e cumulati(e e@ects- or nonadditi(e +\ , $here dominance or epistasis can inNuence the phenotypic (ariance.
•
>n(ironmental
(ariance +\ >n(,
is
the
proportion
of
phenotypic (ariation due to en(ironmental factorsA can be common to family members +\ , or nonshared +\ >,. •
Geneen(ironment interactions can also contribute to (ariance +\ GW>,A this is $hen the en(ironment only has an e@ect on \ P in the presence of certain genotypes- and (ice (ersa.
•
Geneen(ironment correlations can also contribute to (ariance +rG>,A this is $hen certain en(ironments and certain genotypes are correlated +tend to appear together,.
143
Multiple study types are reCuired to get complete picture of genes in(ol(ed in comple% beha(iours- including: 1. "nimal studies 2. =amily4t$in4adoption studies '. asecontrol association studies
>ach study design can re(eal speci&c information about the genetic inNuence on the studied trait
1. A NIMAL
STUDIES
To use animals in studies of human beha(iour- need to e%amine $hether the model is (alid for the disease being studied: 1. Predicti(e (alidity: ho$ $ell does the animal model predict the human condition 2. onstruct (alidity: does the model accurately measure $hat it intends to measure '. =ace (alidity: does the animal model reproduce symptoms of a human condition
The follo$ing aspects of the animal model must be e%amined for (alidity: choice of species- choice of model- choice of test.
12*2*1 C!oice o, species
To select a (alid species for an animal model- need to consider the relation of the model to humans. •
b(ious di@erences in anatomy4function are e%pected but animal model must ha(e su@icient common structural organi9ation and homology to humans in the organstissues- or systems under study. 144
T$o main types of models for beha(iour- in(ertebrate and (ertebrate animal models
17 8nvertebrate ani#al #o'els
ommonly used in(ertebrates include . elegans +nematode, and rosophila +fruit Ny,
n(ertebrates ha(e a disad(antage of lesser homology $ith humans than (ertebrates- but huge ad(antages of greater practicality.
They can still be used as a (alid animal model if there are beha(ioural responses $hich parallels those obser(ed in mammals i.e. rosophila e%hibits many comple% forms of beha(ior including courtship- feeding- circadian
rhythms-
learning- memory
rosophila is the most commonly used due to many ad(antages: •
short generation time
•
small- easy and ine%pensi(e to culture
•
high fecundity +breeding rate,
•
males and females are easily distinguished
•
$ellcharacterised genome
27 Vertebrate ; Ma##alian ani#al #o'els
ommonly used in(ertebrates include primates- cats- dogs- mice
\ertebrates ha(e a ad(antage of greater homology $ith humans than in(ertebrates- but huge disad(antages of practicality and ethical concerns.
Mouse is the most commonly used mammalian model due to: •
shorter generation time than other (ertebrates
145
•
easier and less e%pensi(e to *eep4maintain
•
easy to breed and select beha(iour
•
commercial companies produce strains for purchase
•
used historically in topdo$n approach- used recently in bottomup approach
12*2*2 C!oice o, #o'el
There are t$o di@erent approaches to generating animal models to in(estigate the genes responsible for a particular disease or trait: the topdo$n and bottomup approaches.
17 top.'o)n approac!
This approach focuses on isolating a particular beha(iour then screening the animal?s genes for (ariation that might contribute to the beha(iour.
eha(iour is isolated by arti&cially selecting animals to breed based on beha(iour- $hich establishes t$o strains $ith progressi(ely greater di@erences in beha(ior. •
Termed bidirectional selection
>%amples of studies using the topdo$n approach: 1, Fearning and memory: t$o strains of rats de(eloped based on their ability to XlearnY ho$ to pass through a ma9e •
>rrorprone rats bred $ith each other- errorfree rats bred $ith each other
•
Generated strains of XdullY rats and XbrightY rats
2, "lcohol
preference:
multiple
mouse
strains
de(eloped
demonstrating di@erences in alcohol preference- metabolismand $ithdra$al symptoms.
