Comprehensive Molecular Testing for Fatty Acid Oxidation Disorders. A Guide for Clinicians

Transcription

1 Comprehensive Molecular Testing for Fatty Acid Oxidation Disorders A Guide for Clinicians

2 Cincinnati Children s Hospital Molecular Genetics Laboratory introduces MetaboSeq, our next-generation sequencing panel of 19 genes (see Table 1) associated with fatty acid oxidation disorders (FAOD). MetaboSeq is the first test of its kind for diagnosing any of a large number of FAOD which often have overlapping clinical presentations. Fatty acid oxidation disorders are a group of inherited metabolic conditions. Mutation(s) in any one of a number of specific genes cause defective enzyme production in this key metabolic pathway. During times of fasting, illness, or exertion, fatty acids are normally mobilized in the body, but, in patients with FAOD, an enzyme defect results in the diminished ability to break down fatty acids into the energy necessary to overcome these stressors. Affected patients may show FAOD symptoms (liver failure, rhabdomyolysis, muscular hypotonia, cardiomyopathy with or without arrhythmias and/or renal tubular acidosis) within the first days to weeks of life; these disorders are often fatal if not detected early [Strauss et al. 2009]. Early initiation of treatment leads to good outcomes in most instances. GENETICS of FAOD: Newborn screening has tremendously aided the early diagnosis of FAOD. When an abnormal newborn screening result indicates a specific disorder, Sanger sequencing of the specific gene of interest can identify the underlying genetic cause of the disorder in many patients. However, biochemical screening results may show measurements that overlap with several conditions (e.g. acylcarnitine screening Gene Disorder Gene Disorder ACAD9 acyl-coa dehydrogenase-9 (ACAD9) deficiency uncovers cases of MCADD, M/SCHADD, MCKATD and glutaric acidemia type 2 alike) [Lindner et al. 2010]. Additionally, significant genetic heterogeneity has been reported for long-chain fatty acid oxidation disorders [Spiekerkoetter et al. 2010]. These factors highlight the utility, in some cases, of a comprehensive analysis of multiple genes involved in fatty acid oxidation. The MetaboSeq panel includes FAOD genes in the carnitine cycle and beta-oxidation pathway; as well as other integral pathway genes, including electron transporters (ETFA, ETFB, ETFDH), one gene involved in ketone synthesis (HMGCL), and two genes with phenotypic overlap with traditional FAODs (GLUD1 and TAZ)(see Figure 2). INDICATIONS for MetaboSeq: Abnormal newborn screen Unexplained neonatal hypoglycemia Recurrent maternal fatty liver of pregnancy Reye syndrome Rhabdomyolysis/skeletal myopathy Cardiomyopathy and/or arrhythmias Other nonspecific symptoms of FAOD including: liver failure vomiting lethargy seizures coma Note: Patients with acylcarnitine results or other clinical findings indicating a specific metabolic syndrome may benefit from molecular testing for mutations in that particular gene prior to MetaboSeq testing. Table 1. The MetaboSeq panel for fatty acid oxidation disorders includes tests for all of the genes included here. HADH short-chain 3-hydroxyacyl-CoA dehydrogenase (SCHAD) deficiency ACADM ACADS ACADVL CPT1A medium-chain acyl-coa dehydrogenase (MCAD) deficiency short-chain acyl-coa dehydrogenas (SCAD) deficiency very-long-chain acyl-coa dehydrogenase (VLCAD) deficiency carnitine palmitoyltransferase 1 (CPT1) deficiency CPT2 carnitine palmitoyltransferase 2 (CPT2) deficiency ETFA ETFB ETFDH multiple acyl-coa dehydrogenation (MAD) deficiency multiple acyl-coa dehydrogenation (MAD) deficiency multiple acyl-coa dehydrogenation (MAD) deficiency HADHA HADHB HMGCL HSD17B10 PPARG SLC22A5 SLC25A20 TAZ long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency or general trifunctional protein (TFP) deficiency general trifunctional protein (TFP) deficiency or isolated mitochondrial long-chain ketoacyl- CoA thiolase (LKAT) deficiency 3-hydroxymethyl-3-methylglutaryl-CoA lyase (HMG-CoA lyase deficiency 17-beta-hydroxysteroid dehydrogenase X (HSD10) deficiency peroxisome proliferator activated receptorgamma (PPAR-g) ligand resistance syndrome (PLRS) or familial partial lipodystrophy type 3 primary carnitine deficiency carnitine-acylcarnitine translocase (CACT) deficiency Barth syndrome or familial isolated noncompaction of the left ventricular myocardium GLUD1 congenital hyperinsulinic hyperammonemia (HI/HA) syndrome

