Viktor Kožich

Institute of Inherited Metabolic Disease1.LF UK and VFN Praha

Introduction to biochemical genetics

OutlineAims of the courseBasic features of IEM, population frequencyHistory of IEMGenetic origin of IEMPathogenetic mechanismsSmall molecules and complex moleculesClinical features of IEM

Aims of the course on biochemical genetics

Biochemical genetics = IEMs

genetics

clinical medicine

biochemistry

Case 1-Isovaleric aciduria

failure to thrive, vomitingrepeatedly alteredconsciousnessketoacidosissweaty feet odour

Case 2-adult cystinosis

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

Case 3- MPS I

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

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

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

Case 4-cystinuria

http://graphics8.nytimes.com/images/2007/08/01/health/adam/17046.jpghttp://www.nature.com/ki/journal/v73/n8/images/5002790f1.jpg

Basic features of IEMs

Inborn errors of metabolism

product

substrateusually AR, GR

clinicallyvariable

usually enzyme

Clinical 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

Clinical features: (non)specific signs

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

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

specific non-specific

e.g. NH3, uric acid

Clinical features-multisystemicinvolvement

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

Diagnostic procedures in IEMs

produkt

substrát

DNA/RNA Enzymes Metabolites

|Clinicsspecific-e.g..

smellurine color

nonspecific-e.g.comaPMRdysmorfic featureshepato/myopathieother

Frequency of IEMs

newborn screening 1:1000-1:4000selective screening at least 1:500-1:1000frequency of heterozygotes at least 1:15population specific examples

higher incidence in imbred populations (PKU Turkey, organicacidurias Middle East)tyrosinemia type I- Quebecaspartylglykosaminuria- Finlandlysosomal strorage disorders- Izrael

IEMs in the Czech Republic

peroxisomální4%

sacharidy12%beta-oxidace a

OAU14%

AMK bez HPA18%

HPA a PKU13%

lysosomální16%

mitochondriální20%

puriny/pyrimidiny3%

incidence ~ 1:1000so far ~150 different diseases

ČR, 2005, n=127

Treatment of IEMs

produkt

substrát

History of biochemicalgenetics

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

1857-1936

http://www.thetoque.com/010605/pics/urine.jpg

Trophîme Bigot, 16.-17.století

http://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 fermentation, R.Wirchow describes mitosis1867 dynamit1874 jeans1886 automobil=car1890 underground1895 cinematograf1902 periskop, el.typewriter; Nobel prize fordiscovery of malaria transmission1908 Nobel prize for discovery of immunity1936 tape recored; Nobel prize forneurotransmitter research

Lancet, vol ii, 1902, 1616-1620

Expansion of the field

1908 • 5 diseases(alkaptonuria, cystinuria, porphyria, pentosuria, congenitalsteatorhea)•1909 first textbook•incidence unknown•dx: 4 metabolites in urine•Rx none

2009•at least 500 diseases•Medline 122.797 papers (28/9/2009)•textbook-web based•incidence at least1:1.000•Dx: metabolites, enzymes, DNA•screening MS-MS•Rx for ~ 1/3 diseases

cca 1960 • ca 40-50 diseases•analyticalmethods•incidence of PKU•PKU- 1st screening program (R.Guthrie)•PKU-1st treatableIEM (H.Bickel)

Biochemical genetics in the ČRPKU screening 1975-Doc.Blehová, Prof.Hyánek, Ing.MrskošDx of other IEMs-leader Prof.Hyáneklabs in Prague, H.Králové, Brno, Olomouc, Ostravain 2009:

selective screening ~ 100 pacientů/rok (incidence 1:1000)newborn screening- 10 IEMs since October 1 2009, expected incidence 1:4000

First IEM:

Alkaptonuria

http://www.mja.com.au/public/issues/184_12_190606/sha20286_fm-1b.jpghttp://www.scielo.br/img/revistas/rbr/v46n5/a14fig02.jpg

</< td>

http://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= disturbanceof chemical individuality

Alkaptonuria in 2009retrospective reasearch of mummy HARWA 1500 př.n.l.pateints known before Garrod1902 Garrod1909 tyrosine degradationpathway described1958 enzyme defect in alkaptonuria established1996 HGO gene description2003 treatment with nitisone

http://www.mja.com.au/public/issues/184_12_190606/sha20286_fm-1b.jpghttp://www.scielo.br/img/revistas/rbr/v46n5/a14fig02.jpg

</< td>

http://bjo.bmj.com/content/vol83/issue6/images/large/98532.f1.jpeg

CoxarthrosisValvularinvolvementUrolithiasis

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

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

Liver HGO deficiencyIncidence 1:250 000 až 1:1 000 000High incidence SlovakiaLate complicationsfrequentRx inefficient

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

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

nitison (NTBC)

Novel treatment in alkaptonuria

Genetic origin of IEMs

Genetic origin of IEMMonogenic Mendelian inheritance

AR inheritanceGR/GD inheritancerarely AD inheritance

X

Mitochondrial inheritanceEpigenetic changesX-inactivationEpsistasisGene x environment interactionother