146
• "lcohol preference measured as percentage of alcohol
consumed (oluntarily by mouse strains compared to total liCuids consumed •
Preferred percent of alcohol ranged from 1.R to D.HR in four strains
•
;trains
in(estigated
for
di@erences
in
alcohol
metaboli9ing en9ymes termed alcohol dehydrogenases +"B,
This
approach
ad(antageous
$hen
loo*ing
for
genetic
di@erences $ithin many genes or screening for no(el un*no$n genetic di@erences
17 3otto#.up approac!
This approach focuses on mutagenesis to alter genes &rst- then screening to identify mutations associated $ith beha(ioural changes.
T$o speci&c methods of generating geneticallyaltered animals: •
nserting a gene not usually present or increasing e%pression of a gene not highly e%pressed
•
termed Xtransgenic miceY
isrupting or Kturning o@? one or more genes through a targeted mutation
termed X*noc*out miceY
an ha(e X*noc*do$n miceY $here e%pression is Just decreased
This approach ad(antageous $hen loo*ing for beha(ioural changes resulting from mutation of a speci&c gene or set of genes thought to contribute to beha(iour.
147
12*2* C!oice o, test
nce a model is selected- need to choose appropriate test to measure animal?s beha(iour.
The test must appear to measure reCuested criterion i.e. construct and face (alidity
Bigher function and Xhuman feelingY can not be directly modelled +eg lo$ selfesteem- suicidal thought- depression,
Tests may instead measure comparable basic emotions and comparable4homolog fundamental
beha(ioural4neurochemical
mechanisms +an%iety- stress- memory,
;e(eral beha(iours can be assessed in mice using (arious testsfor e%ample:
1. E/7o"tion 9s -e" "nd Aniety •
pen&eld test
Test
consists
of
recording
mo(ements
and
beha(iour $hen placed in an unfamiliar standard en(ironment
ount number of defecations to indicate an%ietylocomotion to indicate e%ploration
•
>le(ated Plus Ma9e or ^ero Ma9e
oth ma9es consist of a part open- part enclosed ma9e
Montiors open area acti(ity +time spent in open areas and4or numbers of entries into open area,A more open area acti(ity reNects antian%iety beha(iour.
•
Fight4ar* o%
onsists of t$o compartments through $hich the mouse can freely mo(e- one dar*ened and one 148
illuminated
Test monitors amount of time spent in the illuminated compartmentA
=earful animals prefer dar* so more acti(ity in the illuminated area reNects antian%iety
. Soci"7 Inte"ction •
Placing t$o unfamiliar mice in a a freemo(ement en(ironment
onsists of placing the t$o unfamiliar mice in an empty no(el cage
Test monitors the type and number of interactions bet$een the mice- including acti(e a(oidance +one mouse running a$ay from the other,- attac*s or aggressi(e beha(iour- and grooming beha(iour
•
Placing a freemo(ing mouse an threechamber structured en(ironment
onsists of three chambers- &rst chamber $ith an empty $ire cup- an empty second chamber- and third chamber $ith a $ire cup containing an unfamilar mouse.
Test monitors ho$ much time4ho$ many entries the freemo(ing mouse ma*es into each chamberho$ many interactions $ith the unfamilar mouse
Fo$ le(els of social beha(iour4interaction reNects symptoms of se(eral psychiatric disordersincluding autism- depression- or schi9ophrenia
. Le"nin2 "nd Me!oy •
)ater ma9e or Morris test
onsists of placing a mouse in $arm but opaCue
149
$ater $ith a platform in a speci&c location. Mouse aims to reach platform as Cuic*ly as possible as mice don?t li*e $ater. nitially the platform is (isible $ith cues on the $all during training.
uring testing- platform is placed under $ater +in same location, and mice are tested for spatial memory retention by recording time and distance mo(ed to locate the platform as $ell as time spent in the target Cuadrant in relation to the ' other Cuadrants during the test session.
Fonger times to reach the platform or less relati(e time spent in the Cuandrant of platform indicates poorer memory performance
•
bJect recognition test
onsists of placing mouse in an empty open&eldadding an obJect and habituating the mouse to its presence- then remo(ing the old obJect and adding an identical old obJect and nonidentical ne$ obJect.
