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Human Molecular Genetics
IV. Genetics of common diseases/
Multifactorial genetics
genetics of common diseases
• coronary heart disease (CHD)
• atherosclerosis, hypertension
• cancer
• obesitas
• diabetes
• asthma
• schizophrenia
• dementia
genetics of common diseases
• rarely monogenic
• most often (i) polygenic and (ii) multifactorial
a phenotypic trait determined by
(i) interaction between several genes/loci,
each with a small additive effect
(ii) influence of environmental factors
Genetics of ‘common diseases’
multifactorial inheritance
• continuous:
no specific phenotype eg. length
• discontinuous:
specific phenotype eg diabetes,CL/CP
critical balans, treshhold: when crossed, the
phenotype appears, severity phenotype
y
threshold
affected dividuals
Genetics of ‘common diseases’
multifactorial inheritance (discontinuous)
general population liability curve
y
threshold
affected dividuals
displaced liability curve for first-degree relatives
y
threshold
affected dividuals
low but increased risk for family members of affected persons
general population risk for more distant relatives
consequence: one or very few affected persons in a family precludes classical
pedigree analysis for Mendelian traits
Genetics of ‘common diseases’
Evidence for multifactorial inheritence
• family studies
• increased incidence of a disease in particular families
• ‘common environment’: check not related individuals (spouses)
• twin concordance studies
• dizygous (DZ) vs monozygous (MZ) twins
• ‘common environment’: twins raised in different environment
concordant: both affected or neither affected
genetically determined: MZ similarly affected, DZ not
environmental: MZ=DZ
• adoption studies, population and immigration studies
Genetics of ‘common diseases’
Evidence for multifactorial inheritence
Sufficient evidence is obtained for genetic
susceptibility for a given common disorder
Which strategies for disease gene identification
can be followed?
Example of CHD:
what causes do we know for the disease?
Genetics of ‘common diseases’
Identification of genes involved in common diseases
• methods:
linkage analysis using whole genome scans
association studies using SNPs
candidate gene analysis
biochemical analysis
combined approach
• study material:
families/affected sibs – family members
numbers/selection/clinical diagnosis/phenocopies/…
animal models
numbers/more homogeneous genetic background
x2
x1
x1
x2
x1 x2
linkage
• genes are on chromosomes and thus assumed to be
linked during transmission from one generation to
another
• in reality linkage only holds for relative small
distances due to meiotic crossing over
• linkage analysis: follow the pattern of inheritance of
polymorphic markers in pedigrees in which a disease
phenotype segregates
• 1% recombination = 1cM
• RFLP, minisatellites (VNTRs),
microsatellites or (CA)n repeats
linkage
odds of linkage = likelyhood for linkage/no linkage
LOD: logarithm of the odds ratio for linkage
LOD score >3 = significant linkage
<-2 = no significant linkage
haplotypes = sets of alleles on a small chromosome
segment
A
a
B
b
c
c
association studies
association studies and LD
• linkage disequilibrium: combination of closely linked
alleles, referred to as haplotypes, originating from a
single ancestral chromosome
apparently contradictory with the expected random
association assuming the occurrence of random CO
over many generations
• cause: ‘founder’ mutations, recent mutations
• studied by polymorphic markers (RFLPs, CA repeats
, more recently SNPs)
• study of ‘inbred strains’ of mice or rats
Association studies for detection of disease genes
using linkage disequilibrium
Can we use SNPs for association studies in man
• SNPs common and rare
• SNPs coding and non coding
• analysis of haplotypes and LD using SNPs
computer simulation and experimental data
suggest that LD extends only a few kb away
from SNPs
other data suggest > 100 kb
reasons for discrepany
small studies
different populations
Association studies for detection of disease genes
using linkage disequilibrium
Reich et al. Nature Genetics May 2001
rather large blocks of LD interspersed with
recombination hot spots
Association studies for detection of disease genes
using linkage disequilibrium
Study design (Reich et al. Nature Genetics May 2001)
• 19 different chromosomal regions anchored
around a coding SNP
• finished sequence for at least 160 kb (North European)
• frequent minor (less common) allele
allows cross population comparison
possible with modest sample size
useful in search for common diseases
• resequencing of 2 kb region at 0-5-10-20-40-80-160 kb
272 high frequency polymorphisms
calculation of allele frequency and LD
Association studies for detection of disease genes
using linkage disequilibrium
Results (Reich et al. Nature Genetics May 2001)
• relatively large blocks of LD
• why?