Common types of mutations

Molecular consequences of mutations

Protein amountsdecreasedsameincreased

Protein propertieschanges of isolated functionsglobal changesmisfolding

Nonprotein gene productssiRNA

Role of mutations in evolutionSource of variability(less important thanmeiotic recombinationand combination of gametes)

Favourable mutationsare rare and alreadycontained in genomes

Genetic diseases= tip of iceberg (of geneticvariability)

Mutations affect different domains

e.g. PAHcytosolic enzymehomotetramerBH4 as cofaktorcomplicatedmechanism of catalysis

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

Cellular consequences of mutations (e.g.CFTR)

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

Genotype-phenotype (M.Gaucher)

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

severesplenomegalyanemiabone disorderCNS

mildsplenomegalyanemia

unclear

Phenotype

Health-disease threshold

environmentgenes

clinical manifestation

genetic test

preclinical test

IEM treatment- modifying environment

environmentgenes

clinical manifestation

genetic test

preclinical test

Gene x environment interaction (PKU)

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

Disease modification by environment (alpha1-antitrypsin)

Current genetics is not Mendelian

classical genetics1 gene=1 disease

1 gene = more diseases1 gene = diseases withdifferent types of inheritance several genes = same diseasedigenic inheritancetransgenerationaltransmission of epigeneticmarksother ……

Genetics has been and will be full of suprises

Patophysiologicalmechanisms in IEMs

Patophysiology

product

byproduct

enzyme transporter

substrate

precursor

Patophysiology

byproductsubstrate

precursor

Examples:Phe a Phe-derivativesammoniacystine in cystinosiscystine in cystinuriamucopolysaccharides

Patophysiology

product

Examples:glucose in GSDketone bodies in beta-oxidation defectsplasmalogens in peroxisom.diseasescysteine in CBS deficiencyAdoMet in RMATP in mitochondrialdiseases

Local vs. systemic consequences

Local consequence, e.g.:MPS-bones, tendons, spleen, liver, CNScystinuria-urinary tract

Local vs. systemic consequences

produkt

substrát

Distant consequences-e.g.urea cycle disorders- comaorganic acidurias-encephalopathyCBS deficiency- thrombosis, connectivetissue disturbances

Disorders of small andcomplex molecules

Small molecules in biochemicalgenetics

definition: < 1500 Dagases, inorganic ionsamino acidsorganic acidssaccharidespolyolssimple lipidspurines, pyrimidinesvitaminsoligomers: peptides up to 5-10 AA, oligosaccharides

cytosol, mitochondrial stromablood, urine

Diseases of small molecules

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

Isovaleric aciduria

FTT, vomiting, Kussmaulbreathingconsciousness: comawithin 24-48 h after onsetof symptomsmetabolic acidosis, ketonuriasweaty 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.jpghttp://www.nature.com/ki/journal/v73/n8/images/5002790f1.jpg

Complex molecules in biochemicalgenetics

definition: > 1500 Daglykolipidssphingolipidsplasmalogensneutral polysaccharides (glycogen)mucopolysaccharides(other polymers: proteins, nucleic acids...)

usually associated with membranesconcentrations in blood/urine rather low, exceptions exist ( x MS/MS technologie)

Diseases of complex moleculesdisease progresses usually regardless of anyexogenous sources from foodtypical course is progressive (± symptom-freeperiod)dysmorphy at birth possiblefrequent involvement of nervous system andmusculatureorganomegaly due to storage in lysosomalstorage disordersusually untreatable by diet or vitamins

Mukopolysaccharidosis type I

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

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

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

endogenousexogenousOrigin

N YAcute toxicity

inefficiousefficiousRx-diet, vitamins

tissues (U)blood,urineDx

YNImpact on structure

membranescytosol, ECTLocalization

Y±Chronic progression

> 1500 Da< 1500 Da

Patophysiology IEM

product

vedl.produkt

substrate<1500 Da

>1500 Da

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 withhigh risk of IEM

Small moleculesacutelly ill newborn(repeated) atack of long-term uncosciousnessfailure to thrive

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

Food and IEMs (small molecules)

(sub)acute toxicitymilk (lactose)-hepatopathysaccharose/fructose/sorbitol- hepatopathyand hypoglycemiaexcess protein- vomiting, lethargy, coma(urea cycle disorders, organic acidurias)

Fasting and IEMs

hypoglycemia in GSDhypoglycemia with decreased production of ketone bodies (beta-oxidation defects)acidosis, ketonuria and metabolicencephalopathy in prolonged fasting (organicacidurias)respiratory alkalosis and encephalopathy (urea cycle disorders)

Abnormal urinary smell and colorsmell (small volatile molecules):

sweaty feet-isovaleratemaple syrup-branched ketoacidsboiled cabbage-methionine oxidfish-trimethylamineblackcurrant- organic acidsmouse-phenylacetate

colororange-urateblack upon oxidation-homogentisateblue-indoxyl derivatiesgreen-4-OH-butyrate

Common labs in IEMs

BloodglycemiacholesterolTGuric acidMAchyperammonemia, RAlkALT,ASTCKanemia/pancytopenia

Urineketone bodiesuric acidcrystaluriamyoglobinuria

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

1857-1936