Introduction toIntroduction to biochemical geneticsbiochemical genetics

Viktor Kožich

Institute of Inherited Metabolic Disease1.LF UK and VFN Praha

OutlineAims of the course

Basic features of IEM population frequencyBasic features of IEM, population frequency

History of IEM

Genetic origin of IEM

Pathogenetic mechanismsPathogenetic mechanisms

Small molecules and complex molecules

Clinical features of IEM

Aims of the course on biochemical genetics

Biochemical genetics = IEMs

genetics biochemistry

clinical medicine

Case 1-Isovaleric aciduria

failure to thrive, vomiting

repeatedly alteredrepeatedly altered consciousness

k t id iketoacidosis

sweaty feet odoursweaty feet odour

Case 2-adult cystinosis

http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=gene&part=ctns

Case 3- MPS I

http://eyepathologist.com/images/KL1771.jpg

http://deti.msk.ru/plaxin_egor.jpg

http://myweb.lsbu.ac.uk/dirt/museum/margaret/438-1811-2640151.jpg

Case 4-cystinuria

http://graphics8.nytimes.com/images/2007/08/01/health/adam/17046.jpg

http://www.nature.com/ki/journal/v73/n8/images/5002790f1.jpg

Basic features of IEMs

Inborn errors of metabolism

substrateusually AR, GR

d t

clinically variable

usually enzyme

product

Clinical features of IEMs ageClinical features of IEMs-age

http://markandrich.googlepages.com/Old-woman.jpg/Old-woman-full.jpg

http://www.hrr.co.uk/acatalog/crocodile_toddler.jpghttp://www.co.shasta.ca.us/html/DSS/images/FosterParentingAdopt/infant.jpg

Clinical features of IEMs-organs

http://universe-review.ca/I10-82-organs.jpg

Cli i l f t ( ) ifi iClinical features: (non)specific signs

specific non-specificspecific non-specific

e.g. NH3, uric acid

http://img.medscape.com/pi/emed/ckb/pediatrics_genetics/941088-943343-507.jpg

http://gatsome.com/images/iq.gifhttp://www.saratogaschools.org/AcademicServices/MiddleSchool

Clinical features multisystemicClinical features-multisystemic involvement

http://www.istockphoto.com/file_thumbview_approve/5982111/2/istockphoto_5982111-human-internal-organs.jpg

Diagnostic procedures in IEMsDiagnostic procedures in IEMssubstrátsubstrát

d ktprodukt|Clinicsspecific-e.g..

smell

DNA/RNA Enzymes Metabolites

urine colornonspecific-e.g.

comaPMRdysmorfic featureshepato/myopathieother

Frequency of IEMs

newborn screening 1:1000-1:4000

selective screening at least 1:500-1:1000

frequency of heterozygotes at least 1:15q y yg

population specific exampleshigher incidence in imbred populations (PKU Turkey, organichigher incidence in imbred populations (PKU Turkey, organic acidurias Middle East)

tyrosinemia type I- Quebec

aspartylglykosaminuria- Finland

lysosomal strorage disorders- Izrael

IEMs in the Czech RepublicIEMs in the Czech Republic

sacharidy12%beta-oxidace a

OAU14%

puriny/pyrimidiny3%

AMK bez HPA

mitochondriální20%

%

AMK bez HPA18%

peroxisomální4%

HPA a PKU13%

lysosomální16%

ČR, 2005, n=127

incidence ~ 1:1000f 150 diff t di

, ,

so far ~150 different diseases

T t t f IEMTreatment of IEMs

substrát

produkt

History of biochemical genetics

1857-1936

http://www.historiadelamedicina.org/imagenes/garrod.jpg

Trophîme Bigot, 16.-17.století

http://www.thetoque.com/010605/pics/urine.jpghttp://images.google.com/imgres?imgurl=http://upload.wikimedia.org/wikipedia/commons/thumb/8/8c/Bigot,_Trophime_-_A_Doctor_Examining_Urine.jpg/

