Untangling the Spirals ofMetabolic Disease: PrimaryDiagnoses and Secondary Effects:Implications for TreatmentDavid A. H. Whiteman MD1909 Archibald GarrodIn his paper, Inborn Errors ofMetabolism, the disease Alkaptonuria(Ochronosis: Homogentisic AcidOxidase Deficiency) is described asbeing caused by a gene.This landmark is usually cited as thebirth of Biochemical Genetics.CP1022045-49CP1022045-48A 2-week-old boy is admitted to the hospital with sepsisdue to Escherichia coli. He is being breastfed and hasbeen vomiting frequently.Findings include failure to thrive, lethargy, hypotonia,jaundice, hepatomegaly, and positive nonglucose reducingsubstances in the urine. Of the following, the MOST likely explanation for thesefindings is A. galactosemiaB. glycogen storage diseaseC. lactose intoleranceD. maple syrup urine diseaseE. urea cycle defect Classical GalactosemiaMetabolic Cataract andGlaucomaDisaccharide HydrolysisMaltose + H2O >>>maltase>>> D-Glucose+ D-GlucoseLactose + H2O >>>lactase>>> D-Galactose+ D-GlucoseSucrose + H2O >>>sucrase>>> D-Fructose+ D-GlucoseClassical Galactosemia on DietAutosomal Recessive InheritanceGalactosemia VariantsGal-1-P-UDT GalKinase Epimerase9p13 p21 17q21-q22 1p32 pterDuarte PhiladelphiaLos Angeles NegroIndian ChicagoRennesA 2-day-old female infant is refusing to feed and hasbecome increasingly lethargic and hypotonic over the past2 hours. Results of laboratory studies include an arterialblood gas analysis that reveals a pH of 7.13, PCO2 of 27mm Hg, and HCO3 of 14 mEq; positive urine ketones;and an ammonia concentration of 600 mcg/dL. An inbornerror of metabolism is suspected. Of the following, the MOST appropriate laboratory studyto obtain is A. leukocyte lysosomal enzyme activitiesB. plasma long-chain fatty acid levelsC. serum carnitine levelD. urine for reducing substancesE. urine organic acid levelsA 3-month-old infant who has a history of gastroesophagealreflux has had increasing vomiting for 2 days. This morningshe developed rapid, deep, labored breathing; lethargy; andshock. Findings include: serum sodium, 144 mEq/L;potassium, 4.5 mEq/L; chloride, 89 mEq/L; bicarbonate, 5mEq/L; pH, 7.16; glucose, 48 mg/dL; ammonia, 128 mcmol/L;and ketonuria. The MOST likely explanation for these findings is A. aminoacidopathyB. ethylene glycol poisoningC. metoclopramide toxicityD. organic acidemiaE. urea cycle defect A previously healthy 2-year-old boy is brought to the emergencydepartment by his mother, who reports that he has had a coldand fever for the past 2 days. He has been taking only smallamounts of juice and no solid foods. When she tried to arousehim after his nap today, he was lethargic and unresponsive.Results of laboratory studies include a glucose concentration of40 mg/dL, an ammonia level of 200 mcg/dL, and an arterialblood pH of 7.4. At this time, the MOST important study to obtain is A. plasma acylcarnitine profileB. plasma insulin levelsC. serum acetylsalicylic acid concentrationD. urine and stool porphyrinsE. urine organic acidsA Pale Toddler with RecurrentAbdominal PainHistory23 year old G2 mother, uncomplicated term pregnancy, newborn periodChild fed well, with normal growth and development to 2 yearsIntermittent abdominal pain, with fainting and pallorGI consultation and investigations unremarkable3 years: Coma at end of day, with URIHypoglycemia: 24 mg/dlA Pale Toddler with RecurrentAbdominal PainInvestigationCarnitine Deficiency: Total 19 Free 11 umol/LUrine AcylGlcyine Profile:Increased Hexanoyl-, Octanoyl-, Suberyl-GlycinesDNA Analysis MCAD: A985G (K329E) homozygousEnzyme Analysis MCAD: 8% Residual Enzyme Activity compared to controlsA Pale Toddler with RecurrentAbdominal PainManagementTreated with:Riboflavin 50 mg BIDL-Carnitine 50 mg/kg/day intermittentlyReduced fat diet (maximum of 25% of all calories from fatNormal growth and development, no residual neurological