• Study of Yorubans, Nigerian population
common ancestry with NE around 100.000 yrs ago
similar allelic combinations at short distance
half length LD is less than 5 kb
Consequences
• genome wide LD mapping probably possible but
• limited resolution, choose other populations for
refined mapping
Mouse models
• history: ~1900 ‘inbred strains of mice’
• until ~1970s: difficulties in finding the responsible defects
• later: linkage analysis, positional cloning,
genetic maps of mouse and man al lead to
identification of ‘single gene mutants’
• development of powerful statistical programs lead
to ‘quantitative trait locus’ (QTL) analysis
advantages through inbreeding and controlled
environmental factors (eg nutrition)
• knock out and transgenic mice
Genetics of ‘common diseases’
• situation 1 (CHD)
rare monogenic disorders
known (metabolic, biochemical) pathways
• situation 2 (obsesitas)
mouse models with monogenic traits
previously unknown metabolic pathways
Genetics of ‘common diseases’
coronary heart disease
• frequent - high incidence, important impact on public health
• ‘environmental’ and behavioural changes
• increasing age
• single gene disorders (rare) vs polygenic disorders (common)
• multifactorial inheritance
• multiple and complex genetic factors interacting with environment
• Duchenne muscle dystrophy vs infectious disease
Genetics of ‘common diseases’
cardiovascular disease - atherosclerosis
• hart attacks (infarct), stroke (thrombosis) and peripheral
vascular disease
• occlusions in large and middle sized arteries
• late onset as a result of chronic damage of vascular
endothelial cells
• involved elements: LDL, thrombocytes, macrophages, lymphocytes,
smooth muscle cells of intima, invasion of fat particles in blood
vessels and formation of fibrocellular atheromatuos plaques
Genetics of ‘common diseases’
CHD - atherosclerosis
• CHD: coronary heart disease
multifactorial, no Mendelian segregation
genes involved in lipoprotein level, blood pressure,
diabetes, obesitas, blood coagulation, immune system,
blood vessel reactivity
environmental factors: smoking, nutrition, exercise
interindividual variation in disease susceptibility
• predictors of risk:
- blood lipids (cholesterol)
- blood pressure
- blood coagulation factors
Genetics of ‘common diseases’
cardiovascular disease
lipoprotein metabolisme
http://www.mmip.mcgill.ca/unit2/cianflone/lect48nutrition.htm
Exogenous pathway
Esterifaction of
fatty acids and cholesterol
Uptake of remnant particles in liver
by LDL receptor/LRP and chylomicron receptor
Triglycerids - cholesterol
Fat soluble vitamins
phospholipids
apoB48/apoCI-III/apoAIV
Release of fatty acids (FFA) in
peripheral capillaries
Mediated by Lipoprotein lipase (LPL)
and co-factor apoCII
Remnant particle enriched with
cholesterol esters + apoB48 + apoE/C
VLDL particle: central triglycerids and cholesterol packaged
with phospholipiden and one apoB100 molecule
Exogenous pathway
Esterifaction of
fatty acids and cholesterol
Uptake of remnant particles in liver
by LDL receptor/LRP and chylomicron receptor
Triglycerids - cholesterol
Fat soluble vitamins
phospholipids
apoB48/apoCI-III/apoAIV
Release of fatty acids (FFA) in
peripheral capillaries
Mediated by Lipoprotein lipase (LPL)
and co-factor apoCII
Remnant particle enriched with
cholesterol esters + apoB48 + apoE/C
Endogenous pathway
Synthesis of triglycerides and cholesterol
in the liver
Partial conversion of IDL to LDL by hydrolysis of
triglycerides to cholesteryl-ester,
removal of apo’s except apoB100
Partial clearance of IDL in liver by LDLR and apoE
Assembly of triglycerides
and cholesterol with phospholipids/one apoB100 molecule
and many apoC/E molecules
into VLDL particles
IDL
Formation of VLDL remants (IDL)
after removal of triglycerids
Endogenous pathway
excess LDL molecules oxidise
