Viktor Kožich

Ústav dědičných metabolických poruch1.LF UK a VFN Praha

Diagnosis of inbornerrors of metabolism

Structure

Diagnosis in generalNewborn screeningSelective screening

clinical approachlaboratory tests

Inborn errors of metabolism

product

substrateusually AR, GR

clinicallyvariable

usually enzyme

Diagnosis ≈ hypothesis verification

http://farm1.static.flickr.com/21/100467846_832c312de1_o.gif

patient

Linear causality

http://students.ou.edu/Y/Jacob.R.Yandell-1/karma.png

Nonlinear causality

http://www.biomedcentral.com/content/figures/1471-2105-10-122-4-l.jpg

Diagnostic process-iteration

http://www.eservicesperformance.com.au/images/graph_process.gif

Successful diagnosis of IEM

+

3.bp.blogspot.com/.../s320/physicianatdesk.jpg

knowledgeable physician availability of appropriate test

Diagnostic procedures in IEMs

produkt

substrát

DNA/RNA Enzymes Metabolites

|Clinicsspecific-e.g..

smellurine color

nonspecific-e.g.comaPMRdysmorfic featureshepato/myopathieother

Structure

Diagnosis in general

Newborn screeningSelective screening

clinical approachlaboratory tests

Presymptomatic diagnosis

produkt

substrát

at risk relatives

screening of population segment

newborn screening

single disease or groupof diseases

Newborn screening (NBS)

• active search for disease in the entire population

• presymptomatic diagnosis

• sensu stricto- laboratory analyses of diseases usingdry blood spots

NBS

Founder

Prof. Robert Guthrie 1916-1995

University of Buffalo, NY

PKU- bacterial inhibition test

JMG Wilson and G Jungner: Principles and Practice of Screening for Disease, WHO 1968

Classical criteria for NBS

Diseases freqeuncy and severityAsymptomatic latent phaseDisease mechanisms are knownReliable testTest is acceptable by the populationProgram is a continuous processAcceptable treatmetnConditions for dx and rx establishedConsensus on whom and how to treatCost-benefit ratio acceptable by the system

Quality Adjuste Life Yearsusual accepted limit 50.000 USD

QALY for MS-MS (30 IEMs): 6.000-15.000 USD, newborn screening of CF: 13.728 USD

QALY for other situationsdefibrilators in aircrafts/malls 50.000-70.000 USDprostatic ca screening 9.000-23.000 USDmammography 2.000-20.000 (but 232.000 USD under 50 y)MRI screening of breast ca in carriers of BRCA1/2 mutations: 45.000-700.000 USDACE inhibitors in hypertension 5.600 USD

http://www.bioethics.gov/images/new_born_screening.gif

81 diseases

29 diseases

3434

4444

4444 4545

50

5353

3535

51

50

3232

41414747

4646

3737

2929

2929

41414848

4040

DC

51 1331

4545

3535

33

36

2929

50

4141

4848

31

4545

33 4545

4848

52

31

31

3535

54 4949

3232

504949

2424

31

314646

49

51

52

32

5252

31

30

NBS USA-2009

NBS Europe-2009

Disorders screened in ČR 10/2009

~1:4 000

~1:2 900

~1:4 000

Total 1:1 200

Congenital hypothyroidism

1:4000etiology heterogeneous-peripheral diseaseTSH measurement (also T4) Aim: early substitution with thyroxinprevents irreversible CNS damage(+detection of associated congenitalanomalies)

Congenital Adrenal HyperplasiaDeficient activity of 21-hydroxylase- CYP21 gene mutationsAbnormala steroidogenesis in adrenal cortex –hypokortikalism, hypoaldosteronism andhyperandrogenismClassic (salt wasting or simple virilizing) and lateonset formsClassic form 1:10 000, LO up to 1:100. test: 17-hydroxyprogesteroneAim: early substitution prevents life-threateningcortical insufficency and premature puberty