Time spent $ith each obJect during training sessions and test sessions are recorded $ith a (ideotrac*ing system +time spent $ith obJect is de&ned as close in(estigation $ithin 1cm of the obJects,
More time spent $ith the no(el obJect indicates recognition and memory of identical old obJecti.e. better memory performance
1. - AMIL' T%IN ADOPTION
STUDIES
150
"nother method of studying human beha(iour loo*s at inheritance of beha(iours through families.
an directly measure beha(iours in humans +no need for homologous beha(iours as in animal models, but limited to obser(ational analysis as e%perimental inter(ention in human families not ethical4possible.
an loo* at inheritance through family trees +pedigrees,- though e%amination of parents and siblings- through e%amination of t$in siblings- and through e%amination of adopted siblings.
12**1 &a#ily stu'ies
=amily tree studies 4 pedigrees $ere ho$ inherited beha(iour $as &rst studied historically
•
;ir =rancis Galton published a study called Bereditary Genius in 186D loo*ing at the number of Xeminent menY among families of Xeminent menY compared to general population
•
Galton also loo*ed at physical traits by $hether physical strength $as more freCuent in families of prominent arsmen and )restlers.
;trong familial clustering suggests that the trait has a genetic basis- families good for studying Mendelian traits- and families are less susceptible to confounding heterogeneity i.e. same ethnicity- similar en(ironmental inNuences.
isad(antages of family studies include di@iculty separating of shared en(ironment and shared genetics- can be di@icult to obtain largesi9ed families for su@icient statistical po$er.
12**2 )in stu'ies Galton de(eloped t$in studies.
"fter pedigrees- t$in studies $ere used to study inheritance of 151
traits as this study design allo$s partitioning of the e@ects of shared genes4en(ironment.
Mono9ygotic +M^, t$ins / due to clea(age of the embryo at an early stage •
=ormed from same egg and sperm $hich split after fertili9ation
•
Thus
M^
t$ins
share
nearly
1EER
of
genetic
polymorphisms and correlations bet$een M^ t$ins are due to 1EER shared genetic (ariance and common en(ironment: rM^ \ " U \ •
"ny phenotypic (ariation in beha(iour and traits of M^ t$ins are therefore due to nonshared en(ironmental causesA allo$s estimation of nonshared en(ironmental e@ects: \ > 1 rM^
•
Particularly useful in adoption studies $here M^ t$ins ha(e been adopted into separate en(ironments4families from young ages
i9ygotic t$ins +^, / due to polyo(ulation •
=ormed from t$o eggs released simultaneously fertili9ed by t$o di@erent sperm
•
Thus
^
t$ins
share
same
amount
of
genetic
polymorphisms as nont$in siblings +5ER, and correlations bet$een ^ t$ins are due to 5ER shared genetic (ariance and common en(ironment: •
r^ E.5 \ " U \
omparing trait correlation in ^ compared $ith M^ allo$s simple estimation of additi(e genetic (ariance and common en(ironmental (ariance: 2+rM^ / r^,
and
\ "
\ rM^ / \ "
omplications of t$in studies: •
>n(ironmental (ariance +\ >!\ , can be common +\ , or
152
nonshared +\ >, / parenting 4 teaching can be nonshared e(en if sibs are raised in same house or go to the same school if each sib is treated di@erently •
!onadditi(e genetic (ariance +dominance or epistasis, complicates the estimation of genetic and en(ironmental (ariance and reCuires more comple% analysis
•
;eparating
\ G>!
and
\>!\
complicated
by
gene
en(ironment interactions +\ GW>, and geneen(ironment correlations +rG>, •
^ t$ins do share common prenatal en(ironment +in utero,
•
M^ t$ins almost al$ays share a chorion +part of foetal membrane, $hile ^ t$ins almost al$ays do notA this may allo$ o(erestimation of genetic (ariance in M^ t$ins
1.= C ASE>CONTROL ASSOCIATION
STUDIES
More recently- casecontrol association studies used to study inheritance of traits in unrelated indi(iduals
ases and controls should be matched on confounding factors such as age- se%- ethnicity- and any other en(ironmental factors that may cause heterogeneity in the trait being studied
an e%amine indi(idual candidate genes- conduct genome$ide association scans +G)";,- and most recently- e%ome or $hole genome seCuencing.