Attract macrophages, transform into foam cells
upon LDL uptake, oxidation
Membrane and steroid hormone synthesis
Exogenous pathway: dietary lipid absorption and transport
• absorption of fatty acids and cholesterol in intestinal aborptive cell
• esterification to triglycerids and cholesterol-esters, respectively
• transport to lymphatic system and into plasma in the form of
chylomicrons (triglyceride rich lipoproteins)
Exogenous pathway: dietary lipid absorption and transport
• chylomicrons are large particles consisting of
core of triglycerids and cholesterol-esters
apolipoprotein apoB48 and small amounts of
apo CI, CII, CIII en E and A-IV
• metabolised (hydrolyse) in peripheral capillaries to fatty acids as
energy source for skeletal muscle tissue or for storage in fat cells,
through the action of lipoproteine lipase (LPL) and apoCII as co-factor
• following release of triglycerids, apoA en apoC are transferred to HDL by LPL,
chylomicron remnants (cholesterol-rich) are removed from circulation by
LDL receptor en LRP (low density lipoprotein receptor related
protein) mediated pathways in the liver
Endogenous pathway: hepatic lipoproteins
• liver synthesises triglycerids and cholesterol, which together with residual
dietary fat, fat-soluble vitamins and apoB100
(1 molecule per VLDL partikel) are incorporated into VLDL particles and
secreted into circulation
• aim: transport of fatty acids from liver to other tissues
• functional form results through inclusion of apoE and apoCII en CIII from HDL
• hydrolyse and removal of core-triglyceride by LPL
result: VLDL remnants = IDL (intermediate density lipoproteins)
1/2 absorbed by liver via apoB (= ligand for LDL receptor)
1/2 hydrolysed by hepatic lipase to LDL (cholesterol-ester rich)
hepatic lipoproteins (2)
LDL
- carries 60-70% of plasma cholesterol, delivers cholesterol to peripheral
tissues and to the liver for further metabolism and excretion in bile
(receptor mediated process)
- 75% taken up by liver via apoB100 (ligand for LDL receptor)
- 24% to peripheral tissues for membrane and steroid hormone biosynthesis
metabolic consequences of cholestrol uptake by cells
(1) decreased de novo cholesterol synthesis,
(2) increased conversion of cholesterol into cholesterolester
(=storage form of cholesterol)
(3) decreased expression of LDL receptors
- remaining 1% remains in circulation and can be modified by oxidation,
these oxidised LDL particles can attract ‘scavenger’ macrophages which
become foam cells as they ingest these particles
DYSLIPIDEMIAS
• familial LPL and apoCII (=LPL co-factor) deficiency
- no hydrolysis of chilomicrons and VLDL resulting in
hypertriglyceridemia, no increased risk for atherosclerose
- 1/mio except in high risk populations (eg Quebec)
- low fat intake
DYSLIPIDEMIAS
• FH, familial hypercholesterolaemia
- defect in LDL receptor gene: no LDL ‘clearance’ from circulation
(no r, precursor doens’t reach the membrane, r doens’t bind
LDL, hundreds of different mutations)
- HoZ (LDLx4-6) not older than 30 yr, HeZ (1/500)
1/2 heart attack before age of 60 yr (LDLx2)
• familial apoB100 defect
- one single mutation
- no binding of LDL to receptor
- HeZ increased LDL 50-100%
- 1/1000
DYSLIPIDEMIAS
search for ‘common variants’ in genes
influencing LDL content
• linkage studies for three genes involved in LDL metabolism
in 150 families
CYP7: cholesterol 7-hydroxylase, enzyme involved in
bile acid production
• other loci: 1p34, 13q, 15q25
• Hyplip1
- mutant mouse strain for
familial combined hyperlipidemia (FCH) phenotype
- triglycerides and/or cholesterol raised plasma levels
- fine mapping of mouse locus
- 13 candidate genes: mRNA expression and sequencing
- thioredoxin interactin protein
OBESITAS: introduction (1)
• body mass index (BMI) >30
• increased risk for NIDDM, hypertension, CHD,
reproductive problems, etc...
