Untangling the Spirals of Metabolic Disease: Primary Diagnoses and Secondary Effects: Implications for Treatment David A. H. Whiteman MD 1909 Archibald Garrod In his paper, Inborn Errors of Metabolism, the disease Alkaptonuria (Ochronosis: Homogentisic Acid Oxidase Deficiency) is described as being caused by a gene. This landmark is usually cited as the birth of Biochemical Genetics. CP1022045-49 CP1022045-48 A 2-week-old boy is admitted to the hospital with sepsis due to Escherichia coli. He is being breastfed and has been vomiting frequently. Findings include failure to thrive, lethargy, hypotonia, jaundice, hepatomegaly, and positive nonglucose reducing substances in the urine. Of the following, the MOST likely explanation for these findings is A. B. C. D. E. galactosemia glycogen storage disease lactose intolerance maple syrup urine disease urea cycle defect Classical Galactosemia Metabolic Cataract and Glaucoma Disaccharide Hydrolysis Maltose + H2O >>>maltase>>> D-Glucose + D-Glucose Lactose + H2O >>>lactase>>> D-Galactose + D-Glucose Sucrose + H2O >>>sucrase>>> D-Fructose + D-Glucose Classical Galactosemia on Diet Autosomal Recessive Inheritance Galactosemia Variants Gal-1-P-UDT GalKinase Epimerase 9p13 – p21 17q21-q22 1p32 – pter Duarte Los Angeles Indian Rennes Philadelphia Negro Chicago A 2-day-old female infant is refusing to feed and has become increasingly lethargic and hypotonic over the past 2 hours. Results of laboratory studies include an arterial blood gas analysis that reveals a pH of 7.13, PCO2 of 27 mm Hg, and HCO3 of 14 mEq; positive urine ketones; and an ammonia concentration of 600 mcg/dL. An inborn error of metabolism is suspected. Of the following, the MOST appropriate laboratory study to obtain is A. B. C. D. E. leukocyte lysosomal enzyme activities plasma long-chain fatty acid levels serum carnitine level urine for reducing substances urine organic acid levels A 3-month-old infant who has a history of gastroesophageal reflux has had increasing vomiting for 2 days. This morning she developed rapid, deep, labored breathing; lethargy; and shock. Findings include: serum sodium, 144 mEq/L; potassium, 4.5 mEq/L; chloride, 89 mEq/L; bicarbonate, 5 mEq/L; pH, 7.16; glucose, 48 mg/dL; ammonia, 128 mcmol/L; and ketonuria. The MOST likely explanation for these findings is A. B. C. D. E. aminoacidopathy ethylene glycol poisoning metoclopramide toxicity organic acidemia urea cycle defect A previously healthy 2-year-old boy is brought to the emergency department by his mother, who reports that he has had a cold and fever for the past 2 days. He has been taking only small amounts of juice and no solid foods. When she tried to arouse him after his nap today, he was lethargic and unresponsive. Results of laboratory studies include a glucose concentration of 40 mg/dL, an ammonia level of 200 mcg/dL, and an arterial blood pH of 7.4. At this time, the MOST important study to obtain is A. B. C. D. E. plasma acylcarnitine profile plasma insulin levels serum acetylsalicylic acid concentration urine and stool porphyrins urine organic acids A Pale Toddler with Recurrent Abdominal Pain History 23 year old G2 mother, uncomplicated term pregnancy, newborn period Child fed well, with normal growth and development to 2 ½ years Intermittent abdominal pain, with fainting and pallor GI consultation and investigations unremarkable 3 years: Coma at end of day, with URI Hypoglycemia: 24 mg/dl A Pale Toddler with Recurrent Abdominal Pain Investigation Carnitine Deficiency: Total 19 Free 11 umol/L Urine AcylGlcyine Profile: Increased Hexanoyl-, Octanoyl-, Suberyl-Glycines DNA Analysis MCAD: A985G (K329E) homozygous Enzyme Analysis MCAD: 8% Residual Enzyme Activity compared to controls A Pale Toddler with Recurrent Abdominal Pain Management Treated with: Riboflavin 50 mg BID L-Carnitine 50 mg/kg/day intermittently Reduced fat diet (maximum of 25% of all calories from fat Normal growth and development, no residual neurological deficit Now has muscle cramps with exercise (baseball). Her older sister (then 7 years of age) is diagnosed on the basis of DNA analysis, although never symptomatic Two subsequent uncomplicated pregnancies (no HELLP or AFLP) one child affected, the second a carrier. MCAD Deficiency A Child with Multiple Problems The Pregnancy Complicated by abdominal pain, severe nausea and vomiting, “black out spells” Emergency C-section because of maternal hemorrhage (unknown cause) A Child with Multiple Problems The Child Multiple hospital admissions for vomiting and dehydration in first year of life Nissen fundoplication at 18 months of age. No improvement Hospitalization at 2 1/2 years for feeding disorder Episodes of weakness, shaking, decreased activity. No documented hypoglycemia Symptoms worse in the morning, and with routine illness A Child with Multiple Problems Diagnostic Investigation Initial metabolic evaluation negative Challenge testing: Hypoglycemia within12 hours of fasting Fat beta-oxidation intermediates in urine Fibroblast assays: reduced Palmitate oxidation and reduced LCHAD activity A Child with Multiple Problems Subsequent Pregnancy Prenatal diagnosis of LCHAD Mother developed HELLP syndrome A Twitchy Floppy Baby History Uncomplicated pregnancy and birth: 75th percentile 2 weeks: Colic 6 weeks: Muscle twitches Dramatic Hypotonia Tires when feeding Startles easily 8 weeks: Severe constipation Growth at 5th percentile A Twitchy Floppy Baby Examination Head circumference and weight below 5th percentile: length at 10th Dysmorphic face: telecanthus and prominent forehead, Flat occiput, ridged sutures Hypoplastic distal digital phalanges with arch fingerprint forms Severe hypotonia, no lateralizing neurological signs Fever of unknown origin (103F max) Spontaneous twitches of all extremities A Twitchy Floppy Baby Investigations Dysmorphologist: ? FG, ? Schinzel-Gideon Syndromes, Rule out chromosome anomaly, micro deletions. Karyotype and multiple FISH micro deletions: Negative Metabolic Screening: Mild Lactic Acidemia (Max 2.5 umol/L) EEG: Normal CK: 107 Carnitine: Total 33 Free 24 umol/L Skin Fibroblasts: Modest (55 %) reduction in Palmitate and Myristate Oxidation LCHAD and LCAT Enzyme activities normal Mitochondrial DNA deletion/mutation analysis: Unremarkable A Twitchy Floppy Baby The Crisis : Diagnosis and Treatment Anticonvulsants for twitching ineffective Klonopin Keppra Controversy: Seizures vs. Myoclonus Muscle Biopsy for Enzyme Analysis NADH Dehydrogenase 37% of controls Treatment: L-Carnitine Vitamins A,B-complex,C,E,K Ubiquinone (CoEnzyme Q) Sodium Succinate RRF Amino Acids Glucose (primarily alanine) LDH Pyruvate NADH + H+ (reduced) PC Gluconeogenesis LACTATE NAD+ (oxidized) PDH Acetyl CoA CO2 + H2O TCA Cycle Pyruvate Lactate NADH + H+ NAD+ (reduced form) (oxidized form) NADH + H+ + 1/2O2 NAD+ + H2O + energy Mitochondrial Metabolism CP1022045-76 Mitochondrial Chromosome “Not a Well Mitochondrion” H+ ATP ADP H+ NADH Succinyl-CoA H+ H+ NAD+ 1/2 O 2 H2 O SDH COMPLEX III COMPLEX I COMPLEX IV CoQ Cyt C V Primary Hyperlactacidemia • Disorders of Pyruvate Metabolism • • • • Krebs Cycle Defects Disorders of Gluconeogenesis • • • • • Pyruvate carboxylase deficiency Disorders affecting the pyruvate dehydrogenase complex (PDH) 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 