1.? P AT&OLOGICAL ,ARIATIONS
IN )E&A,IOUR
#esearch into beha(ioural genetics has focused mostly on pathological changes in beha(iour- including ;chi9ophrenia or "l9heimer?s isease.
1.?.1 GENETIC
STUDIES IN SC&IHOP&RENIA
153
;chi9ophrenia +;^, is a comple% brain disorder a@ecting `1 percent of the population.
;ymptoms include delusions- paranoia- hallucinations- social $ithdra$al- thought disorder- altered speech- catalepsystereotypies- echopra%ia and unusual posturing and mannerisms.
i(ided into positi(e +e%cess of, and negati(e +loss of, symptoms
T$in studies analy9ing concordance suggest a genetic component
Main strategies for identifying ;^ genes include: •
Fin*age studies: Foo* for inheritance of common genes in pedigrees $ith high number of a@ected people
•
asecontrol association studies: loo* for genetic di@erences in group of ;^ patients (ersus controls
•
Microarray e%pression analysis: "nalyse di@erential gene e%pression in autopsy brain tissue
•
Pharmacologic "nimal Models: rug induced state in animal that mimics symptoms in humans- producing a beha(iour model $hich is useful for detecting e@ecti(eness of antischi9ophrenia medications i.e. "mphetamineinduced stereotypy
•
Topdo$n approach "nimal Models: reeding of strains that mimic schi9ophrenia symptoms in terms of abnormal startle response +termed prepulseinhibition de&cit,
•
ottomup approach "nimal Models: opamine receptor *noc*out mice and glutamate receptor *noc*do$n mice
"nimal models helped establish three of the most prominent theories of schi9ophrenia: the dopamine hypothesis- serotonin +or serotonin/dopamine, hypothesis- and glutamate hypothesis.
154
Schio/heni" Do/"!ine &y/othesis
;^ patients thought to ha(e dopamine +", hypersensiti(ity
opaminergic system in(ol(ed in locomotor acti(ity- emotionmoti(ation and cognition
"mphetamine causes e%cess " release at the synapse
an cause psychotic li*e symptoms in healthy people
hlorproma9ine +an antipsychotic drug, bloc*s dopamine receptor sites and relie(es symptoms of ;^ in humans and animal models
opamine receptor *noc* out mice ha(e also been sho$n to ha(e reduced response to pharmacologicallyinduced ;^li*e state compared to $ildtype mice
Schio/heni" Seotonin &y/othesis
;^li*e symptoms can be induced in pharmacologic animal models and humans through hallucinogenic drugs +F;mescalin, $hich mediate their e@ects (ia serotonin receptors.
lo9apine is e@ecti(e in reducing the negati(e symptoms of schi9ophrenia +e.g. disturbances of speech and Nattening of a@ect,- $hich seems to $or* by bloc*ing serotonin sites in the brain.
Schio/heni" G7ut"!"te &y/othesis
PP and *etamine produce beha(iour in healthy humans that closely resembles symptoms of schi9ophrenia $hich $or*s through glutamate receptors.
Positi(e symptoms are paranoia- agitation- and auditory hallucinationsA negati(e symptoms include apathy- social $ithdra$al and cogniti(e de&cits- such as impaired attention and $or*ing memory.
Glutamate receptor *noc*out or *noc*do$n mice appear to sho$ abnormal locomotor patterns- prepulse inhibition de&citand poorer performance in spatial memory tests. 155
1.?.1 GENETIC
STUDIES IN A LH&EIMER +S DISEASE
"l9heimer?s disease +", is the most common form of dementiaresulting in a decline or degeneration of cogniti(e functioning from brain atrophy- leading to a $ide range of symptoms.
;ymptoms include confusion- aggression- mood s$ings- speech problems- and memory loss.