• 1/3 Amerikan population, increasing problem
in children
• interaction between genetic, environmental and
psychosocial factors
• energy homeostasis
OBESITAS: introduction(2)
• obesitas genes identified
• genetic predisposition
• availability of food, composition,
excersise
• “thrifty gene” hypothesis (Neel, 1999)
OBESITAS: introduciton(3)
energy balance
• energy storage when energy intake is higher than total
expenditure
• E-expenditure through physical activity,
basal metabolism and adaptive thermogenesis
OBESITAS:
control of energy-intake and body weight
• behaviour, autonomous nervous system and
neuroendocrine
• short term: start and stop eating due to hunger and
saturation, controled by neural and endocrine factors
• long term eg by leptine = hormone produced
by fat cells
• CNS ligand-receptor signaltransduction pathways
OBESITAS GENEN
‘single gene disorders’ mouse models for obesitas:
causative genes are identified
(agouti, fat, tubby, obese, diabetes)
Db/Db
Ob/Ob
obesitas
hyperfagie
hypogonadisme
orexigenic
agouti
MC4R
Ob
anorexigenic
leptine gereguleerde centrale melanocortine circuit
neuropeptide Y/Argp (Agouti related peptide)
- endogenous regulator of energy balance
- “feeding-inducing” neuropeptides
- strong expression at nucleus arcuatus
(hypothalamus)
- leads to suppression of MC4R
(melanocortin 4 receptor)
- causes increased food intake
- decreases energy expenditure
- link with insuline is unclear, not the dominant
peripheral signal molecule
- first discovered orexigenic factor
neuropeptide Y/NPY receptors
- KO mouse: normal
NPY/leptin dubble KO: reduced effect of leptin KO
- KO for 6 known receptors
obesity instead of expected anorexigenic effect
reveals complexity of control mechanisms
and multifactorial control
agouti/Agrp
- gain-of-function Argp mutant mouse:
obesity phenotype comparable with loss-of-function
for Pomc of Mc4r
- Ay mutation: ectopic expression of agouti
color, dominant obesity syndrome,
increased growth and yellow hair color
- related to Argp
leptin-leptin receptor
- leptos = thin
- hormone primarily produced by adipocyt
- belongs to cytokine family of proteins
- is responsible for complex neural respons incl
hunger, behavioural changes (search for food),
decreased metabolism, infertility...
leptin-leptin receptor
- communication concerning lon term energy storage
- other effects outside CNS: decreased
triglyceride accumulation in tissues other than fat
tissue (eg muscle, liver), contributes to
insuline resistance
- abscence of leptin signal in the presence of food
causes obesitas
- causes decreased expression of NPY/Argp
- induces starvation respons
leptin
- treatment by subcutaneous leptin injection
- 2 families with leptin mutatie
- AR
- HoZ for loss-of-function mutation
leptin-receptor
-1 family
- HoZ mutation responsable for truncation of the
cytoplasmatic domain
- class I cytokine receptor
CART/POMC/-MSH
- CART: cocaine and amphetamine related transcript
-MSH: derived from proopiomelanocortin (POMC)
- CART and POMC are induced by leptin (anorexigenic)
- produced by two neuronal populations within the
hypothalamus
- POMC: twchildren with HoZ or compound HoZ
for loss-of-function mutation
Mc4r
- hypothalamic homologue of MC1R (receptor
in melanocytes)
- KO: melanocortin obesity syndrome = agouti
but without yellow hair color
- mutation in humans are responsible for
4-5%of obesity cases (haploinsufficiency,
not dominant negative)
Other genes
• neuropeptide processing enzyme
- carboxypeptidase E: exclusively in mouse (fat)
- PC-1: discovered in man
complex obesity syndromes in mouse and man
probably as a results of POMC processing
MC3R: obesity in mouse
UCP and BAT (brown adipose tissue)
QTL analysis in mice: search for obesity genes
• effect of individual genes on energy expenditure,
hyperphagia and fat storage
• study of the effect of dietary composition
• more than 70 loci identified in mice
blue: whole genome scan human
red: mouse QTLs
green: human monogenic mutations
Breakthrough in genetic studies on common diseases
• ADAM33 gene in asthma (Nature 418:426, 2002)
• G72 in schizophrenia (PNAS 99:13675, 2002)