deficitNow has muscle cramps with exercise (baseball).Her older sister (then 7 years of age) is diagnosed on the basis of DNA analysis, although never symptomaticTwo subsequent uncomplicated pregnancies (no HELLP or AFLP)one child affected, the second a carrier.MCAD DeficiencyA Child with Multiple ProblemsThe PregnancyComplicated by abdominal pain, severe nausea and vomiting,black out spellsEmergency C-section because of maternal hemorrhage(unknown cause)A Child with Multiple ProblemsThe ChildMultiple hospital admissions for vomiting and dehydration in first year of lifeNissen fundoplication at 18 months of age.No improvementHospitalization at 2 1/2 years for feeding disorderEpisodes of weakness, shaking, decreased activity.No documented hypoglycemiaSymptoms worse in the morning, and with routine illnessA Child with Multiple ProblemsDiagnostic InvestigationInitial metabolic evaluation negativeChallenge testing:Hypoglycemia within12 hours of fastingFat beta-oxidation intermediates in urineFibroblast assays: reduced Palmitate oxidation and reduced LCHAD activityA Child with Multiple ProblemsSubsequent PregnancyPrenatal diagnosis of LCHADMother developed HELLP syndromeA Twitchy Floppy BabyHistoryUncomplicated pregnancy and birth: 75th percentile2 weeks: Colic6 weeks: Muscle twitchesDramatic HypotoniaTires when feedingStartles easily8 weeks: Severe constipationGrowth at 5th percentileA Twitchy Floppy BabyExaminationHead circumference and weight below 5th percentile: length at 10thDysmorphic face: telecanthus and prominent forehead, Flat occiput, ridged suturesHypoplastic distal digital phalanges with arch fingerprint formsSevere hypotonia, no lateralizing neurological signsFever of unknown origin (103F max)Spontaneous twitches of all extremitiesA Twitchy Floppy BabyInvestigationsDysmorphologist: ? FG, ? Schinzel-Gideon Syndromes, Rule out chromosome anomaly, micro deletions.Karyotype and multiple FISH micro deletions: NegativeMetabolic Screening:Mild Lactic Acidemia (Max 2.5 umol/L)EEG: Normal CK: 107Carnitine:Total 33 Free 24 umol/LSkin Fibroblasts: Modest (55 %) reduction in Palmitate and Myristate OxidationLCHAD and LCAT Enzyme activities normalMitochondrial DNA deletion/mutation analysis: UnremarkableA Twitchy Floppy Baby The Crisis : Diagnosis and TreatmentAnticonvulsants for twitching ineffectiveKlonopinKeppraControversy: Seizures vs. MyoclonusMuscle Biopsy for Enzyme AnalysisNADH Dehydrogenase 37% of controlsTreatment:L-CarnitineVitamins A,B-complex,C,E,KUbiquinone (CoEnzyme Q)Sodium SuccinateRRFAmino Acids(primarily alanine)GlucosePyruvateGluconeogenesis Acetyl CoALACTATELDHNADH + H+NAD+(reduced) (oxidized)CO2+ H2OTCACyclePDH PCPyruvate LactateNADH+H+(reduced form)NAD+(oxidized form)NADH+ H+ +1/2O2 NAD++H2O+ energyMitochondrial MetabolismCP1022045-76Mitochondrial ChromosomeNot a Well MitochondrionSDHCoQCyt CCOMPLEX ICOMPLEX IIICOMPLEX IV VH +H +H +H +ADPATPNADHNAD+1/2 O2H2OSuccinyl-CoAPrimary Hyperlactacidemia Disorders of Pyruvate Metabolism Pyruvate carboxylase deficiency Disorders affecting the pyruvate dehydrogenasecomplex (PDH) Krebs Cycle Defects Disorders of Gluconeogenesis Fructose-1,6-diphosphatase deficiency Glycogen storage disease type I Phosphoenolpyruvate carboxykinase deficiency Respiratory Chain Disorders Secondary to Other Inborn Errors of Metabolism Organic acid disorders Fatty acid oxidation disordersMMBID 8th ed. 2001, chapter 100, p 2289Zschocke/Hoffmann: Vademecum Metabolicum. Schattauer 1999, p 13Pedigree Assembly Pedigree: A graphical representation of afamily, showing relationships amongindividuals and medical and demographicinformation Example:Maternal Inheritance PedigreeHeteroplasmy can lead tovariable phenotypes in daughtercellsDisease Map of theMitochondrial GenomeFactors that Complicate Analysisof Traditional Inheritance Reduced penetrance Variable expressivity Pleiotropy