disorders MMBID 8th ed. 2001, chapter 100, p 2289 Zschocke/Hoffmann: Vademecum Metabolicum. Schattauer 1999, p 13 Pedigree Assembly • Pedigree: A graphical representation of a family, showing relationships among individuals and medical and demographic information • Example: Maternal Inheritance Pedigree Heteroplasmy can lead to variable phenotypes in daughter cells Disease Map of the Mitochondrial Genome Factors that Complicate Analysis of Traditional Inheritance • • • • • • Reduced penetrance Variable expressivity Pleiotropy New mutations Gonadal mosaicism Genetic heterogeneity Gonadal (Germline) Mosaicism • One parent is is presumed to have a mixture of cells: mostly normal, but with at least some mutant germ cells. • Proportion of mutant gametes depends on when mutation occurred during mitotic expansion. • Presents counseling difficulty: What is the risk in the next pregnancy? A de novo mutation in a germ cell lineage produces mosaicism Case Report • Male infant born at 34 weeks gestational age to a G3/P1 mother • Maternal serologies were negative • Prenatal course revealed a fetus thought to be IUGR as well as oligohydramnios • Delivery was via C-section due to fetal decelerations Family History • Sibling who died at 25 weeks gestation secondary to non-immune hydrops • Healthy 18 month old sister • Both parents are in good health and unrelated Case 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) at 4 hrs of life and increased to 24 mM over the next 24 hrs • Electrolytes, glucose, NH3 normal • Sepsis work-up initiated Case Report 2nd 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 99 Physical Examination • Weight and length in the 10-25th percentile. Head circumference 5-10th percentile • Mild dysmorphic features including short palpebral fissures, mild micrognathia, long thumbs • No organomegaly • Muscle hypotonia Typical Organic Acid Profile in LCHAD Deficiency Dicarboxylic aciduria 3 Hydroxy dicarboxylic aciduria * *Internal standard TFP/LCHAD Deficiency Genes: - α-subunit gene (chromosome: 2p23) - β-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 death Diagnosis: TFP/LCHAD Deficiency - 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%) Treatment: - avoidance of fasting; - low-fat diet high in complex carbohydrates - fat primarily as medium chain triglycerides Elevated Lactic Acid in MTFP/LCHAD Deficiency • Direct inhibition of mitochondrial oxidative phosphorylation by 3-hydroxypalmitoyl-CoA (Ventura J Inher Met Dis 1996) • Long-chain acyl-carnitines may inhibit the pyruvate dehydrogenase complex (Moore Int J Biochem 1992) • Long-chain acyl-CoA esters inhibit the mitochondrial ATP/ADP carrier and the dicarboxylic carrier in vitro (Halperin PNAS 1972) Molecular Testing • Molecular studies revealed a homozygous 5 bp deletion in exon 4 of the alpha subunit of the MTFP gene involving base pairs 274 to 278 • This novel mutation creates a premature stop codon • Possible uniparental disomy Isovaleric 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 feeding ISOVALERIC ACIDEMIA Siblings: Healthy newborn girl, elevated C5-AcylCarnitine Increased urine isovalerylglycine Older brother ? Mild motor delays Increased Urine Isovalerylglycine “Self treats” – avoids excessive protein One parent found to have A282V mutation in IVCoADehydrogenase gene, now known to be associated with attenuated disease. Other parent’s mutation “classical”. MULTIFACTORIAL ? Siblings: Boy from a difficult pregnancy with low tone, failure to gain weight, recurrent illness with fluctuating blood sugars, unusual odor leg pains, muscle cramps, cardiomyopathy, pancreatic insufficiency. 