3 Clinical utility of testing: A genetic diagnosis of an FAOD using MetaboSeq can assist in the treatment and management of a patient, provide prognostic information about the patient s condition, as well as supply information on which to base accurate genetic counseling and further testing recommendations for at-risk family members, perhaps before symptoms appear. Figure 1. Diagnostic algorithm for fatty acid oxidation disorders. Patient Symptoms Abnormal newborn screen Specific disorder suspected? Genetic testing for specific disorder Unknown which FAO disorder Unexplained neonatal hypoglycemia Negative Positive FAO clinical symptoms Maternal acute fatty liver of pregnancy (AFLP) DIAGNOSIS DIAGNOSIS

4 Figure 2. Pathway for mitochondrial fatty acid beta-oxidation. The letters represent key enzymatic steps and specific enzyme and corresponding gene names are in the key below the diagram. Long-chain Fatty Acid A Fatty Acid CoA Acyl-CoA CoA B Acyl- D CoA C Acyl- Dicarboxylic Acids M Acyl-CoA E F G H 2-enoyl 3-hydroxyacyl 3-ketoacyl -CoA -CoA -CoA I J K L Outside Cell Glucose Inside Cell (Cytosol) Glucose-6-P Mitochondrion Acyl-CoA Acyl-CoA (-2C) + Ketones TCA Cycle Electron Transfer Chain ATP KEY A B C D E SLC22A5 (OCTN2) CPT1A (CPT1) SLC25A20 (CACT) CPT2 (CPT2) ACADVL (VLCAD) F G H I J ACADM (MCAD) ACADS (SCAD) ACAD9 (ACAD9) ECHS1 (2-enoyl-CoA hydratase) HADHA (LCHAD, TFP) K L M HADH (SCHAD) HADHB (LCKAT, TFP) (TFP) References: Berardo, A., S. DiMauro, et al. (2010). A diagnostic algorithm for metabolic myopathies. Curr Neurol Neurosci Rep 10(2): Houten, S. M. and R. J. Wanders (2010). A general introduction to the biochemistry of mitochondrial fatty acid beta-oxidation. J Inherit Metab Dis 33(5): Lindner, M., G. F. Hoffmann, et al. (2010). Newborn screening for disorders of fatty-acid oxidation: experience and recommendations from an expert meeting. J Inherit Metab Dis 33(5): Lund, A. M., F. Skovby, et al. (2010). Clinical and biochemical monitoring of patients with fatty acid oxidation disorders. J Inherit Metab Dis 33(5): Spiekerkoetter, U. (2010). Mitochondrial fatty acid oxidation disorders: clinical presentation of long-chain fatty acid oxidation disorders before and after newborn screening. J Inherit Metab Dis 33(5): Strauss AW, Andresen BS, Bennett MJ (2009). Mitochondrial Fatty Acid Oxidation Disorders. Pediatric Endocrinology and Inborn Errors of Metabolism. Ed K Sarafoglou. New York, McGraw Hill. Tewhey, R., J. B. Warner, et al. (2009). Microdroplet-based PCR enrichment for large-scale targeted sequencing. Nat Biotechnol 27(11): Wanders, R. J., J. P. Ruiter, et al. (2010). The enzymology of mitochondrial fatty acid beta-oxidation and its application to follow-up analysis of positive neonatal screening results. J Inherit Metab Dis 33(5): Wilcken, B. (2010). Fatty acid oxidation disorders: outcome and long-term prognosis. J Inherit Metab Dis 33(5): M

5 Palmitoyltransferase CPT2 Deficiency (CPT2 Deficiency) Disorder: CPT2 deficiency is a disorder of long-chain fatty acid oxidation (FAOD) which may result in either of two distinct presentations based upon the affected individual s age and body systems affected. The infantile / neonatal (hepatocardiomuscular) form typically presents before one year of age with hypoketotic hypoglycemia, acute liver failure, cardiomyopathy, and/or sudden death, triggered by fasting or fever. A more severe neonatal form of CPT2 deficiency, including dysmorphic features, brain dysplasia, respiratory distress, hypoglycemia, cardiomegaly, heart rhythm disorders, cystic renal dysplasia, hepatomegaly, and neonatal death, has also been described. The adult (myopathic) form typically presents in young adults as episodes of muscle pain, weakness, and rhabdomyolysis triggered by prolonged exercise, or occasionally by illness or fasting. Complications which may arise from the rhabdomyolysis are acute renal failure, respiratory insufficiency, and paroxysmal arrhythmias. In the intervals between episodes, individuals with the classical adult CPT2 deficiency appear healthy. Males are more likely to be affected than females. CPT2 deficiency is inherited in an autosomal recessive manner. Indications: Confirmation of diagnosis in a symptomatic individual Abnormal newborn screen or acylcarnitine profile suggesting CPT2 deficiency Presymptomatic testing of at-risk siblings Carrier identification in individuals with a family history of CPT2 deficiency Prenatal diagnosis of an at-risk fetus, after confirmation of biallelic mutations in the parents (by prior arrangement only) Additional information and test requisitions are available at: Shipping Instructions Please enclose test requisition with sample. All information must be completed before sample can be processed. Place samples in styrofoam mailer and ship at room temperature by overnight Federal Express to arrive Monday through Friday Ship to: Cytogenetics and Molecular Genetics Laboratories 3333 Burnet Avenue NRB 1042 Cincinnati, OH Molecular Genetics Laboratory CLIA#: 36D Phone: (513) Fax: (513) moleculargenetics@cchmc.org