New mutations Gonadal mosaicism Genetic heterogeneityGonadal (Germline) Mosaicism One parent is is presumed to have a mixtureof cells: mostly normal, but with at leastsome mutant germ cells. Proportion of mutant gametes depends onwhen mutation occurred during mitoticexpansion. Presents counseling difficulty: What is therisk in the next pregnancy?A de novo mutation in a germcell lineage produces mosaicismCase Report Male infant born at 34 weeks gestationalage to a G3/P1 mother Maternal serologies were negative Prenatal course revealed a fetus thought tobe IUGR as well as oligohydramnios Delivery was via C-section due to fetaldecelerationsFamily History Sibling who died at 25 weeks gestationsecondary to non-immune hydrops Healthy 18 month old sister Both parents are in good health andunrelatedCase Report Mild respiratory distress postnatally. ABG showed a pH of 7.33, CO2 was 33,HC03 was 17 Lactic acid was 10 mM (normal: < 2.2) at4 hrs of life and increased to 24 mM overthe next 24 hrs Electrolytes, glucose, NH3 normal Sepsis work-up initiatedCase Report2nd day: Mottled appearance Blood pressure low Lactic acid remained at 22 mM Echocardiogram was normal (anatomy/fxn)3rd day:Transfer to Mayo ABG on arrival: pH 7.13, pC02 99Physical Examination Weight and length in the 10-25th percentile.Head circumference 5-10th percentile Mild dysmorphic features including shortpalpebral fissures, mild micrognathia, longthumbs No organomegaly Muscle hypotonia**Internal standardDicarboxylicaciduria3 Hydroxy dicarboxylicaciduriaTypical Organic Acid Profile in LCHAD DeficiencyGenes: - -subunit gene (chromosome: 2p23)- -subunit gene (chromosome: 2p23)Incidence: once considered same as MCAD def. (1:15,000 live births)Symptoms: Hypoketotic hypoglycemia Reye-like syndrome: hypoglycemia hyperammonemia elevated transaminases brain edema fatty liver with microvesicular steatosis elevated uric acid Lactic acidosis Myopathy/rhabdomyolysis Cardiopathy hypertrophic/dilated cardiomyopathy AV-block ventricular arrhythmias Retinitis pigmentosa (RP) Peripheral neuropathy Cholestasis Maternal pregnancy complications Sudden unexplained deathTFP/LCHAD DeficiencyTFP/LCHADDeficiencyTreatment:- avoidance of fasting;- low-fat diet high in complex carbohydrates- fat primarily as medium chain triglycerides- urine organic acids (hypoketotic dicarboxylic aciduria, hydroxy dicarboxylic aciduria)- plasma/blood spot acylcarnitines- in vitro probe assay in fibroblasts- molecular genetic analysis (allele frequency of 1528G>C mutation: 87%)Diagnosis:Elevated Lactic Acid inMTFP/LCHAD Deficiency Direct inhibition of mitochondrial oxidativephosphorylation by 3-hydroxypalmitoyl-CoA(Ventura J Inher Met Dis 1996) Long-chain acyl-carnitines may inhibit thepyruvate dehydrogenase complex (Moore Int J Biochem1992) Long-chain acyl-CoA esters inhibit themitochondrial ATP/ADP carrier and thedicarboxylic carrier in vitro (Halperin PNAS 1972)Molecular Testing Molecular studies revealed a homozygous 5bp deletion in exon 4 of the alpha subunit ofthe MTFP gene involving base pairs 274 to278 This novel mutation creates a prematurestop codon Possible uniparental disomyIsovaleric acidemia Defect in breakdown of Isovaleryl-CoA Product of leucine catabolism Build up of isovaleric acid 2 clinical manifestations Severe neonatal presentation 3-6 days 50% mortality Chronic intermittent type < 1year Precipitated by URI or high protein intake Frequency of episodes decreases with age Often unveiled when mother stops breast feedingISOVALERIC ACIDEMIASiblings:Healthy newborn girl, elevated C5-AcylCarnitineIncreased urine isovalerylglycineOlder brother? Mild motor delaysIncreased Urine IsovalerylglycineSelf treats avoids excessive proteinOne parent found to have A282V mutation in IVCoADehydrogenase gene, now known to be associated with attenuated disease.Other parents mutation classical.MULTIFACTORIAL ?Siblings:Boy from a difficult pregnancy with low tone, failure to gain weight, recurrent illness with fluctuating blood sugars,unusual odorleg pains, muscle cramps, cardiomyopathy, pancreaticinsufficiency.Brother with low tone, feeding problems, failure to gain weight, hypertrophic cardiac septum, cyclical low white blood cells,gastroesophageal reflux, intermittent lethargy with illness elevated blood ammonia, fatty liverMother with chronic fatigue, neurological symptoms with brain scanleukodystrophyMULTIFACTORIAL ?Both boys:NBS suggested Glutaric Aciduria II: not confirmed by enzymesUnusual pattern of fat oxidation in skin cellsUrinary hexanoylglycine: MCAD A985GHeterozygous for an Unusual new mutation in SPINK1, (Pancreatitis)A common variant in CFTR (Cystic Fibrosis)No mtDNA mutationsNormal Electron Transport Chain AnalysisSome Response to Carnitine, Glycine, Riboflavin and Low FatWhere Are Genes and What Do They Do?DNA)(GeneRNA ProteinFunctionGene Expression(Transfer of GeneticInformation)NucleusCellmRNALocationof GenesNucleusCellDNA)(One GeneRegulatoryRegionProtein CodingRegionChromosome46 TotalOut of ~3000/chromosomeA.B.Example ofGeneticInformationTransferC.Gene Expression(Transfer of genetic information)C GCGTTGATGGCAACTACaa1aa2aa3aa4aa5aa6Codeforaa2Codeforaa1Codeforaa3etc.Double Stranded DNA ProteinExonsTTTG CTTCTGACAC121 AACTGTGTTC ACTAGCAACC TCAAACAGAC ACCATGGTGC ACCTGACTCC TGAGGAGAAG181 TCTGCCGTTA CTGCCCTGTG GGGCAAGGTG AACGTGGATG AAGTTGGTGG TGAGGCCCTG241 GGCAGgttgg tatcaaggtt acaagacagg tttaaggaga ccaatagaaa ctgggcatgt301 ggagacagag aagactcttg ggtttctgat aggcactgac tctctctgcc tattggtcta361 ttttcccacc cttagGCTGC TGGTGGTCTA CCCTTGGACC CAGAGGTTCT TTGAGTCCTT421 TGGGGATCTG TCCACTCCTG ATGCTGTTAT GGGCAACCCT AAGGTGAAGG CTCATGGCAA481 GAAAGTGCTC GGTGCCTTTA GTGATGGCCT GGCTCACCTG GACAACCTCA AGGGCACCTT541 TGCCACACTG AGTGAGCTGC ACTGTGACAA GCTGCACGTG GATCCTGAGA ACTTCAGGgt601 gagtctatgg gacccttgat gttttctttc cccttctttt ctatggttaa gttcatgtca661 taggaagggg agaagtaaca gggtacagtt tagaatggga aacagacgaa tgattgcatc721 agtgtggaag tctcaggatc gttttagttt cttttatttg ctgttcataa caattgtttt781 cttttgttta attcttgctt tctttttttt tcttctccgc aatttttact attatactta841 atgccttaac attgtgtata acaaaaggaa atatctctga gatacattaa gtaacttaaa901 aaaaaacttt acacagtctg cctagtacat tactatttgg aatatatgtg tgcttatttg961 catattcata atctccctac tttattttct tttattttta attgatacat aatcattata1021 catatttatg ggttaaagtg taatgtttta atatgtgtac acatattgac caaatcaggg1081 taattttgca tttgtaattt taaaaaatgc tttcttcttt taatatactt ttttgtttat1141 cttatttcta atactttccc taatctcttt ctttcagggc aataatgata caatgtatca1201 tgcctctttg caccattcta aagaataaca gtgataattt ctgggttaag gcaatagcaa1261 tatttctgca tataaatatt tctgcatata aattgtaact gatgtaagag gtttcatatt1321 gctaatagca gctacaatcc agctaccatt ctgcttttat tttatggttg ggataaggct1381 ggattattct gagtccaagc taggcccttt tgctaatcat gttcatacct cttatcttcc1441 tcccacagCT CCTGGGCAAC GTGCTGGTCT GTGTGCTGGC CCATCACTTT GGCAAAGAAT1501 TCACCCCACC AGTGCAGGCT GCCTATCAGA AAGTGGTGGC TGGTGTGGCT AATGCCCTGG1561 CCCACAAGTA TCACTAAGCT CGCTTTCTTG CTGTCCAATT TCTATTAAAG GTTCCTTTGT1621 TCCCTAAGTC CAACTACTAA ACTGGGGGAT ATTATGAAGG GCCTTGAGCA TCTGGATTCT1681 GCCTAATAAA AAACATTTAT TTTCATTGCa atgatgtatt taaattattt ctgaatattt1741 tactaaaaag ggaatgtggg aggtcagtgc atttaaaaca taaagaaatg atgagctgtt1801 caaaccttgg gaaaatacac tatatcttaa actccatgaa agaaggtgag gctgcaacca1861 gctaatgcac attggcaaca gcccctgatg cctatgcctt attcatccct cagaaaagga1921 ttcttgtaga ggcttgattt gcaggttaaa gttttgctat gctgtatttt acattactta1981 ttgttttagc tgtcctcatg aatgtctttt cactacccat ttgcttatcc tgcatctctc2041 tcagccttga ct0.00006% of the human genome sequenceFrom LehningerEach spot containsDNA from a knowncDNA or an ESTEgg TadpoleIdentifying differences in gene expression with microarraysHigher in EggHigher in tadpoleNormal Tissue Malignant Tissue/Malignant Tissue/Normal TissueFrom Lodish et al.