>arly onset " +onset occurs bet$een 'E to 6E years old, •
also called familial " +or =", due to direct inheritance
•
Fate onset +onset occurs after 6E years old, •
rare form of " 5R " cases
common form of "
!o de&niti(e " diagnosis e%cept during autopsy. Medical history- neurological e(aluation +memory and mental tests etc, can be used to diagnose " as a cause of beha(ioural and cogniti(e changes.
rain atrophy can be assessed through imaging +M#- P>T scan,- and mar*ers of degeneration may be tested for in cerebrospinal Nuid +;=, analysis.
" aetiology cause un*no$n but most important ris* factors are age and family history +# 2.',- suggesting genetic component.
There are many di@erent theories for " pathophysiology- but t$o main hypotheses: •
amyloid plaCues normal neuronal protein +amyloid precursor protein, ly9ed into " fragments
•
!euro&brillary Tangles +!=T, normal microtubule stabilising Tau protein hyperphosphorylated-
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dissociates from microtubules
ottomup approach "nimal Models de(eloped to test role of protein phosphatases +PPs, in " as dysregulation of the PPs may cause hyperphosphorylation of the Tau protein found in ". •
Protein phosphatases +PPs, remo(e phosphate groups from proteins +opposite to phosphorylases,
•
PPs play a role in memory- are essential for synaptic functioning
PP *noc*out mouse model produced- then tested for memory tas* beha(ioural tests +spatial memory tested $ith $ater ma9eA associati(e memory tested $ith obJect recognition tas*, •
)ater ma9e sho$ed no di@erence bet$een $ildtype and PP *noc*out mice in time spent in the platform Cuadrant
•
bJect recognition test sho$ed a di@erence in associati(e memory bet$een $ildtype and PP *noc*out mice recognition inde% greater for PP $ildtype than *noc*out- suggesting that PP function is important for memory processes
1.@ NON>PAT&OLOGICAL ,ARIATIONS
IN )E&A,IOUR
!onpathological beha(iours also $idely studied in beha(ioural genetics.
;tudy into nonpathological beha(iours e%amines the genetic causes of normal (ariation in certain beha(iours.
ncludes study into ntelligence- "ntisocial beha(iour +"ggression- rritability,- and Personality traits +>%tra(ersion4ntra(ersion- !o(eltysee*ing or #e$ardsee*ing beha(iours,
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1[email protected] GENETIC
STUDIES IN INTELLIGENCE
ntelligence one of the &rst beha(ioural traits studied for inheritance patterns: Galton?s 0eredtary e'us 186D •
Be compared the freCuency of Xudges of >ngland;tatesmen- >nglish PremiersY and other Xeminent menY among families of the $ell*no$n Xeminent menY of his time- compared to the general population
utj Galton de&ned XintelligenceY and XsuccessY as ha(ing a particular type of JobA this is not an obJecti(e measure of intelligence. n Galton?s time sur(i(ing and Nourishing as a family o(er time reCuired considerable e@ort.
ntelligence is de&ned no$ as: the ability to reason- plan- sol(e problems- thin* abstractly- comprehend comple% ideas- learn Cuic*ly and learn from e%perience. • These things are more testable and measurable compared
$ith Galton?s de&nition Psycho!etic !ethods of !e"suin2 inte77i2ence
Psychometric tests aim to measure intelligence in a standardi9ed $ay and ha(e been found to ha(e high statistical reliability- but di@erent tests measure intelligence di@erently
Main measure of general intelligence is S/e"!"n+s 2 f"cto •
;pearman?s g measures general intelligence irrespecti(e of subJect matter +people $ith high (alues of g $ill be reasonably successful in most subJects,
•
orrelated to large numbers of physiological characteristics thought to be lin*ed to intelligence
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•
Bigh heritability estimates H8 8ER
•
;igni&cantly associatied $ith the (olume of gray matter in the brain and higher g (alues correlated $ith a higher (olume of gray matter +r2 HER,
• "lso correlated $ith the follo$ing brain characteristics:
prefrontal lobe- o(erall brain mass- rate of brain glucose metabolism- and cortical thic*ness
ther psychometric methods de(eloped after$ards $hich test intelligence in di@erent $ays and sometimes by separate subJects or separate types of thin*ing4reasoning. These include:
•
;tanford/inet ntelligence ;cales
•
#a(enIs Progressi(e Matrices
•
;cholastic "ptitude Tests +;"T,
•
ntelligence uotient +,
>ach test has gloading score $hich measures of general mental ability +estimating ;pearman?s g,
Most $idely*no$n and tested measure is
Inte77i2ence uotient 5I6
alculated by setting median result from general population sample to an arbitrary point on scale +1EE points,.