1857 L.Pasteur demonstrates microbial origin of gfermentation, R.Wirchow describes mitosis

1867 dynamit1867 dynamit

1874 jeans

1886 t bil1886 automobil=car

1890 underground

1895 cinematograf

1902 periskop el typewriter; Nobel prize for1902 periskop, el.typewriter; Nobel prize for discovery of malaria transmission

1908 Nobel prize for discovery of immunity1908 Nobel prize for discovery of immunity

1936 tape recored; Nobel prize for t itt hneurotransmitter research

Lancet, vol ii, 1902, 1616-1620

Expansion of the field

1908 • 5 diseases

2009•at least 500 diseases

cca 1960 • ca 40 50• 5 diseases

(alkaptonuria, cystinuria, porphyria,

t i it l

•at least 500 diseases •Medline 122.797 papers (28/9/2009)t tb k b b d

• ca 40-50 diseases•analytical

th dpentosuria, congenital steatorhea)•1909 first textbook

•textbook-web based•incidence at least 1:1.000

methods•incidence of PKU•PKU- 1st

•incidence unknown•dx: 4 metabolites in urine

•Dx: metabolites, enzymes, DNA•screening MS-MS

screening program (R.Guthrie)•PKU-1st treatable

•Rx noneg

•Rx for ~ 1/3 diseasesIEM (H.Bickel)

ČBiochemical genetics in the ČRPKU i 1975 D Bl h á P f H á kPKU screening 1975-Doc.Blehová, Prof.Hyánek, Ing.Mrskoš

Dx of other IEMs-leader Prof.Hyánek

labs in Prague, H.Králové, Brno, Olomouc, g , , , ,Ostrava

in 2009:in 2009:selective screening ~ 100 pacientů/rok (incidence 1:1000)1:1000)

newborn screening- 10 IEMs since October 1 2009, expected incidence 1:4000p

First IEM:

AlkaptonuriaAlkaptonuria

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http://www.mja.com.au/public/issues/184_12_190606/sha20286_fm-1b.jpghttp://www.scielo.br/img/revistas/rbr/v46n5/a14fig02.jpghttp://bjo.bmj.com/content/vol83/issue6/images/large/98532.f1.jpeg

1859 Boedeker- alkapton in urine1891 alkapton=homogentisic acid

http://www.bioinformatics.nl/webportal/background/images/mendelexperiment.gif

Garrodův revolutionary concept

Chemical individuality is determined by genes

Inborn errors of metabolism= disturbance of chemical individualityof chemical individuality

Alkaptonuria in 2009retrospective reasearch of mummy HARWA 1500 př.n.l.pateints known before Garrod1902 Garrod1909 tyrosine degradation pathway describedp y1958 enzyme defect in alkaptonuria establishedp1996 HGO gene description2003 treatment with nitisone2003 treatment with nitisone

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CoxarthrosisCoxarthrosisValvular involvementUrolithiasis

http://www.mja.com.au/public/issues/184_12_190606/sha20286_fm-1b.jpghttp://www.scielo.br/img/revistas/rbr/v46n5/a14fig02.jpghttp://bjo.bmj.com/content/vol83/issue6/images/large/98532.f1.jpeg

http://www.natuurlijkerwijs.com/english/b4f4ca00.gif

Liver HGO deficiencydeficiency

Incidence 1:250 000 ž 1 1 000 000000 až 1:1 000 000

High incidence Slovakia

Late complications pfrequent

Rx inefficientRx inefficient

http://ourworld.compuserve.com/homepages/sbrillanti/liver2.jpg

http://en.wikipedia.org/wiki/

Novel treatment in alkaptonuria

nitison (NTBC)

http://www.natuurlijkerwijs.com/english/b4f4ca00.gif

Genetic origin of IEMs

Genetic origin of IEM

Monogenic Mendelian inheritanceAR inheritanceGR/GD inheritancerarely AD inheritance

X

Mitochondrial inheritanceMitochondrial inheritanceEpigenetic changesX-inactivationX inactivationEpsistasisGene x environment interactionother