IEMs of amino acids

accumulation of toxic products: chronic toxicity: e.g. Pheacute toxicity: BCKA, glutarate, isovalerate

clinical featureschronic toxicity: PMR, seizuresacute toxicity: encephalopathy with coma

therapy aim: decrease the load of affectedpathway + supplement other AA

Beta-oxidation defects

common mechanism: decreased production of ketone bodies upon fastingaccumulation of toxic acylCoAs

clinical features:common-hypoglycemia under fastingin some FAO defects-myopathy (especially after prolongedexercise), cardiomyopathy

treatment: prevention of hypoglycemia in catabolic situations, prevention of fasting

Cystic fibrosisAR inheritance1: 2500 – 4000 Mutations in CFTR gen

abnormal chloride channelabnormally thick mucus-bronchi, pancreas, male GU

Severe chronically debilitating disease leading to respiratory failureMedian survival in developed countries: 37yAim ot therapy: slowing down the progression by ATB treatment

Good sampling practice

http://cms.ich.ucl.ac.uk/website/imagebank/images/Neonatal_appendix.gif

correct drying3 hres, no direct heat

Why is correct sampling crucial?

Laboratory analysis of DBS

http://nl.ijs.si/et/talks/esslli02/metadata_files/Haystack-FINALb.jpg

Immunological metods

CH and CAH: hormonesCF: IRT

Tandem mass spectrometrymodern analytical methodprofile of analyteswide spectrum of compounds: amino acids, acylcarnitines, sugars......enzyme activitiesused for NBS since mid1990s

www.nature.com/.../v2/n2/fig_tab/nrd1011_F5.html

http://www.ch.cam.ac.uk/staff/pics/es1.jpg

Structuralinformation

http://cache.boston.com/resize/bonzai-fba/Globe_Photo/2008/01/22/1201060537_1951/300h.jpg

MS/MS- verygood identificationof analyteshigh sensitivityfast analysis of many metabolitesin a single assay

suchá krevní kapka

200 µl methanolu

se standardy

odstranění terčíku25 min extrakce

vyražení

3 mm terčíku

odpaření methanolu

butylace 30 min při 60 °Codpaření derivatizačního

činidla

analýza

1 vzorku 2,5 min

100µl

derivatizačního

činidla

NBS for IEMs by MS/MS

200 µl

1:1 acetonitril a voda + 0,02 % kys.

mravenčí

Optimal screening test

•http://www.connectseward.org/shs/da1/review07/Right%20Skewed.jpg

e.g. classical PKU and mild HPA

Low PPV

•http://www.connectseward.org/shs/da1/review07/Right%20Skewed.jpg

e.g. MSUD-Xle, CF-IRT

solution to problem: second tier test

Results from NBS programsNormal

cave!!!!- atypical form of screened disease is not excluded

Borderlineusually non-specificrecall needed-MS/MS on a new sample

Positivevarying PPV (5-100%)diagnostic workup initiated

Structure

Diagnosis in generalNewborn screening

Selective screeninglaboratory testsclinical approach

Diagnostic procedures in IEMs

produkt

substrát

DNA/RNA Enzymes Metabolites

|Clinicsspecific-e.g..

smellurine color

nonspecific-e.g.comaPMRdysmorfic featureshepato/myopathieother

IEM- metabolite analysis

produkt

Substrát

Metabolite analysis- IEMs

specialized testsusually not available in routinely labsmostly profile analysesmostly chromatographic techniques, expensiveequipment neededlaborious methods, lack of kits and controlmaterialcomplex interpretation (e.g. organic acids) by specialist

Small molecules in biochemicalgenetics

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

cytosol, mitochondrial stromablood, urine

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)

http://ustl1.univ-lille1.fr/chimie/html/Enseignement/ATE_web/chrom/Tswett_final.jpg

Sensitivity

Alkaptonuria: 1-5 g homogentisate /dCystinuria: 1-5 g cystine /d

PKU: 0.1 g Phe /l blood

MCAD: C8 acylcarnitin 0.0001 g / l bloodDBS punch 0.003 ml blood

0.2 – 1 ml serum

urine- liters

HPTLC- oligosaccharides in urine

courtesy Dr.Ledvinová

α L- iduronidase deficiency

(excretion of dermatan sulphate/DS and heparan sulphate/HS )

courtesy Dr.Ledvinová

AA- citrullinemia

patient

control

GC-MS: methylmalonic aciduria

0,0 2,5 5,0 7,5 10,0 12,5 15,0 17,5 20,0Time [min.]