"n indi(idual?s result from the test is ran*ed compared to the results of normali9ation sample. =or e%ample- 1 standard de(iation in the population tested may eCual 15 points
has been sho$n to be a Cuantitati(e trait $ith a normal distribution in populations $hich is also highly heritable
I t3in "nd "do/ti9e studies
i@erent heritability +h2, and common en(ironment analyses +c 2, ha(e been done $ith
M^ t$ins (s ^ t$ins reared together ha(e h 2 of 5EDER
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M^ t$ins reared apart ha(e h 2 of 5R
^ t$ins reared apart ha(e h 2 of HR
0nrelated indi(iduals reared together ha(e c 2 +estimation of en(ironmental component, eCual to only HR
orrelations bet$een di@erent sibs +t$in- nont$in- adopti(e, sho$ that shared en(ironment doesn?t contribute a lot of (ariance in . n other $ords- a shared en(ironment but no shared genes $ill gi(e a lo$ correlation in scores.
The se"ch fo I 2enes
an e%amine the genetic basis of diseases in(ol(ing mental retardation to identify genes that may be essential for functioning intelligence- ho$e(er many genetic syndromes result in mental retardation
;ome candidate genes identi&ed through studies: •
Wlin*ed nonspeci&c mental retardation and G1
•
Microcephaly and ";PM
Bo$e(er- candidate gene approach failed to yield replicable results +i.e. genes that are consistently associated $ith intelligence in di@erent populations,.
an also e%amine biological path$ays $hich relate to normal brain function +i.e. neurotransmitters, • "P>kH and MT ha(e been marginally associated
$ith
an also conduct genome$ide scans +G)";, for genes • uantitati(e Trait Foci +TF, ProJect in 2EE1 aimed
to identify TFs of high +[16E, compared $ith a(erage +`1EE, by e%amining 18H2 simple seCuence repeat +;;#, mar*ers spaced at 2 cM throughout the genome • =ound no signi&cantly associated regions
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• " G)"; G)"; in 2EE8 scanned 5EE-EEE 5EE-EEE ;!Ps from from -E8D
children and found 6 ;!Ps associated $ith • Bo$e(er- no polymorphisms $ere found in protein
coding regions and o(erall the (ariation in the 6 detected ;!Ps ;!Ps accounted for only 1R of the (ariance in / problem called Xmissing heritabilityY
1.@. GENETIC
STUDIES IN A NTI NTI>SOCIAL )E&A,IOUR
=irst studies into aggression and genetics in the late 1D6Es loo*ed at a cytogenetic abnormality WVV males / in hospitali9ed and institutionali9ed men •
=irst study in ;cotland 1D65 found patients out of 2E' +'.5R, to be WVV- compared to E out of 2ED randomly selected males +ER,- but no statistical analysis
•
=alsely characterised them as aggressi(e and (iolent based only on their presence in the hospital
More recently studies into antisocial beha(iour loo* at genes related to brain acti(ity i.e. neurotransmitters
"ntisocial beha(iour beha(iour is de&ned as: deliberate interpersonal interpersonal aggression or lac* of consideration to others $hich may cause damage to societyA includes lo$le(el nuisance- minor o@ensesserious (iolence- and criminal beha(iour
Methods of !e"suin2 "nti>soci"7 #eh"9iou$
Multiple measures and scales used to monitor 4 rate antisocial beha(iour
riginally based on criminal or (iolent crime con(ictions +loo*ing at indi(iudals already con(icted of crimes,
;elfreported ;elfreported antisocial or (iolent acts $hich may or may not
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led to a con(iction
"ttitude "ttitude Cuestionnaires $hich are are predicti(e of (iolent (iolent or criminal beha(iour
i@erent heritability and genetic associations $ill be found depending on beha(iour measures
&eit"#i7ity esti!"tes usin2 t3in "nd "do/ti9e studies$
