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’ 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) Genetics of ‘common diseases’ threshold affected dividuals y general population liability curve threshold affected dividuals y 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 displaced liability curve for first-degree relatives

Genetics of ‘common diseases’ 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 Evidence for multifactorial inheritence

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?

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/pheno- copies/… animal models numbers/more homogeneous genetic background Genetics of ‘common diseases’ Identification of genes involved in common diseases

x1 x2 x1 x2 x1x2

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 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 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 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

Esterifaction of fatty acids and cholesterol 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 Uptake of remnant particles in liver by 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 - 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 Uptake of remnant particles in liver by LDL receptor/LRP and chylomicron receptor Exogenous pathway

Synthesis of triglycerides and cholesterol in the liver Assembly of triglycerides and cholesterol with phospho- lipids/one apoB100 molecule and many apoC/E molecules into VLDL particles IDLFormation of VLDL remants (IDL) after removal of triglycerids 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 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

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 % - 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 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 obesitas hyperfagie hypogonadisme

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 receptors obesity instead of expected anorexigenic effect reveals complexity of control mechanisms and 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 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)