Common types of mutationsCommon types of mutations

Molecular consequences of mutationsMolecular consequences of mutations

Protein amountsProtein amounts decreasedsamesameincreased

Protein propertieschanges of isolated functionsglobal changesmisfolding

Nonprotein gene productssiRNAsiRNA

Role of mutations in evolutionRole of mutations in evolution

Source of variabilitySource of variability(less important than meiotic recombination

d bi ti fand combination of gametes)

Favourable mutations are rare and already

t i d icontained in genomes

Genetic diseases= tip ofGenetic diseases tip of iceberg (of genetic variability)

Mutations affect different domains

e.g. PAH

cytosolic enzyme

homotetramer

BH4 as cofaktor

complicatedcomplicated mechanism of catalysiscatalysis

Erlandsen H and Stevens RC, The Structural Basis of Phenylketonuria. Mol Genet Metab. 1999 Oct; 68(2):103-125.

Cellular consequences ofCellular consequences of mutations (e.g.CFTR)

http://student.biology.arizona.edu/honors97/group7/Hallick1.gif

Genotype-phenotype (M.Gaucher)

Phenotype

severesplenomegalyanemiaanemiabone disorderCNS

mildsplenomegalyanemia

unclear

http://www.nature.com/embor/journal/v4/n7/thumbs/embor873-f4.gif

unclear

Health-disease threshold

clinical manifestation

i t

clinical manifestation

environment

genes

preclinical test

genetic test

IEM treatment- modifying environment

clinical manifestation

en ironment

clinical manifestation

environment

genes

preclinical test

genetic test

Gene x environment interaction (PKU)

http://www.pkunews.org/adults/image005.gif

Disease modification byDisease modification by environment (alpha1-antitrypsin)( p yp )

C t ti i tCurrent genetics is not MendelianMendelian

1 gene = more diseases

1 gene = diseases with1 gene = diseases with different types of inheritance

several genes = same diseaseseveral genes = same disease

digenic inheritance

transgenerationaltransgenerational transmission of epigenetic marks

classical genetics1 gene=1 disease

other ……g

Genetics has been and will be full of suprises

Patophysiological mechanisms in IEMs

Patophysiology

precursor

byproductsubstrate byproductsubstrate

enzyme transporter

productproduct

Patophysiology

precursor

byproductsubstrate byproductsubstrate

Examples:Examples:Phe a Phe-derivativesammoniacystine in cystinosiscystine in cystinuriamucopolysaccharides

PatophysiologyExamples:

glucose in GSDk t b di i b tketone bodies in beta-oxidation defectsplasmalogens inplasmalogens in peroxisom.diseasescysteine in CBS deficiencyAdoMet in RMATP in mitochondrial di

product

diseases

product

Local vs. systemic consequences

Local consequence, e.g.:MPS b t d l liMPS-bones, tendons, spleen, liver, CNScystinuria-urinary tractcystinuria urinary tract

Local vs. systemic consequences

Distant consequences-e.g.q gurea cycle disorders- coma organic acidurias-encephalopathyCBS deficiency thrombosis connectiveCBS deficiency- thrombosis, connective tissue disturbances

substrát

produkt

Disorders of small and complex molecules

Small molecules in biochemicalSmall molecules in biochemical geneticsg

definition: < 1500 Dagases inorganic ionsgases, inorganic ionsamino acidsorganic acidssaccharidessaccharidespolyolssimple lipids

i i idipurines, pyrimidinesvitaminsoligomers: peptides up to 5-10 AA, oligosaccharides

cytosol, mitochondrial stromablood, urine

Diseases of small molecules

usually dependent on exogenous supplymanifestation: (repeated) acute toxicity usuallymanifestation: (repeated) acute toxicity, usually with encephalopathy/comahepatopathy commonhepatopathy commoncommon disturbances in routine labs-ammonia, Astrup, ketone bodies, glycemia, uric acid...p, , g y ,symptoms develop due to specific type of food, fasting, catabolismchronic course possible (if toxicity low)usually good therapeutic response to diet and/or it ivitamins

Isovaleric aciduria

FTT, vomiting, Kussmaul b thibreathing

consciousness: coma within 24-48 h after onset of symptoms

metabolic acidosis, ketonuria

sweaty feet syndrome

http://images.google.com/imgres?imgurl=http://upload.wikimedia.org/wikipedia/commons/8/8b/Isovaleric_acid_structure.png