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 liver Mother with chronic fatigue, neurological symptoms with brain scan leukodystrophy MULTIFACTORIAL ? Both boys: NBS suggested Glutaric Aciduria II: not confirmed by enzymes Unusual pattern of fat oxidation in skin cells Urinary hexanoylglycine: MCAD A985G Heterozygous for an Unusual new mutation in SPINK1, (Pancreatitis) A common variant in CFTR (Cystic Fibrosis) No mtDNA mutations Normal Electron Transport Chain Analysis Some Response to Carnitine, Glycine, Riboflavin and Low Fat Where Are Genes and What Do They Do? Chromosome One Gene Out of ~3000/chromosome Nucleus Location of Genes ) Regulatory Region 46 Total Cell B. mRNA Gene Expression (Transfer of genetic information) ) Function DNA C. Example of Genetic Information Transfer Nucleus ( Gene Expression (Transfer of Genetic Information) Gene RNA Code C G for C G aa 1 G C T A Code T A for aa 2 G C A T Code T A for aa 3 G C Protein aa 1 aa 2 aa 3 aa 4 aa aa 5 6 etc. Double Stranded DNA Protein ( A. Cell Protein Coding Region DNA 0.00006% of the human genome sequence 121 181 241 301 361 421 481 541 601 661 721 781 841 901 961 1021 1081 1141 1201 1261 1321 1381 1441 1501 1561 1621 1681 1741 1801 1861 1921 1981 2041 AACTGTGTTC TCTGCCGTTA GGCAGgttgg ggagacagag ttttcccacc TGGGGATCTG GAAAGTGCTC TGCCACACTG gagtctatgg taggaagggg agtgtggaag cttttgttta atgccttaac aaaaaacttt catattcata catatttatg taattttgca cttatttcta tgcctctttg tatttctgca gctaatagca ggattattct tcccacagCT TCACCCCACC CCCACAAGTA TCCCTAAGTC GCCTAATAAA tactaaaaag caaaccttgg gctaatgcac ttcttgtaga ttgttttagc tcagccttga ACTAGCAACC CTGCCCTGTG tatcaaggtt aagactcttg cttagGCTGC TCCACTCCTG GGTGCCTTTA AGTGAGCTGC gacccttgat agaagtaaca tctcaggatc attcttgctt attgtgtata acacagtctg atctccctac ggttaaagtg tttgtaattt atactttccc caccattcta tataaatatt gctacaatcc gagtccaagc CCTGGGCAAC AGTGCAGGCT TCACTAAGCT CAACTACTAA AAACATTTAT ggaatgtggg gaaaatacac attggcaaca ggcttgattt tgtcctcatg ct TCAAACAGAC GGGCAAGGTG acaagacagg ggtttctgat TGGTGGTCTA ATGCTGTTAT GTGATGGCCT ACTGTGACAA gttttctttc gggtacagtt gttttagttt tctttttttt acaaaaggaa cctagtacat tttattttct taatgtttta taaaaaatgc taatctcttt aagaataaca tctgcatata agctaccatt taggcccttt GTGCTGGTCT GCCTATCAGA CGCTTTCTTG ACTGGGGGAT TTTCATTGCa aggtcagtgc tatatcttaa gcccctgatg gcaggttaaa aatgtctttt ACCATGGTGC AACGTGGATG tttaaggaga aggcactgac CCCTTGGACC GGGCAACCCT GGCTCACCTG GCTGCACGTG cccttctttt tagaatggga cttttatttg tcttctccgc atatctctga tactatttgg tttattttta atatgtgtac tttcttcttt ctttcagggc gtgataattt aattgtaact ctgcttttat tgctaatcat GTGTGCTGGC AAGTGGTGGC CTGTCCAATT ATTATGAAGG atgatgtatt atttaaaaca actccatgaa cctatgcctt gttttgctat cactacccat TTTG ACCTGACTCC AAGTTGGTGG ccaatagaaa tctctctgcc CAGAGGTTCT AAGGTGAAGG GACAACCTCA GATCCTGAGA ctatggttaa aacagacgaa ctgttcataa aatttttact gatacattaa aatatatgtg attgatacat acatattgac taatatactt aataatgata ctgggttaag gatgtaagag tttatggttg gttcatacct CCATCACTTT TGGTGTGGCT TCTATTAAAG GCCTTGAGCA taaattattt taaagaaatg agaaggtgag attcatccct gctgtatttt ttgcttatcc CTTCTGACAC TGAGGAGAAG TGAGGCCCTG ctgggcatgt tattggtcta TTGAGTCCTT CTCATGGCAA AGGGCACCTT ACTTCAGGgt gttcatgtca tgattgcatc caattgtttt attatactta gtaacttaaa tgcttatttg aatcattata caaatcaggg ttttgtttat caatgtatca gcaatagcaa gtttcatatt ggataaggct cttatcttcc GGCAAAGAAT AATGCCCTGG GTTCCTTTGT TCTGGATTCT ctgaatattt atgagctgtt gctgcaacca cagaaaagga acattactta tgcatctctc Exons Identifying differences in gene expression with microarrays Egg Normal Tissue Tadpole Malignant Tissue Higher in Egg /Normal Tissue Higher in tadpole /Malignant Tissue Each spot contains DNA from a known cDNA or an EST From Lehninger From Lodish et al.
© Copyright 2020