6 Specimen: At least 3 mls of whole blood in purple/lavender top (EDTA) tube. Label tube with patient s name, birth date, and date of collection. Phlebotomist must initial tube to verify patient s identity. Testing Methodology: Testing is performed by PCR-based sequencing of all 5 exons and exon/intron boundaries of the CPT2 gene. Test Sensitivity: PCR-based sequencing of all 5 exons and exon/intron boundaries of the CPT2 gene detects >95% of patients with CPT2 deficiency. The sensitivity of DNA sequencing is over 99% for the detection of nucleotide base changes, small deletions and insertions in the regions analyzed. Mutations in regulatory regions or other untranslated regions are not detected by this test. Large deletions involving entire single exons or multiple exons, large insertions and genetic recombinational events may not be identified using these methods. Rare primer site variants may lead to erroneous results. CPT2 is the only gene associated with CPT2 deficiency. MetaboSeq fatty acid oxidation defects panel detects mutations in CPT2 as well as 18 other genes involved with FAOD. Please see our website for details. Turn-Around Time: Four weeks Cost: Please call for current pricing, assistance with precertification or with any billing questions. Results: Each test report includes a detailed interpretation of the genetic findings, the clinical significance of the result, and specific recommendations for clinical management and additional testing, if warranted. Results will be reported to the referring physician or health care provider as specified on the test requisition form. References: Bonnefont, J. P., F. Djouadi, et al. (2004). palmitoyltransferases 1 and 2: biochemical, molecular and medical aspects. Mol Aspects Med 25(5-6): Bonnefont, J. P., F. Taroni, et al. (1996). Molecular analysis of carnitine palmitoyltransferase II deficiency with hepatocardiomuscular expression. Am J Hum Genet 58(5): Illsinger, S., T. Lucke, et al. (2008). -palmitoyltransferase 2 deficiency: novel mutations and relevance of newborn screening. Am J Med Genet A 146A(22): Orngreen, M. C., M. Duno, et al. (2005). Fuel utilization in subjects with carnitine palmitoyltransferase 2 gene mutations. Ann Neurol 57(1): Spiekerkoetter, U. (2010). Mitochondrial fatty acid oxidation disorders: clinical presentation of long-chain fatty acid oxidation defects before and after newborn screening. J Inherit Metab Dis 33(5): Taroni, F., E. Verderio, et al. (1993). Identification of a common mutation in the carnitine palmitoyltransferase II gene in familial recurrent myoglobinuria patients. Nat Genet 4(3): Wieser, T. (2004; updated 2011). Palmitoyltransferase II Deficiency. GeneReviews. R. A. Pagon, T. D. Bird, C. R. Dolan, K. Stephens and M. P. Adam. Seattle (WA). CPT Codes: CPT2 full gene sequence analysis: CPT2 family-specific mutation analysis: M

7 LEGEND '=Abnormal, C=Cntical, A= Interpretive Data, c=corrected, L=Low, H=High,!=Footnote, T =Textual Result Result Comments 113: Glucose Level Critical action: Notiiy physician Critical notiiy: dane mejas Read back: Yes Notiiy date: c&hedt!>dtfote 4112\' t/2ofs.. Cbller.:ted Time 05:00 POT PDT Procedure Units Sodium Level meq/l Potassium Level Chloride Lvl C02 Lvl Anion Gap Glucose Level BUN Crea1inine Estimated GFR.(Schwartz:.Bedside) Magnesium Serum Phosphorus.se.ru.m... Bilirubin Conjugated Bilirub1n Unconjugated AST(SGOT) ALT(SGPT) Alkaline Phosphatase,Serum Gamma Glutamyltransferase Ammonia,Blood Venous meq/l meq/l meq/l meq/l mg/dl mg/dl rngldl ml/min/1.73 m2 mg/dl rng/dl mg/dl mg/dl IU/L IU/L IU/L IU/L mcmol/l.la.ctic.. Acid.. Blood,Ve.nous.. Albumin Level 0.7 MMOL!L g/dl Reference Range [1,35.:.1.45J [ ] [96-110] [18-27] [ OJ [60-105] [6-20],,, 10 1:0 4:] [ ],,, [3 9:6 5.l [ ] [ ] [3-74] [6-45] [95-380],, [15-85] [9-33] [0.5:2 2J.. [ ] Seattle Children's Hospital NAME: XIE, J IANHUA DRACO PO Box 5371 DOB: 9/16/2012 Print Date: Seattle, Washington MRN: RRID: 9/ :52 PDT of 2081

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