25

50

75

100

125

150

175

200Vo

ltage

[mV]

3,26

5,54

5,86

6,61

6,64

7,21 7,

77

10,3

610

,63

11,5

112

,08

12,2

8

13,7

213

,80

14,0

0

14,7

014

,81

18,3

90,0 2,5 5,0 7,5 10,0 12,5 15,0 17,5 20,0

Time [min.]

50

100

150

200

250

300

350

Vol

tage

[mV

]

3,57

4,24

5,07

5,93

6,63

10,6

4

12,0

812

,29

13,4

713

,72

13,8

1

14,7

014

,80

14,9

6

17,8

0

18,4

0

GC-MS: methylmalonic aciduria

http://biochemgenetics.stanford.edu/images/Fig%204%20(MMA).jpg

GC-MS: MCAD deficiency

0,0 2,5 5,0 7,5 10,0 12,5 15,0 17,5 20,0Time [min.]

50

100

150

200

250

Vol

tage

[mV

]

3,29

5,12

5,94

6,67

10,6

8

12,2

812

,33

13,7

613

,85

14,7

414

,88

18,4

718

,69

0,0 2,5 5,0 7,5 10,0 12,5 15,0 17,5 20,0Time [min.]

50

100

150

200

250

300

350

Vol

tage

[mV

]

3,57

4,24

5,07

5,93

6,63

10,6

4

12,0

812

,29

13,4

713

,72

13,8

1

14,7

014

,80

14,9

6

17,8

0

18,4

0

before treatment

after treatment

In vivo metabolite measurement

brain creatine deficiency

Metabolite analysis- IEMs

usually not available in routinely labsmostly profile analyses by chromatographic techniquecomplex interpretation (e.g. organic acids)

Diagnosis of IEM

produkt

Substrát

enzym

Principles of enzyme assays

substrate* product*

cofactor altered cofactor

•separation of substrate from product•quentitation of change

•quentitation of change

Enzyme assays in IEM diagnosisCells are usually needed

leukocytes, fibroblastschorion, amniocytes

Fluorimetry and radiometry (photometry)Measured variable:

substrate/product concentration changecofactor concentration change

ÚDMP: 46 enzyme assays

Typical results

Diagnosis of IEM

produkt

SubstrátDNA

http://www.biostudio.com/images/anim_freeman_dna_sequencing.gif

http://openwetware.org/images/6/61/DNA_sequencing.JPG

http://www.mun.ca/biology/scarr/fluorescent_dideoxy_sequencing.jpg

TCGGACCTCGGT/CGAAAAAGA

OTC: Mutation c. 829 C>T (R277W)

A GC T T CCAG G G CAC T T C T A T T T G T T T T T G T G G AA A G A CT G G CBases5 10 15 20 25 30 35 40

A GC TT CC AG G G C AC T T CT WT C T R T T T KT KT t GTG AWR G AC WG R C

Bases10 15 20 25 30 35 40 45 50

Heterozygous deletion

Wild type sequence

http://www.nature.com/nbt/journal/v26/n10/images/nbt1486-F1.gif

Structure

Diagnosis in generalNewborn screening

Selective screeninglaboratory tests

clinical approach

Patophysiology IEM

product

vedl.produkt

substrate<1500 Da

>1500 Da

1 32

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-multisystemicinvolvement

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

Clinical features of IEMs-organs

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

endogenousexogenousOrigin

N YAcute toxicity

inefficiousefficiousRx-diet, vitamins

tissues (U)blood,urineDx

YNImpact on structure

membranescytosol, ECTLocalization

Y±Chronic progression

> 1500 Da< 1500 Da

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

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

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)

Organic acidurias

several dozens of diseasescommon feature: excretion of carboxylic acids(test-organic acids in urine) orginin usually from carbon skeleton degradationof AAs (or saccharides or lipids)usually acute presentation- „intoxication type“metabolic acidosis common (combination withhyperammonemia frequent)

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

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

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

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

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)

Successful diagnosis of IEM

+

3.bp.blogspot.com/.../s320/physicianatdesk.jpg

knowledgeable physician availability of appropriate test