Cystinuria

http://graphics8.nytimes.com/images/2007/08/01/health/adam/17046.jpg

http://www.nature.com/ki/journal/v73/n8/images/5002790f1.jpg

Complex molecules in biochemicalComplex molecules in biochemical geneticsg

definition: > 1500 Daglykolipids

sphingolipids

plasmalogensplasmalogens

neutral polysaccharides (glycogen)

mucopolysaccharides

(other polymers: proteins, nucleic acids...)

usually associated with membranesy

concentrations in blood/urine rather low, exceptions exist ( x MS/MS technologie)exceptions exist ( x MS/MS technologie)

Diseases of complex moleculesdisease progresses usually regardless of any exogenous sources from foodgtypical course is progressive (± symptom-free period)p )dysmorphy at birth possiblefrequent involvement of nervous system andfrequent involvement of nervous system and musculatureorganomegaly due to storage in lysosomalorganomegaly due to storage in lysosomal storage disordersusually untreatable by diet or vitaminsusually untreatable by diet or vitamins

Mukopolysaccharidosis type I

http://eyepathologist.com/images/KL1771.jpg

http://deti.msk.ru/plaxin_egor.jpg

http://myweb.lsbu.ac.uk/dirt/museum/margaret/438-1811-2640151.jpg

< 1500 Da > 1500 Da< 1500 Da > 1500 Da

Acute toxicity Y NAcute toxicity Y N

Chronic progression ± Y

Localization cytosol, ECT membranes

Impact on structure N Y

Dx blood,urine tissues (U)

Origin exogenous endogenous

Rx diet vitamins efficious inefficiousRx-diet, vitamins efficious inefficious

Patophysiology IEM

substrate

vedl.produkt<1500 Da

vedl.produkt

>1500 Da

product

1 32

Clinical manifestation of IEMs

Clinical picture-age

http://markandrich.googlepages.com/Old-woman.jpg/Old-woman-full.jpg

http://www.hrr.co.uk/acatalog/crocodile_toddler.jpghttp://www.co.shasta.ca.us/html/DSS/images/FosterParentingAdopt/infant.jpg

Clinical features-organs

http://universe-review.ca/I10-82-organs.jpg

Selected common situations withSelected common situations with high risk of IEMg

Small moleculest ll ill bacutelly ill newborn

(repeated) atack of long-term uncosciousnessfailure to thrivefailure to thrive

Complex moleculesComplex moleculesprogressive CNS and musculature involvementfacial dysmorphyfacial dysmorphyorganomegaly (liver, spleen, heart)

Food and IEMs (small molecules)

(sub)acute toxicitymilk (lactose)-hepatopathy

saccharose/fructose/sorbitol- hepatopathysaccharose/fructose/sorbitol hepatopathy and hypoglycemia

excess protein vomiting lethargy comaexcess protein- vomiting, lethargy, coma (urea cycle disorders, organic acidurias)

Fasting and IEMs

hypoglycemia in GSD

hypoglycemia with decreased production of ketone bodies (beta-oxidation defects)

acidosis, ketonuria and metabolic encephalopathy in prolonged fasting (organic p p y p g g ( gacidurias)

respiratory alkalosis and encephalopathy (urearespiratory alkalosis and encephalopathy (urea cycle disorders)

Abnormal urinary smell and colorsmell (small volatile molecules):

sweaty feet-isovalerate

maple syrup-branched ketoacids

boiled cabbage-methionine oxid

fish trimethylaminefish-trimethylamine

blackcurrant- organic acids

mouse-phenylacetate p y

colororange-urateg

black upon oxidation-homogentisate

blue-indoxyl derivaties

green-4-OH-butyrate

Common labs in IEMs

Blood

l i

Urine

k t b diglycemia

cholesterol

TG

ketone bodies

uric acid

t l iTG

uric acid

crystaluria

myoglobinuria

MAc

hyperammonemia, RAlk

ALT,AST

CK

anemia/pancytopenia

1857-1936

http://www.historiadelamedicina.org/imagenes/garrod.jpg