Human Molecular Genetics IV. Genetics of common diseases/ Multifactorial genetics

Human Molecular GeneticsIV. 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’

• continuous: no specific phenotype eg. length

• discontinuous: specific phenotype eg diabetes,CL/CP

critical balans, treshhold: when crossed, the phenotype appears, severity phenotype

threshold

affected dividuals

y

multifactorial inheritance

multifactorial inheritance (discontinuous)


threshold

affected dividuals

y

general population liability curve

threshold

affected dividuals

y

low but increased risk for family members of affected personsgeneral population risk for more distant relativesconsequence: one or very few affected persons in a family precludes classical

pedigree analysis for Mendelian traits

displaced liability curve for first-degree relatives


• 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 notenvironmental: MZ=DZ

• adoption studies, population and immigration studies

Evidence for multifactorial inheritence


Evidence for multifactorial inheritence

Sufficient evidence is obtained for geneticsusceptibility 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?

• methods:linkage analysis using whole genome scans

association studies using SNPscandidate gene analysisbiochemical analysiscombined approach

• study material:families/affected sibs – family membersnumbers/selection/clinical diagnosis/pheno-copies/…

animal modelsnumbers/more homogeneous genetic background


Identification of genes involved in common diseases

x1

x2

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

c c

B

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 genesusing 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 SNPscomputer simulation and experimental datasuggest that LD extends only a few kb away from SNPs

other data suggest > 100 kb

reasons for discrepanysmall studiesdifferent populations


Reich et al. Nature Genetics May 2001

rather large blocks of LD interspersed with recombination hot spots


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 comparisonpossible with modest sample sizeuseful in search for common diseases

• resequencing of 2 kb region at 0-5-10-20-40-80-160 kb272 high frequency polymorphismscalculation of allele frequency and LD


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


• situation 1 (CHD)rare monogenic disorders

known (metabolic, biochemical) pathways

• situation 2 (obsesitas)mouse models with monogenic traitspreviously unknown metabolic pathways


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

Esterifaction of fatty acids and cholesterol

Triglycerids - cholesterolFat soluble vitaminsphospholipidsapoB48/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

Uptake of remnant particles in liverby LDL receptor/LRP and chylomicron receptor

Exogenous pathway

VLDL particle: central triglycerids and cholesterol packaged with phospholipiden and one apoB100 molecule

Esterifaction of fatty acids and cholesterol

Triglycerids - cholesterolFat soluble vitaminsphospholipidsapoB48/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

Uptake of remnant particles in liverby LDL receptor/LRP and chylomicron receptor

Exogenous pathway

Synthesis of triglycerides and cholesterolin the liver

Assembly of triglycerides and cholesterol with phospho-lipids/one apoB100 moleculeand many apoC/E moleculesinto VLDL particles

IDL Formation of VLDL remants (IDL)after removal of triglycerids

Partial conversion of IDL to LDL by hydrolysis oftriglycerides to cholesteryl-ester, removal of apo’s except apoB100

Partial clearance of IDL in liver by LDLR and apoE

Endogenous pathway

Endogenous pathway

excess LDL molecules oxidiseAttract 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-estersapolipoprotein 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

• 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)

Endogenous pathway: hepatic lipoproteins

- 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

hepatic lipoproteins (2)

LDL

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

• 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

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 signal- transduction pathways

OBESITAS GENEN

‘single gene disorders’ mouse models for obesitas: causative genes are identified (agouti, fat, tubby, obese, diabetes)

Ob/Ob

Db/Db

obesitashyperfagiehypogonadisme

leptine gereguleerde centrale melanocortine circuit

Ob

agouti

MC4R

orexigenic

anorexigenic

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 receptorsobesity instead of expected anorexigenic effectreveals complexity of control mechanismsand multifactorial control

- 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

agouti/Agrp

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...

- 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-leptin receptor

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 manprobably 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 humanred: mouse QTLsgreen: 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)

Documents

Human Molecular Genetics IV. Genetics of common diseases/ Multifactorial genetics