i@erent types of antisocial beha(iour and di@erent methods of measuring antisocial beha(iour can ha(e $idely di@erent heritability measures- especially if not controlling for G%> interactions and correlations •
Beritability estimates ha(e ranged from h 2 E to 1R
Metaanalysis Metaanalysis of 51 t$in and adoption studies in 2EE2 sho$ed the follo$ing estimates of (ariance components: • \ '2R +additi(e genetic e@ects, " • \ DR +nonadditi(e +nonadditi(e genetic e@ects, • \ 16R +common en(ironmental en(ironmental e@ects, • \ > H'R +nonshared en(ironmental en(ironmental e@ects,
The se"ch fo "nti>soci"7 #eh"9iou 2enes$
Many genes e%amined for association $ith antisocial beha(iourmost studies focus on polymorphisms in the follo$ing genes or $hich may regulate the follo$ing genes: •
MT +cathecholmethyltra +cathecholmethyltransferase, nsferase,
•
5BTT +;erotonin transporter, transporter,
•
TPB +tryptophan hydro%ylase,
•
#H +dopamine H receptor gene,
•
M"" +monoamine o%idase ", metaboli9es neurotransmitters neurotransmitters such as norepinephrine- serotoninand dompamine
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(ey study in "nti>soci"7 #eh"9iou$ C"/si et "7. 00 5 Science ,F?=6
;eminal $or* by apsi et al. 2EE2 e%amining both genetic polymorphism polymorphism in M"" promoter and en(ironmental factor of maltreatmentmaltreatment- as $ell as G%> interactions
Foo*ed at four di@erent measures of antisocial beha(iour $hich $ere sho$n to correlate $ell: •
iagnosed $ith onduct isorder according to diagnostic criteria
•
on(iction for a (iolent crime by age 26
•
Psychological assessment using the isposition To$ard \iolence ;cale at age 26
•
Peer assessment using "ntisocial Personality isorder symptom scale +completed by someone that *no$s them $ell,
hildhood maltreatment maltreatment sho$n to induce changes to neurotransmitter neurotransmitter systemsA hypothesised that di@erences in M"" $ill handle these changes di@erently
=indings: •
!o maltreatment both lo$ and high M"" e%pressors had 9ero or negati(e inde% for antisocial beha(iour
•
Probable maltreatment maltreatment lo$ and high M"" e%pressors had ele(ated but similar inde%es for antisocial beha(iour
•
;e(ere maltreatment both lo$ and high M"" e%pressors had ele(ated inde% for antisocial beha(iour 0T lo$ M"" e%pressors had much larger inde% of antisocial beha(iour than high M"" e%pressors
onclusion: G%> e@ect of lo$ M"" e%pression and se(ere maltreatment
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Othe Qndin2s fo 7in #et3een MAOA "nd "nti>soci"7 #eh"9iou$
"nimal models M"" *noc*out and transgenic mice associated $ith increased aggression
"ssociation studies null M"" allele lin*ed $ith antisocial beha(iour in males $ithin a utch family
TOPIC 1$ )E&A,IOURAL GENETICS PRACTICE UESTIONS 1. Fist the important considerations $hen choosing an animal model to study human beha(iour. 2. escribe the type of approach to creating an animal model that focuses on gene manipulation. '. )hat *ind of tests can be used to measure an%iety in mouse models and $hat *ind of (alidity should these tests ha(e if they are appliable to human beha(iour H. True or =alse: rosophila beha(iour is not comple% enough to be a good model to study beha(iour genetics. 5. utline one of the methods that has been used to study a pathological change in beha(iour and $hat it has disco(ered about the disease. 6. escribe t$o di@iculties in studying the genetic basis of intelligence or antisocial beha(iour. . )hy are neurotransmitter or neurotransmitterrelated genes studied most in relation to beha(iour 8. )hat is does G%> mean and $hy is it important in beha(ioural research
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